Kinetics and dynamics of electrophoretic translocation of polyelectrolytes through nanopores

The idea of sequencing a DNA based on single-file translocation of the DNA through nanopores under the action of an electric field has received much attention over the past two decades due to the societal need for low cost and high-throughput sequencing. However, due to the high speed of translocation, interrogating individual bases with an acceptable signal to noise ratio as they traverse the pore has been a major problem. Experimental facts on this phenomenon are rich and the associated phenomenology is yet to be fully understood. This thesis focuses on understanding the underlying principles of polymer translocation, with an emphasis on pore-polymer interactions, polymer architecture, and polymer chain fluctuations. Langevin dynamics simulations are used to study a variety of polymer and pore designs. For a uniformly charged linear polymer, a nanopore with charge patterns along its length is proposed. Variation in the charge pattern length reveals the existence of a critical length at which the polymer is trapped, causing a significant delay during the pore emptying stage. This trapping is modeled using an appropriate free energy landscape and the Fokker-Planck formalism. The predictions of this theory are in qualitative agreement with the simulation results across different pore and polymer lengths. Moreover, a linear polymer with charge patterns along its backbone passing through such a charge-patterned pore shows rich kinetic behavior; a significant delay is introduced even in the pore entrance and threading stages due to pattern matching, suggesting the use of pore-polymer interactions to slow down translocation. In a related study, the translocation of charged star polymers through an uncharged pore is simulated. Star polymers with different functionalities show rich translocation kinetics while passing through such a pore. The mean translocation time varies non-monotonically with the polymer functionality, suggesting the use of nanopores as a filtering and analytical technique for star polymers. Recent experiments have suggested the use of phi29 polymerase in conjunction with a protein pore (α-Hemolysin) in the presence of an electric field to slow down the polymer translocation speed, enabling reasonably successful base-calling. The role of polymer chain fluctuations inside the nanopore is evaluated using Langevin dynamics simulations on models of this construct. By monitoring the contributions of the conformational fluctuations of the polymer, the diffusional behavior of monomers of the chain under the speed resulting from the polymerase activity and externally imposed voltage gradients is computed. The simulations show that even if the translocation speed is slowed down considerably by using the polymerase-nanopore construct, the conformational fluctuations of ssDNA inside the pore are always present at high levels, resulting in high levels of noise in the detection signal

Study of variance, heritability and genetic advance for various yield contributing and quality traits in spring wheat (Triticum aestivum L.)

An experiment was conducted to study the coefficient of variance, heritability and genetic advance for different traits in spring wheat .The investigation comprised of 7 lines of wheat and their 21 crosses in half diallel fashion was carried out in RBD with three replications. Data were recorded for days to 75% heading, days to maturity,plant height, number of productive tillers per plant, spike length, number of spikelets per spike, number of grains per spike, grain weight per spike, 1000-grain weight, biological yield per plant, harvest index, grain yield per plant, protein content and sedimentation value. The mean squares of the analysis of variance revealed significant and highly significant differences among genotypes for characters studied. Both PCV and GCV (21.8% and 21.3%) were highest for biological yield per plant followed by grain yield per plant (20.9%) and harvest index (19.7%) respectively. Whereas, ECV was maximum (15.2%) for grain yield per plant followed by harvest index (12.2%) and lowest value was recorded for days to 75% heading (0.85%). Days to 75% heading was highly heritable (90.94%) trait followed by plant height (87.23%) while least heritability (17.73%) was noticed for number of grain per spike. The highest genetic advance shown by the biological yield per plant (48.33g) followed by grain yield per plant (19.75g), however, the greatest genetic value percent mean 43.084 for the days to maturity and minimum (2.10) for spike length. Hence, these statistical parameters might be given top priority to strengthen the successful breeding program

Combining ability and gene interaction study for yield, its attributing traits and quality in common wheat

Combining ability and nature of gene interactions that contribute to yield and its attributing traits were investigated using 21 wheat hybrids developed by crossing 7 varieties in a half diallel mating design. Estimate of GCA effects exhibited that the parents UP2672, UP2526 and WH542 were identified as good general combiners revealing their ability in transmitting additive genes in desirable direction to their progenies. Hybrid viz., PBW 621 × UP 2425 (15.125) found to be the best specific crosses for grain yield plant-1, whether, WH 542 × HD 2967 (22.587) and UP 2526 × UP 2425 (14.490) had the highest SCA for biological yield plant-1 and harvest index, respectively. However, the best specific cross combinations for other characters were found for WH 542 × QLD 40 (-3.694) for days to maturity, PBW 621 × UP 2526 (-3.819) for plant height, HD 2967 × UP 2526 (7.527) for 1000 grain weight and WH 542 × UP 2672 (2.077) for sedimentation value. While crosses PBW 621 × UP 2425, UP 2526 × UP 2425 and QLD 40 × UP 2425 were found to be the best specific combiner for the characters number of productive tillers plant-1, grain yield plant-1, spike length, grain weight spike-1, harvest index, days to 75 % heading and protein content

Modelling and Characterization of Magnetic Microfibers

Polymer fibers of varying microfluidic properties can be fabricated in a lab setting. Fibers coated with paramagnetic particles act like slender paramagnetic beams. These fiber moves in a magnetic field. Thus polymer fibers coated with paramagnetic particles can be used as actuators in various microfluidic applications, such as DNA separation, droplet manipulation and liquid transport. In order to use the fibers as actuators, it is necessary to model the fiber and develop control strategies. A static model of the paramagnetic fiber based on energy methods is presented in [1]. The model relies on a demagnetizing factors approximation to determine the magnetic field inside the fiber. The first part of the thesis examines the conditions under which the demagnetizing factors approximation holds. The model allows for implementation of simple feedforward control strategies to control the position of the fiber. For implementation of better control algorithms, methods to sense the shape and the tip position of the fiber are required. These sensing methods are also presented here. The model depends on the bending rigidity and magnetic susceptibility of the fiber. Since the fibers can be synthesized in a lab setting, these properties are usually not known. This thesis also presents methods to characterize the bending rigidity of the fiber, based on the sensing methods. The bending rigidity and the magnetic susceptibility of the fiber, along with the model can be used to implement a basic feedforward control strategy to accurately position the tip of the fiber. This enables the use of the fiber as a microfluidic actuator

Online Parameter Estimation and Adaptive Control of Magnetic Wire Actuators

Cantilevered magnetic wires and fibers can be used as actuators in microfluidic applications. The actuator may be unstable in some range of displacements. Precise position control is required for actuation. The goal of this work is to develop position controllers for cantilevered magnetic wires. A simple exact model knowledge (EMK) controller can be used for position control, but the actuator needs to be modeled accurately for the EMK controller to work. Continuum models have been proposed for magnetic wires in literature. Reduced order models have also been proposed. A one degree of freedom model sufficiently describes the dynamics of a cantilevered wire in the field of one magnet over small displacements. This reduced order model is used to develop the EMK controller here. The EMK controller assumes that model parameters are known accurately. Some model parameters depend on the magnetic field. However, the effect of the magnetic field on the wire is difficult to measure in practice. Stability analysis shows that an inaccurate estimate of the magnetic field introduces parametric perturbations in the closed loop system. This makes the system less robust to disturbances. Therefore, the model parameters need to be estimated accurately for the EMK controller to work. An adaptive observer that can estimate system parameters on-line and reduce parametric perturbations is designed here. The adaptive observer only works if the system is stable. The EMK controller is not guaranteed to stabilize the system under perturbations. Precise tuning of parameters is required to stabilize the system using the EMK controller. Therefore, a controller that stabilizes the system using imprecise model parameters is required for the observer to work as intended. The adaptive observer estimates system states and parameters. These states and parameters are used here to implement an indirect adaptive controller. This indirect controller can stabilize the system using imprecise initial parameter estimates. The indirect adaptive controller overcomes the limitations of the EMK controller by stabilizing the closed loop system despite inaccurate initial parameter estimates. The experiment setup used to test the controllers is also presented. Experiments were performed to test the adaptive controller using cantilevered cobalt and nickel wires. The closed loop system using the indirect controller is stable. The wire tracks continuous desired trajectories up to 30Hz. Experiments were also performed to test the robustness of the adaptive and EMK controllers when the wire is interacting with water. The adaptive controller performs poorly when unmodeled disturbances are encountered, necessitating fall back to the EMK controller in some applications. The adaptive controller functions as an EMK controller if observer gain is set to 0. Thus, the indirect adaptive controller estimates model parameters, stabilizes the wire in the unstable region and can be switched into a non-adaptive mode for applications

Structural and functional characterization of tautomerase and aspartase/fumarase superfamily enzymes

Biocatalysis is a branch of biochemistry that exploits the ability of an enzyme to convert a substrate into a compound of economic value in an environmentally friendly manner. Enzymes are known to catalyze complex chemical reactions in their active site pockets and it is important to understand the intricacies of the reaction mechanism at the molecular level. X-ray crystallography, a powerful technique, is used to calculate the position of individual atoms comprising an enzyme in the three-dimensional space. Solving structures of enzymes in complex with a ligand tells us how a compound binds in the enzyme active site and the role of the different amino acid residues during catalysis. This knowledge further enables the engineering of enzymes for efficient and selective catalysis. The thesis of Harshwardhan Poddar describes the structural, functional and mechanistic characterization of three enzymes belonging to the tautomerase and aspartase/fumarase superfamily of enzymes. 4-Oxalocrotonate tautomerase (4-OT) is a versatile enzyme capable of promiscuously catalyzing synthetically useful C-C bond-forming reactions. The mechanism of these important reactions has been elucidated by solving structures of the wild-type and engineered 4-OT variants in complex with substrates. In addition, a new member of the tautomerase superfamily, RhCC, was identified and shown to catalyze a cofactor-independent oxygenation reaction, previously unseen in any other member of this superfamily. Finally, a new enzyme capable of catalyzing C-N bond forming reactions, called ethylenediamine-N,N’-disuccinic acid lyase (EDDS lyase), was identified in the aspartase/fumarase superfamily of enzymes. Crystal structures in complex with substrates and product gave important insights into the mechanism and synthetic potential of this robust enzyme. These three enzymes have shown their potential in the production of valuable compounds such as precursors for pharmaceuticals and various food additives, and the work described in this thesis brings forth new opportunities for further research into the optimization of the properties of these enzymes for industrial applications