FUNCTIONAL CHARACTERIZATION OF HEXOKINASE-LIKE 1 (HKL1) FROM ARABIDOPSIS THALIANA

Arabidopsis hexokinase1 (AtHXK1) is a moonlighting protein with roles in both glucose signaling and catalysis. In this study, we first cloned and characterized the six HXK related genes from Arabidopsis. Three of the six encoded proteins were shown not to phosphorylate hexoses and thus, are designated as hexokinase-like (HKL) proteins. Though they are only 50% identical to HXK1, the amino acid sequences of HKL1 and HKL2 both have well conserved glucose binding domains and other recognized structural elements. The possible basis for their lack of catalytic activity was further probed by site-directed mutagenesis and ultimately was attributed to a suite of amino acid substitutions. Gene expression studies showed that transcripts of HKL1 and HKL2 occur in most plant tissues, thus supporting the hypothesis that they have regulatory functions. The function of AtHKL1 was more closely examined using a reverse genetics approach. We identified a T-DNA knockout mutant for HKL1 and made HKL1 overexpression lines in different genetic backgrounds. Their phenotypes showed that HKL1 is a negative regulator of plant growth. Interestingly, many of the phenotypes required the presence of HXK1 protein. Both HKL1-GFP and HXK1-GFP are expressed at mitochondria and both were shown to interact with each other by coimmunoprecipitation assays. However, even though the HKL1 phenotypes included some dependence on glucose treatments, we conclude that HKL1 likely does not have a direct role in glucose signaling. Instead, we found from seedling signaling assays and a novel root hair phenotype that HKL1 mediates plant growth responses at least in part by promoting ethylene biosynthesis and/or signaling. Overall, these studies have helped to identify, characterize, and define the function of non-catalytic HXKs from Arabidopsis

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

FUNCTIONAL CHARACTERIZATION OF CALCIUM DEPENDENT PROTEIN KINASE 32 FROM ARABIDOPSIS

Calcium-dependent protein kinases (CPKs) are major plant Ca2+ sensors, many of which have roles in plant stress responses. The Arabidopsis genome encodes 34 CPK isoforms. Here we report characterization of AtCPK32 gene function. Analysis of transgenic plants expressing pCPK32-GUS shows that CPK32 is highly expressed in roots, pollen and embryo, as well as leaf hydathodes, and the abscission zone of mature siliques. Real time RT-PCR and promoter expression patterns show that CPK32 is responsive to abiotic and biotic stresses. Plants treated with salt, ABA, osmotic stress (PEG), wounding, and flagellin 22 peptide show up-regulation of CPK32 upon these stress treatments. The overexpression of CPK32 results in ABA and salt insensitive phenotypes whereas disruption of CPK32 gene by T&ndashDNA insertion leads to ABA and salt hypersensitive phenotypes in seed germination and early seedling growth assays. Interestingly, CPK32 overexpression plants are sensitive to drought whereas cpk32&ndash1 mutant plants are drought resistant suggesting that ABA and salt might be operating independent of drought stress tolerance. In a protoplast transient expression assay CPK32 is localized to the plasma membrane. Upon ABA treatment, CPK32 quickly moves from the plasma membrane to the cytosol and nucleus. Two key posttranslational modifications, myristoylation and palmitoylation play a crucial role in sub-cellular targeting of CPK32 to the plasma membrane. Mutation of these acylation sites leads to cytosolic and nuclear localization of CPK32 protein. Together our data provides evidence that CPK32 is a negative regulator of ABA signaling and is involved in multiple stress signaling pathways

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

Role of pyridoxine 5\u27-phosphate oxidase in metabolism and transfer of pyridoxal 5\u27-phosphate

Deficiency of vitamin B6 due to mutations in key B6 metabolizing enzymes is suspected to contribute to several pathologies. Vitamin B6 in its active form, pyridoxal 5’-phosphate (PLP) is a cofactor for over 140 known B6 requiring (or PLP-dependent) enzymes, that serve vital roles in many biochemical reactions. There are three primary vitamin B6 forms, pyridoxine (PN), pyridoxamine (PM) and pyridoxal (PL) which are phosphorylated to pyridoxine 5’-phosphate (PNP), pyridoxamine 5’-phosphate (PMP) and PLP respectively. Pyridoxal kinase (PLK) and pyridoxine 5’-phosphate oxidase (PNPO) are the key enzymes involved in both salvage and de novo pathways of PLP biosynthesis. Mutations in these enzymes are one of the most important causes of PLP deficiency, apart from dietary insufficiency of vitamin B6 and drug inhibition of PLK and PNPO. One of our objectives is to understand the molecular basis of reduced catalytic activity of PNPO in case of the R95C homozygous missense natural mutant, which leads to the PLP deficiency and the debilitating disease, neonatal epilepsy encephalopathy. Using site-directed mutagenesis, circular dichroism, enzyme kinetics and fluorescence spectroscopy, we have shown that the reduced enzymatic activity exhibited by PNPO R95C mutant is due to reduced binding affinity of the oxidase cofactor, flavin mononucleotide (FMN), which is required by the enzyme for oxidizing the inactive B6 vitamers into the active PLP. High concentrations of B6 are linked to neurotoxic effects, which can be attributed to the highly reactive aldehyde group of PLP which reacts with many nucleophiles in the cell. This reactivity is most likely why the in vivo concentration of “free” PLP is about 1 μM, raising the intriguing question of how the cell supplies sufficient PLP to meet the requirements of the numerous B6 dependent enzymes. Our second objective is to determine how despite the low in vivo concentration of free PLP, enough of this co-factor is made available to activate PLP-dependent enzymes. We have used affinity pull down assays, fluorescence polarization and enzyme kinetics to show that PNPO forms specific interactions with B6 enzymes with dissociation constants less than 1 µM. We also show that transfer of PLP from PNPO possibly occurs by compartimentalization or channeling. Although, channeling is a controversial subject, it offers an efficient, exclusive, and protected means of delivery of the highly reactive PLP. High concentrations of B6 are linked to neurotoxic effects, which can be attributed to the highly reactive aldehyde group of PLP which reacts with many nucleophiles in the cell. This reactivity is most likely why the in vivo concentration of “free” PLP is about 1 ?M, raising the intriguing question of how the cell supplies sufficient PLP to meet the requirements of the numerous B6 dependent enzymes. Our second objective is to determine how despite the low in vivo concentration of free PLP, enough of this co-factor is made available to activate PLP-dependent enzymes. We have used affinity pull down assays, fluorescence polarization and enzyme kinetics to show that PNPO forms specific interactions with B6 enzymes with dissociation constants less than 1 µM. We also show that transfer of PLP from PNPO possibly occurs by compartimentalization or channeling. Although, channeling is a controversial subject, it offers an efficient, exclusive, and protected means of delivery of the highly reactive PLP

Money handling influences BMI: a survey of cashiers

Money is a recent phenomenon in the evolutionary history of man and therefore no separate brain centre to handle money is likely to have evolved. The brain areas activated by food reward and money reward are extensively overlapping. In an experimental set-up, hunger was demonstrated to influence money related decisions and money related thoughts to influence hunger. This suggests that the brain areas evolved for handling food related emotions are exapted to handle money and therefore there could be a neuronal cross-talk between food and money. If this is true then attitude and behavior related to money and wealth could influence obesity. We conducted a survey of 211 individuals working as full time cashiers in order to test whether ownership over the cash, the amount of cash handled per day and the duration of cash handling work affected their body mass index (BMI). Cashiers who had ownership over the money had a significantly higher age corrected mean BMI than salaried cashiers. The BMI correlated positively with duration of service as cashier even after correcting for age and duration of sedentary job in males. Among salaried cashiers of both sexes, bank cashiers whose mean daily cash handling was one or two orders of magnitude greater than that of shop cashiers, had a significantly higher BMI. The effects of amount of money handled per day, years of service as cashier and ownership over the money handled could be shown to influence BMI independent of each other. The results support the exaptation hypothesis and suggest that the changing economy and attitudes towards money may be a contributing factor to the current obesity epidemic

Environmental complexity is more important than mutation in driving the evolution of latent novel traits in E. coli

Recent experiments show that adaptive Darwinian evolution in one environment can lead to the emergence of multiple new traits that provide no immediate benefit in this environment. Such latent non-adaptive traits, however, can become adaptive in future environments. We do not know whether mutation or environment-driven selection is more important for the emergence of such traits. To find out, we evolve multiple wild-type and mutator E. coli populations under two mutation rates in simple (single antibiotic) environments and in complex (multi-antibiotic) environments. We then assay the viability of evolved populations in dozens of new environments and show that all populations become viable in multiple new environments different from those they had evolved in. The number of these new environments increases with environmental complexity but not with the mutation rate. Genome sequencing demonstrates the reason: Different environments affect pleiotropic mutations differently. Our experiments show that the selection pressure provided by an environment can be more important for the evolution of novel traits than the mutational supply experienced by a wild-type and a mutator strain of E. coli