The work in this thesis focuses on the study of three main aspects of proteins, viz, Protein structure, stability, and dynamics. Chapter 1 is a general introduction to the topics studied in this thesis. Chapter 2 deals with the first aspect, i.e., protein structure in which we describe an approach to use saturation mutagenesis phenotypes to guide protein structure prediction. Chapters 3 and 4 discuss how to increase protein stability using surface electrostatics, and Chapter 5 details a method to predict whether a proline substitution in a given protein would be stabilizing or destabilizing. Hence, Chapters 3-5 can be associated with the second aspect, i.e., protein stability. The third aspect, namely protein dynamics, is dealt with in Chapters 6 and 7 which study conformational dynamics of adenylate kinase. Protein structure prediction is a difficult problem with two major bottlenecks, namely, generation of accurate models and the selection of the most appropriate models from a large pool of decoys. In Chapter 2, the problem of model discrimination is addressed using mutant phenotype information derived from saturation mutagenesis library. A library of ~1500 single-site mutants of the E. coli toxin CcdB (Controller of Cell Division or Death B) has been previously constructed in our lab. The pooled library was characterized in terms of individual mutant phenotypes at various expression levels which were derived from the relative populations of mutants at each expression level. The relative populations of mutants were estimated using deep sequencing. Mutational tolerances were derived from the phenotypic data and were used to define an empirical parameter which correlated with a structural parameter, residue depth. We further studied how this new parameter can be used for model discrimination. Increasing protein stability in a rational way is a challenging problem and has been addressed by various approaches. One of the most commonly used approaches is optimization of protein core residues. Recently, optimization of protein surface electrostatics has been shown to be a useful approach for increasing stability of proteins. In Chapter 3, from analyses of a dataset of ~1750 non-homologues proteins, we show that proteins having a pI away from physiological pH, possess a significant fraction of unfavorably placed charged amino acids on their surface. One way to increase protein stability in such cases might be to alter these surface charges. This hypothesis was validated experimentally by making charge reversal mutations at putative unfavorable positions on the surface of maltose binding protein, MBP. The observed stabilization can potentially be increased by combining multiple individually stabilizing mutations. Different combinations of such mutations were made and tested in Chapter 4 to decide which mutants can be combined to achieve net stabilization. Ideas were tested through systematic experimentation which involved generation of two-site, three-site, and four-site mutations. A maximum increase in melting temperature (Tm) of 3-4 °C over wild-type protein was achieved upon combination of individually stabilizing mutants. Proline (Pro) has two special stereo-chemical properties when it is a part of a polypeptide chain. First the φ value of Pro has a very constrained distribution and second, Pro lacks an amide hydrogen. Due to these properties, introduction of Pro might perturb stability/activity of the protein. In Chapter 5 we describe a procedure to accurately predict the effects of Pro introduction on protein stability. Pro scanning mutagenesis was carried out on the model protein CcdB and the in vivo activity of the individual mutants was also examined. A decision tree was constructed, using the special stereo-chemical properties of Pro to maximize correlation of predicted phenotype with the in vivo activity. Binary classification as perturbing or non-perturbing of every Pro substitution was possible using the decision tree. The performance of the decision tree was assessed on various test systems, and the average accuracy was found to be ~75%. The role of conformational dynamics in enzyme catalysis has been explored in great detail in the literature. In Chapter 6, with the help of very long (350 ns), fully atomistic, explicit solvent molecular dynamics simulations, we studied conformational dynamics of adenylate kinase. We found the existence of a relatively stable state which lies intermediate between the open and closed conformations of the enzyme. The finding was further confirmed by computing a two dimensional configurational free energy surface when motions along each of the two movable domains (LID and NMP) are considered as reaction coordinates. We also discussed possible roles of the intermediate state during enzyme catalysis. The role of water in stabilization of intermediate states was also discussed. In Chapter 7, we studied dynamical coupling between LID and NMP domains of adenylate kinase during domain opening. Our observation suggests that the LID domain should start opening prior to the NMP domain. On the domain opening trajectory, the free energy surface of LID domain was found to be very rugged. We discuss a possible role of water in the ruggedness of the domain motions. The Appendix contains 3 supplementary parts of the thesis. Appendix I is a mutant dataset obtained from 454 sequencing analysis. It includes the normalized number of reads per mutation at each expression level along with mutational sensitivity score. Appendix II is parameters used for one of the electrostatic calculations. Appendix III contains a list of PDB ids used for database analysis in surface electrostatics work discussed in Chapter 3.

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Particulate contamination of the optical surfaces of solar collectors, often called "soiling", can have a significant deteriorating impact on energy yield due to the absorption and scattering of incident light. Soiling has more destructive effect on concentrated solar systems than on flat-plate photovoltaic panels, as the former are incapable of converting scattered sunlight. The first part of this thesis deals with the soiling losses of flat-plate photovoltaic (PV), concentrated solar power (CSP), and concentrated photovoltaic (CPV) systems in operation in several regions of the world. Influential parameters in dust accumulation losses, as well as different cleaning mechanisms in pursuit of restoring the efficiency of soiled systems, have been thoroughly investigated. In lieu of the most commonly-practiced manual cleaning method of using high-pressure water jets, the concept of automatic dust removal using the electrostatic forces of electrodynamic screen (EDS) technology is in a developmental stage and on its way toward commercialization. This thesis provides comprehensive analytical solutions for the electric potential and electric field distribution in EDS devices having different configurations. Numerical simulations developed using finite element analysis (FEA) software have corroborated the analytical solutions which can easily be embedded into software programs for particle trajectory simulations while also providing flexibility and generality in the study on the effect of different parameters of the EDS on the electric field and ensuing dust-removal performance. Evaluation and comparison of different repelling and attracting forces exerted on dust particles is of utmost importance to a detailed analysis of EDS performance in dust removal. Hence, the balance of electrostatic and adhesion forces, including van der Waals and capillary forces, have received significant attention in this dissertation. Furthermore, different numerical analyses have been conducted to investigate the potential causes of observed failures of EDS prototypes that functioned well in a laboratory environment but failed after outdoor exposure. Experimental studies form the last two chapters of this dissertation. Different tests have been conducted on an EDS sample integrated with a PV cell to restore the efficiency of the cell after dust deposition. In order to evaluate the performance of the EDS in dust-particle removal, we have studied the particle size distribution on the EDS surface after each dust deposition and EDS cleaning cycle using a custom-built dust-deposition analyzer. Furthermore, we have pursued several experiments to examine how the geometric and operational EDS parameters affect particle charge via charge-to-mass-ratio measurements.

This study investigated the relationship between teachers' pedagogical content knowledge (PCK) about electrostatics and learners' performance in the topic. Two in-service and two pre-service physical science teachers, together with their learners, agreed to participate in the study. The PCK of the teachers was viewed as two manifestations; the personal PCK, which is static in nature and the enacted PCK, which is dynamic. A content representation (CoRe) tool and lesson plans were used to collect data that reflected the personal PCK of the teachers. The data for the enacted PCK was collected using classroom observations, and video stimulated recall (VSR) interviews. A topic specific PCK model was adopted as the framework for this study. The model asserts that specific content is transformed for instruction through five components, namely; learners' prior knowledge, curricular saliency, what is difficult to teach, representations including analogies, and conceptual teaching strategies. Guided by the model, I developed two rubrics to assess and quantify the quality of the teachers' personal and enacted PCK on a four-point scale. Learners, on the other hand, wrote a test developed specifically for this study which explored their performance in the fundamental concepts chosen for this study. The performance of the learners was then related to the personal and the enacted PCK of the teachers separately. The results revealed that the personal and the enacted PCK of the teachers, as well as the performance of the learners, varied across fundamental concepts of electrostatics. The variations in the personal and enacted PCK provided empirical evidence that supports the notion that PCK has a concept specific nature. The results also showed that the performance of the learners was better related to the enacted PCK of the teachers compared to the personal PCK. These results imply that it is important to make teaching practice the centre of pre-service teacher education given the direct impact of enacted PCK on learning. Furthermore, exploring PCK at concept level reveals the strengths and weaknesses of the concepts. As such, pre-service teacher education and in-service teacher professional development may be tailored in a manner that addresses the concepts that require intervention. / Thesis (PhD)--University of Pretoria, 2020. / Science, Mathematics and Technology Education / PhD / Unrestricted

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CLIC1 is an intracellular membrane protein that is unusual in that it can exist in both a soluble and an integral membrane form. The manner in which this protein inserts into membranes is unknown although it is proposed to undergo a change in structure whereby it initially experiences a degree of unfolding and then refolds into its new membrane-bound conformation. This study focuses on the characterisation of CLIC1 in terms of its secondary, tertiary and quaternary structure, the determination of its conformational stability at equilibrium and the establishment of its unfolding kinetics, all under conditions of varying pH, polarity, redox conditions, temperature and ionic strength. CLIC1 was found to be most stable at pH 7.0 / 20oC. The unfolding process is two-state and cooperative, producing a DG(H2O) of ~10 kcal/mol and a m-value of ~2 kcal/mol per molar urea. A decrease in pH to 5.5 or an increase in temperature to 37oC resulted in the stabilisation of an equilibrium intermediate species under mild denaturing conditions and a destabilisation of the native state. This was further evidenced by an increase in the rate of unfolding of CLIC1 from the native state to the denatured state under these conditions. A state with similar properties to the intermediate species was detected in the absence of urea at pH 5.5 / 37oC and under non-reducing conditions at both pH 7.0 / 20oC and pH 5.5 / 20oC. The intermediate species is more hydrophobic than either the native or denatured state; it is stabilised by salts, has a reduced secondary structure, increased flexibility and a buried Trp35 relative to the native state. The rate of formation of the intermediate species is a slow process which may involve an oligomerisation step. The results from this study provide an interpretation for the structure and mechanism of CLIC1 pore formation in vivo by comparing the effects of the environment on the structure and stability of the protein.

Electrostatic charge generation in gas-solid fluidized beds is a significant industrial problem. Associated problems include particle agglomeration and particle wall fouling. In the polymerization industry this may result in "sheets" of fused polymer, due to exothermic reaction causing the melting of the polymer, which can fall off and block the distributor plate disrupting fluidizing gas flow. Additionally, blockage of the catalyst feed or the polymer removal system can take place or the product can become non-uniform. All of these problems require shut-down of the reactor which results in lost production time. While this phenomena has been identified for many years, the mechanisms involved are not well understood, especially wall fouling and the distribution of charge within the bed. Isolation of individual parameters such as hydrodynamics, operating conditions, and material involved is necessary to evaluate how each parameter impacts charge generation during fluidization. In this thesis, the fluidization system consisted of a stainless steel column, two online Faraday cups, and a retractable distributor plate. This system allowed for the simultaneous measurement of charge within different regions of the bed: the entrained fine particles, the particles adhered to the column wall, and the bulk of the bed. Additionally, mass and particle size distributions were measured and images of the layer of particles adhered to the column wall were taken for comparison. This allowed for a charge distribution comparison and evaluation of wall fouling. Three different parameters were investigated: duration of fluidization, column wall material, and relative humidity of fluidizing gas. Fluidization time was studied for 15, 30, 60, 120, 180, and 360 min; relative humidity was investigated for 0%, 20%, 40%, 60%, and 80% relative humidity. Both fluidization time and relative humidity were evaluated at four different fluidization gas velocities, two each in the bubbling and slugging flow regimes. Column wall material was evaluated for a stainless steel and carbon steel column at two gas velocities, one each in the bubbling and slugging flow regimes. Fluidization time was found to influence wall fouling in the bubbling flow regime as the particle layer continued to build as fluidization progressed. In the slugging flow regime, the particle layer developed within 15 minutes of the onset of fluidization. The bubbling flow regime was shown to have a greater capacity for charge generation than the slugging flow regime. This was due to the vigorous mixing in the bubbling flow regime resulting in more particle-particle interactions. Column wall material was shown to influence wall fouling in the slugging flow regime due to the differences in surface roughness of the columns. This was due to the particle-wall contacts resulting in frictional charging which is the predominant charging mechanism in this flow regime. Charge was also impacted in the bubbling flow regime in those particles that were adhered to the column wall. Relative humidity was found to influence wall fouling at the lowest gas velocity tested. However, variations in generation of charge occurred at all fluidization gas velocities tested; the charge-to-mass ratios for the particles adhered to the column wall in the slugging flow regime decreased with high relative humidities. This was due to either the formation of a water film layer on the column wall or instantaneous surface water films on the particles throughout fluidization.

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