Development Of Tools For Phosphosite-Specific Kinase Identification And Discovery Of Phosphatase Substrates

Phosphorylation is a ubiquitous post translational modification implicated in many diseases, such as cancer. The phosphorylation status of cellular proteins is regulated by the activity of kinases and phosphatases. The biological significance of many phosphorylation events remain unknown because the methods to determine which kinase or phosphatase is responsible for phosphorylation are limited. Previously, we established kinase-catalyzed labeling where kinases accept γ-modified ATP analogs, such as ATP-arylazide and ATP-biotin, to label phosphoproteins. To study substrates of kinases and phosphatases, here we developed two new methods using kinase-catalyzed labeling. As one application, we developed K-CLASP (Kinase-catalyzed CrossLinking And Streptavdin Purification) to identify the in-cellulo kinase of a phosphorylated site on a protein. In this case, we used ATP-arylazide to mediate crosslinking between a biotin tagged peptide carrying a phosphosite of interest and the respective kinase. Using Protein kinase A (PKA) and its known peptide substrate kemptide, we demonstrated that K-CLASP is capable of identifying PKA as the kinase responsible for kemptide phosphorylation in cell lysates. Then we used K-CLASP to identify the kinases that phosphorylate S178 of the Miz1 protein in a collaboration project. For phosphatase substrate identification, we developed K-BIPS (Kinase-catalyzed Biotinylation to Identify Phosphatase Substrates). In prior work, we observed that labeling of phosphoproteins by ATP-biotin is reduced when phosphatases are inactive. The phosphatase dependency of biotinylation is due to the presence of already existing phosphorylation, which prevents ATP-biotin labeling. Therefore, in K-BIPS, ATP-biotin labeling is carried out after the inactivation of a particular phosphatase. The loss in biotinylation can then be analyzed to reveal substrates. To establish K-BIPS as a viable method, we carried out ATP-biotin labeling in lysates treated with the general phosphatase inhibitor okadaic acid. Many known phosphatase substrates were observed validating our method. Then as further applications, we used K-BIPS to explore substrates of PP1-Gadd34 and PP1-MYPT1 phosphatase complexes. The results demonstrate that K-BIS-Phos is a feasible method for phosphatase substrate identification. In summary, we have developed two chemical tools based on kinase-catalyzed labeling that will enhance our understanding of phosphorylation events mediated by kinases and phosphatases

SPIN-ORBIT COUPLING EFFECTS IN InSb QUANTUM WELL STRUCTURES

Indium antimonide (InSb) is a narrow band gap material which has the smallest electron effective mass (0.014m0) and the largest electron Lande g-facture (-51) of all the III-V semiconductors. Spin-orbit effects of III-V semiconductor heterostructures arise from two different inversion asymmetries namely bulk inversion asymmetry (BIA) and structural inversion asymmetry (SIA). BIA is due to the zinc-blende nature of this material which leads to the Dresselhaus spin splitting consisting of both linear and cubic in-plane wave vector terms. As its name implies SIA arises due to the asymmetry of the quantum well structure, this leads to the Rashba spin splitting term which is linear in wave vector.Although InSb has theoretically predicted large Dresselhaus (760 eVÅ3) and Rashba (523 eÅ2) coefficients there has been relatively little experimental investigation of spin-orbit coefficients. Spin-orbit coefficients can be extracted from the beating patterns of Shubnikov-de Haas oscillations (SdH), for material like InSb it is hard to use this method due to the existence of large electron Lande g-facture. Therefore it is essential to use a low field magnetotransport technique such as weak antilocalization to extract spin-orbit parameters for InSb.The main focus of this thesis is to experimentally determine the spin-orbit parameters for both symmetrically and asymmetrically doped InSb/InxAl1-xSb heterostructures. During this study attempts have been made to tune the Rashba spin-orbit coupling coefficient by using a back gate to change the carrier density of the samples. Dominant phase breaking mechanisms for InSb/InxAl1-xSb heterostructures have been identified by analyzing the temperature dependence of the phase breaking field from weak antilocalization measurements. Finally the strong spin-orbit effects on InSb/InxAl1-xSb heterostructures have been demonstrated with ballistic spin focusing devices

Diagnostics and modeling of polymer electrolyte membrane water electrolysers

Proton exchange membrane water electrolyser (PEMWE) technology can be used to produce hydrogen from renewable energy sources; the technology is therefore a promising component in future national power and transportation fuel systems. The main challenges faced by the technology include prohibitive materials costs, maximising efficiency and ensuring suitable longevity. Therefore, research is needed to understand the internal operation of the systems so that cell design can be optimised to obtain maximum performance and longevity. PEMWE is a low temperature electrolysis system that consists of cell components such as end plates, current collectors, bipolar plates, gas diffusion layers (GDLs) and membrane electrode assemblies (MEAs). Cell performance is strongly reliant on the materials and designs of each of the components. Three cell designs were used to study different aspects of PEMWE operation: commercial cell, optically transparent cell and combined optical and current mapping cell. Polarisation measurements performed on a commercially available lab-scale test cell at ambient conditions illustrated an increase in mass transport limitations with increasing water flow rate which was confirmed using electrochemical impedance spectroscopy (EIS) measurements. A transparent cell was constructed to allow optical access to the flow channels. Measurements made on the cell showed a transition from bubbly to slug flow that affects mass transport limitations and consequently the electrochemical performance. Thermal imaging measurements supported a mass and energy balance of the system. Finally, a combined transparent and current mapping cell was constructed using PCB technology that indicated higher current densities closer to the exit of the channel. Optical measurements showed that this increase in current was associated with larger bubbles and a transition to slug flow which led to enhanced mass transport of water to the electrode surface. A model developed for the system showed that the cell potential is dominated by the anode activation overpotential. Experimental data obtained at similar conditions with the commercially available lab-scale test cell agreed well with the model and the fitted parameters were in close proximity with values published in literature

Cyclotron motion and magnetic focusing in semiconductor quantum wells with spin-orbit coupling

We investigate the ballistic motion of electrons in III-V semiconductor quantum wells with Rashba spin-orbit coupling in a perpendicular magnetic field. Taking into account the full quantum dynamics of the problem, we explore the modifications of classical cyclotron orbits due to spin-orbit interaction. As a result, for electron energies comparable with the cyclotron energy the dynamics are particularly rich and not adequately described by semiclassical approximations. Our study is complementary to previous semiclassical approaches concentrating on the regime of weaker fields.Comment: 14 pages, 8 figures included, version to appear in Phys. Rev.

Detection of spin polarized currents in quantum point contacts via transverse electron focusing

It has been predicted recently that an electron beam can be polarized when it flows adiabatically through a quantum point contact in a system with spin-orbit interaction. Here, we show that a simple transverse electron focusing setup can be used to detect such polarized current. It uses the amplitude's asymmetry of the spin-split transverse electron focusing peak to extract information about the electron's spin polarization. On the other hand, and depending on the quantum point contact geometry, including this one-body effect can be important when using the focusing setup to study many-body effects in quantum point contacts.Comment: 5 pages, 5 figure

An Experimentally Validated Steady State Polymer Electrolyte Membrane Water Electrolyser Model

A simple electrochemical model is developed to understand the overpotentials associated with a polymer electrolyte membrane water electrolyser (PEMWE) operating at room temperature (20 °C) and atmospheric pressure (1 atm). The model is validated using experimental results and fitted parameter values are reported

Patterns of Drug Use and Serum Sodium Concentrations in Older Hospitalized Patients : A Latent Class Analysis Approach

Peer reviewedPublisher PD

A proposal for the measurement of Rashba and Dresselhaus spin-orbit interaction strengths in a single sample

We establish an exact analytical treatment for the determination of the strengths of the Rashba and Dresselhaus spin-orbit interactions in a single sample by measuring persistent spin current. A hidden symmetry is exploited in the Hamiltonian to show that the spin current vanishes when the strength of the Dresselhaus interaction becomes equal to the strength of the Rashba term. The results are sustained even in the presence of disorder and thus an experiment in this regard will be challenging.Comment: 5 pages, 5 figure

Two-phase flow behaviour and performance of polymer electrolyte membrane electrolysers: Electrochemical and optical characterisation

Understanding gas evolution and two-phase flow behaviour are critical for performance optimization of polymer electrolyte membrane water electrolysers (PEMWEs), particularly at high current densities. This study investigates the gas-bubble dynamics and two-phase flow behaviour in the anode flow-field of a PEMWE under different operating conditions using high-speed optical imaging and relates the results to the electrochemical performance. Two types of anode flow-field designs were investigated, the single serpentine flow-field (SSFF) and parallel flow-field (PFF). The results show that the PFF design yielded a higher cell performance than the SSFF design at identical operating conditions. Optical visualization shows a strong relationship between the flow path length and the length of gas slugs produced, which in turn influences the flow regime of operation. Longer flow path length in the SSFF results in annular flow regime at a high current density which degrades cell performance. The annular flow regime was absent in the PFF design. It was found the effect of flow rate on performance depends strongly on operating temperature in both flow patterns. Results of this study indicate that long channel length promotes gas accumulation and channel-blocking which degrades performance in PEMWEs

Investigation of natural material to reduce industrial noise

Today with rapid industrialization, industrial noise has become a serious environmental problem in Sri Lanka. The noise which is generated by machines in industry is called as industrial noise. This excessive noise can interfere with communication between supervisors and employees. Continuous exposure to noise can cause fatigue, which often results in accidents and reduces the pace and quality of work. Noise affects negatively on the day today life of surrounding people, who have faced with many problems mentally and physically. Therefore, noise control is one of the major requirements to improve the living environment. Most of the developed countries use practical techniques to minimize the nuisance such as barrier walls, duct silencers, acoustical wall panel, soundproof curtains, sound enclosures for industrial machinery and other similar noise control treatments that are installed near the source to effectively reduce the sound level. However, Sri Lanka has not yet yielded much into this issue as noise reduction methods are costly. Therefore, it is necessary to find out cost effective methods to control industrial noise. This research was conducted to investigate the potential uses of Salvinia dust as natural sound reduction material, to give a solution for the existing industrial noise problems. Specimens having a size of 75 mm (diameter) x 25 mm (thickness) were made by using Salvinia dust. Salvinia dust was mixed with cement at a ratio of 1:1 and water was added. Noise Reduction Coefficient of these specimens were investigated by using an experimental set-up including signal generator, speaker and noise level meter. Noise reduction ability of the materials was quantified by using Noise Reduction Co-efficient (NRC). Variation of NRC with particle size, mix proportion and sample thickness were also investigated. Effectiveness of Salvinia dust to control industrial noise is discussed in this paper