Motif affinity and mass spectrometry proteomic approach for the discovery of cellular AMPK targets: identification of mitochondrial fission factor as a new AMPK substrate

AMP-activated protein kinase (AMPK) is a key cellular energy sensor and regulator of metabolic homeostasis. Although it is best known for its effects on carbohydrate and lipid metabolism, AMPK is implicated in diverse cellular processes, including mitochondrial biogenesis, autophagy, and cell growth and proliferation. To further our understanding of energy homeostasis through AMPK-dependent processes, the design and application of approaches to identify and characterise novel AMPK substrates are invaluable. Here, we report an affinity proteomicstrategy for the discovery and validation of AMPK targets using an antibody to isolate proteins containing the phospho-AMPK substrate recognition motif from hepatocytes that had been treated with pharmacological AMPK activators. We identified 57 proteins that were uniquely enriched in the activator-treated hepatocytes, but were absent in hepatocytes lacking AMPK. We focused on two candidates, cingulin and mitochondrial fission factor (MFF), and further characterised/validated them as AMPK-dependent targets by immunoblotting with phosphorylation site-specific antibodies. A small-molecule AMPK activator caused transient phosphorylation of endogenous cingulin at S137 in intestinal Caco2 cells. Multiple splice-variants of MFF appear to express in hepatocytes and we identified a common AMPK-dependent phospho-site (S129) in all the 3 predominant variants spanning the mass range and a short variant-specific site (S146). Collectively, our proteomic-based approach using a phospho-AMPK substrate antibody in combination with genetic models and selective AMPK activators will provide a powerful and reliable platform for identifying novel AMPK-dependent cellular targets

The role of CDC48 in the retro-translocation of non-ubiquitinated toxin substrates in plant cells

When the catalytic A subunits of the castor bean toxins ricin and Ricinus communis agglutinin (denoted as RTA and RCA A, respectively) are delivered into the endoplasmic reticulum (ER) of tobacco protoplasts, they become substrates for ER-associated protein degradation (ERAD). As such, these orphan polypeptides are retro-translocated to the cytosol, where a significant proportion of each protein is degraded by proteasomes. Here we begin to characterise the ERAD pathway in plant cells, showing that retro-translocation of these lysine-deficient glycoproteins requires the ATPase activity of cytosolic CDC48. Lysine polyubiquitination is not obligatory for this step. We also show that while RCA A is found in a mannose-untrimmed form prior to its retro-translocation, a significant proportion of newly synthesised RTA cycles via the Golgi and becomes modified by downstream glycosylation enzymes. Despite these differences, both proteins are similarly retro-translocated

Development and application of a high-performance framework for high-fidelity simulations of plasma-assisted ignition of hydrocarbon fuels using nanosecond pulsed discharges

The application of non-equilibrium plasma (NEP) pulses to ignite hydrocarbon/air mixtures has emerged as a promising technology for ensuring reliable ignition and combustion stability in difficult regimes. Despite its promise, major challenges and limitations still remain, particularly in the realm of conducting high-fidelity multidimensional numerical studies. The aim of this thesis is to develop, implement, and apply a robust and efficient computational framework that addresses some of these shortcomings. As a preliminary step, the ignition of hydrocarbon/air mixtures by nanosecond pulsed discharges (NSPD) is investigated using a zero dimensional isochoric adiabatic reactor. A state-of-the-art two-temperature kinetics model, comprised of an experimentally-verified NEP plasma mechanism coupled with a hydrocarbon/air oxidation mechanism, is used. Simulations are performed to assess the impact of changing initial pressure (which varies from 1 to 30 atm) and fuel type (methane and ethylene). It is found that at lower pressures, plasma-assisted ignition (PAI) imparts a benefit over thermal ignition for both fuel types, through the creation of combustion radicals O, H, and OH. At higher pressures, PAI of methane loses efficiency compared to ethylene, due to a lack of available H radicals (which are swept up by O₂), which limits the conversion of formaldehyde to formyl. Next, a robust and efficient framework for simulating NSPD in multiple dimensions is developed. The reactive Navier-Stokes equations are extended to include a drift-diffusion plasma-fluid model with a local field approximation (LFA) in a finite-volume solver, which uses an adaptive mesh refinement (AMR) strategy to address the wide separation of length scales in the problem. A two-way coupling strategy is used whereby the plasma-fluid model and reactive Navier-Stokes equations are integrated simultaneously. An effective grid refinement approach is developed in order to ensure that the physical structures that arise during and after the NSD (including the propagating streamer heads, electrode sheaths, and expansion wave during the inter-pulse period) are resolved efficiently. Severe time step size restrictions that arise from the explicit temporal integration of the transport terms are mitigated through use of a semi-implicit approach for solving Poisson's equation for the electric potential, and an implicit strategy for evaluating electron diffusion terms. A series of numerical studies are then conducted to investigate the ignition and propagation phases of atmospheric air streamers in axisymmetric discharge configurations. A range of conditions and configurations are explored to characterize the streamer, with an emphasis on the cathode sheath region, which supports steep gradients in charged species number densities as well as strong electric fields. The formation of the cathode sheath is shown to be a consequence of processes at the cathode surface, driven by electron losses at the boundary, and a strong dependence on the emission of secondary electrons. Finally, the oxidation of ethylene/air mixtures mediated by NSPD is simulated in a pin-to-pin configuration. All phases of the plasma discharge are simulated explicitly (including streamer ignition, propagation, and connection, as well as the subsequent spark phase), along with the evolution of the plasma during the inter-pulse period. Temporally and spatially-resolved results are presented, with an emphasis on the analysis of heating and energy deposition, as well as of the evolution of the concentration of active particles generated during the NSPD and their influence on ignition. The impact of pin thickness is discussed, and it is shown that the use of thin pins limits the regions of energy deposition and temperature increase near the pin tips, hindering ignition. The application of multiple pulses is explored and it is shown that multiple voltage pulses of the same strength leads to substantial energy deposition and temperature increases O(1,000 - 10,000 K) near the pin tips. Discussion is rounded out by addressing how pulse frequency and initial mixture control the generation of active particles and combustion products. Finally, recommendations for future work are provided.Aerospace Engineerin

Plasma-assisted ignition of methane/air and ethylene/air mixtures: Efficiency at low and high pressures

The ignition of methane/air and ethylene/air mixtures by nanosecond pulsed discharges (NSPD) was numerically studied using a zero-dimensional isochoric adiabatic reactor. A combustion kinetics model was combined with a non-equilibrium plasma mechanism, which features vibrational and electronic excitation, dissociation, and ionization of neutral particles (O2 and N2) via electron impact. A time to ignition metric τ was defined, and ignition simulations encompassing a wide range of pressures (0.5-30 atm) and pulsing conditions for each fuel were executed. For each fuel, τ depended primarily on initial pressure and energy deposition rate, and scaling laws were derived. The benefit gained from plasma-assisted ignition (PAI) was quantified by comparing τ with a thermal ignition time. For both fuels, PAI resulted in a faster ignition at low pressures, while at higher pressures (p0 ≥ 5 atm), methane/air ignition became inefficient (meaning a longer ignition time for the same input energy compared to thermal ignition). Ethylene/air PAI showed only a modest deterioration. The drop in performance with pressure was due to the mean electron energy achieved during the pulse, which exhibited an inverse relationship with pressure, leading to fewer excited species and combustion radicals. The poor performance of methane/air mixture ignition at high pressure was explained by an analysis of the reaction pathways. At high pressures (p0 ~30 atm), H is consumed mostly to form hydroperoxyl (HO2), leading to a bottleneck in the formation of formyl (HCO) from formaldehyde (CH2O). Instead, for ethylene/air ignition, at both low and high pressures there exist several bypass pathways that facilitate the formation of HCO and CO directly from various intermediates, explaining the more robust performance of PAI for ethylene at pressure.SCOPUS: cp.jinfo:eu-repo/semantics/publishe

Ignition of methane and ethylene via nanosecond pulsed discharges

info:eu-repo/semantics/publishe

Development of skeletal kinetics mechanisms for plasma-assisted combustion via principal component analysis

info:eu-repo/semantics/publishe

Skeletal Chemical Kinetics Mechanisms for Plasma-Assisted Combustion

info:eu-repo/semantics/publishe