FCS Analysis of Pore Formation by the Human Protein Bax in Lipid Membranes

Bax is a pro-apoptotic member of the BCL-2 protein family and its paramount function during apoptosis is to form pores in the mitochondrial outer membrane: MOM). In a simplified apoptosis model, upon apoptotic stimulation, Bax is activated via an unresolved mechanism, translocates to the MOM, where it changes conformation, inserts into the membrane as a monomer, and finally undergoes in-membrane oligomerization to form a pore. Once the Bax pore is formed, cytochrome c and other mitochondria-resident proteins escape into the cytoplasm where they activate the cascade of caspases, which dismantle the cell. To date, the structure of the inactive cytoplasmic form of Bax is known; however, the structure of the membrane-integrated, active form of Bax remains unsolved. The absence of membrane structure for Bax leaves the mechanism of Bax pore formation poorly understood. To investigate the mechanism of Bax pore formation, we developed and used approaches to study fluorescently labeled Bax in solution and in lipid membranes using single-molecule sensitivity fluorescence techniques based on fluorescence correlation spectroscopy - FCS. These single-molecule fluorescence techniques provide us with a non-invasive method to study the mechanism of Bax pore formation reconstituted in giant unilamellar vesicles: GUVs). Our results show, that in the environment of a GUV lipid membrane, Bax can form a complex distribution of coexisting pore sizes ranging from 1 nm to 20 ┬╡m in diameter. Evidence is provided by directly examining oligomerization and mobility changes of Bax molecules in GUVs by fluorescence cross-correlation spectroscopy: FCCS) and observing the large complexes by confocal microscopy. We show that in the presence of Bcl-xL, an inhibitor of Bax pore formation, membrane-bound Bax was primarily monomeric. We also show, that in the large length-scale format of the GUV, Bax forms mega-pores - structures that reveal its affinity for highly curved membranes. Analysis of the line tension in the rim of Bax mega-pores indicates that Bax dramatically reduces line tension while stabilizing these lipidic pores. Furthermore, our results demonstrate that Bax forms pores by increasing membrane surface energy and by changing the curvature of lipid membranes, thus manifesting an ability to sculpt lipid bilayers. In addition to studying Bax in lipid membranes, we have investigated the in vitro mechanism of Bax activation in detergent micelles, where, due to the formation of large hydrodynamic volume protein-detergent micelles, Bax has been proposed to form homo-oligomers upon activation prior to membrane insertion, which is in contrast to the in vivo studies which reported that Bax binds MOM as a monomer and then undergoes homo-oligomerization. Our studies using fluorescence intensity distribution analysis: FIDA) have shown that Bax is a monomer before, during and after interaction with detergent micelles; however, it does form large hydrodynamic volume complexes with detergent micelles. The results of our FIDA study establish the connection between the in vitro and in vivo mechanism of Bax activation, showing that, prior to the formation of an oligomeric pore in a lipid membrane, Bax binds lipid membranes as a monomer

On-line computer control of turbine generators using state estimation and optimal feedback

Imperial Users onl

Transitioning power distribution grid into nanostructured ecosystem : prosumer-centric sovereignty

PhD ThesisGrowing acceptance for in-house Distributed Energy Resource (DER) installations at lowvoltage level have gained much significance in recent years due to electricity market liberalisations and opportunities in reduced energy billings through personalised utilisation management for targeted business model. In consequence, modelling of passive customers’ electric power system are progressively transitioned into Prosumer-based settings where presidency for Transactive Energy (TE) system framework is favoured. It amplifies Prosumers’ commitments into annexing TE values during market participations and optimised energy management to earn larger rebates and incentives from TE programs. However, when dealing with mass Behind-The-Meter DER administrations, Utility foresee managerial challenges when dealing with distribution network analysis, planning, protection, and power quality security based on Prosumers’ flexibility in optimising their energy needs. This dissertation contributes prepositions into modelling Distributed Energy Resources Management System (DERMS) as an aggregator designed for Prosumer-centered cooperation, interoperating TE control and coordination as key parameters to market for both optimised energy trading and ancillary services in a Community setting. However, Prosumers are primarily driven to create a profitable business model when modelling their DERMS aggregator. Greedy-optimisation exploitations are negative concerns when decisions made resulted in detrimental-uncoordinated outcomes on Demand-Side Response (DSR) and capacity market engagements. This calls for policy decision makers to contract safe (i.e. cooperative yet competitive tendency) business models for Prosumers to maximise TE values while enhancing network’s power quality metrics and reliability performances. Firstly, digitalisation and nanostructuring of distribution network is suggested to identify Prosumer as a sole energy citizen while extending bilateral trading between Prosumer-to- Prosumer (PtP) with the involvements of other grid operators−TE system. Modelling of Nanogrid environment for DER integrations and establishment of local area network infrastructure for IoT security (i.e. personal computing solutions and data protection) are committed for communal engagements in a decentralise setting. Secondly, a multi-layered Distributed Control Framework (DCF) is proposed using Microsoft Azure cloud-edge platform that cascades energy actors into respective layers of TE control and coordination. Furthermore, modelling of flexi-edge computing architecture is proposed, comprising of Contract-Oriented Sensor-based Application Platform (COSAP) employing Multi-Agent System (MAS) to enhance data-sharing privacy and contract coalition agreements during PtP engagements. Lastly, the Agents of MAS are programmed with cooperative yet competitive intelligences attributed to Reinforcement Learning (RL) and Neural Networks (NN) algorithms to solve multimodal socio-economical and uncertainty problems that corresponded to Prosumers’ dynamic energy priorities within the TE framework. To verify the DERMS aggregator operations, three business models were proposed (i.e. greedy-profit margin, collegial-peak demand, reserved-standalone) to analyse comparative technical/physical and economic/social dimensions. Results showed that the proposed TE-valued DERMS aggregator provides participation versatility in the electricity market that enables competitive edginess when utilising Behind-The-Meter DERs in view of Prosumer’s asset scheduling, bidding strategy, and corroborative ancillary services. Performance metrics were evaluated on both domestic and industrial NG environments against IEEE Standard 2030.7-2017 & 2030.8-2018 compliances to ensure deployment practicability. Subsequently, proposed in-house protection system for DER installation serves as an add-on monitoring service which can be incorporated into existing Advance Distribution Management System (ADMS) for Distribution Service Operator (DSO) and field engineers use, ADMS aggregator. It provides early fault detections and isolation processes from allowing fault current to propagate upstream causing cascading power quality issues across the feeder line. In addition, ADMS aggregator also serves as islanding indicator that distinguishes Nanogrid’s islanding state from unintentional or intentional operations. Therefore, a Overcurrent Current Relay (OCR) is proposed using Fuzzy Logic (FL) algorithm to detect, profile, and provide decisional isolation processes using specified OCRs. Moreover, the proposed expert knowledge in FL is programmed to detect fault crises despite insufficient fault current level contributed by DER (i.e. solar PV system) which conventional OCR fails to trigger

Delineating the Steps of BAX Pore Activation

The BCL2 protein family is the primary gatekeeper of mitochondrial apoptosis and governs integrity of the organelles\u27 outer membranes. Permeabilization of mitochondrial outer membranes permits egress of cytochrome c and other apoptogenic factors, resulting in apoptosome formation, caspase activation, and subsequent proteolytic demolition of cells. Proapoptotic BAX & BAK effect the release of cytochrome c while their antiapoptotic counterparts like BCL-2, BCL-XL, & MCL-1 oppose this permeabilization. A third class of the BCL2 family, the prodeath BH3-only proteins, act as sentinels of cell stress and exert their influences by occupying antiapoptotic BCL2 members and/or activating BAX/BAK. Cell-free reconstitution assays have revealed that BAX/BAK undergo significant conformational changes to oligomerize and form pores in membranes. Previously unresolved was the basis for the outer cell membranes\u27 escape from BAX poration during apoptosis. Unlike outer cell membranes, which are roughly 40% cholesterol, mitochondrial outer membranes are only 5-10% cholesterol. Vesicle leakage assays demonstrated that BAX pore activation is severely inhibited by the sterol. Inclusion of the total enantiomer of cholesterol in our assays uncovered that this BAX functional suppression was due to bilayer structure alteration rather than a stereospecific protein-cholesterol interaction. Real-time observation of BAX-vesicle binding showed that cholesterol curbs membrane integration by the protein, thus suppressing oligomerization and pore formation. Oxysterols and bile acids are physiological derivatives of cholesterol. Further employment of our vesicle leakage regime revealed that 25-hydroxycholesterol at low micromolar concentrations accelerates BAX pore formation, suggesting a compensatory mechanism for BAX inhibition by cholesterol. Bile acids lithocholic and chenodeoxycholic acids are toxic and induce apoptosis at high concentrations, thus we reasoned that the physiological detergents may directly activate BAX. While truncated BAX: ΔC) was effectively activated by monomeric detergent, the full-length protein required micellar bile acids, implying that bile acids could play at most only an amplification role in BAX-mediated apoptosis. In nonstressed cells, BAX exists as a soluble, cytosolic monomer or loosely affiliated with mitochondria while cellular recognition of death signals induces BAX transition to a membrane-integral state. The physical basis of this translocation and locale of BAX activation are poorly characterized. Assemblage of a cell-free scheme comprising full-length BAX, antiapoptotic BCL-XL, and BH3-only activators cBID & BIMS, and synthetic vesicles revealed that BIMS more effectively activates BAX and is less suppressible by BCL-XL. Each of these four proteins can independently adsorb to membranes and that BCL-XL outraces BAX to their in-membrane functional sites. Membrane-bound cBID and BIMS robustly accelerate the bilayer integrations of BAX & BCL-XL; the two activators recruit equivalently at the membrane surface, however, suggesting that BIMS, unlike cBID, can activate BAX prior to interaction on a bilayer scaffold

A study of a space communication system for the control and monitoring of the electric distribution system. Volume 2: Supporting data and analyses

It is technically feasible to design a satellite communication system to serve the United States electric utility industry's needs relative to load management, real-time operations management, remote meter reading and to determine the costs of various elements of the system. The functions associated with distribution automation and control and communication system requirements are defined. Factors related to formulating viable communication concepts, the relationship of various design factors to utility operating practices, and the results of the cost analysis are discussed The system concept and several ways in which the concept could be integrated into the utility industry are described

SynerCrete’18: interdisciplinary approaches for cement-based materials and structural concrete: synergizing expertise and bridging scales of space and time, vol. 2

info:eu-repo/semantics/publishedVersio

The 1991 International Aerospace and Ground Conference on Lightning and Static Electricity, volume 2

The proceedings of the conference are reported. The conference focussed on lightning protection, detection, and forecasting. The conference was divided into 26 sessions based on research in lightning, static electricity, modeling, and mapping. These sessions spanned the spectrum from basic science to engineering, concentrating on lightning prediction and detection and on safety for ground facilities, aircraft, and aerospace vehicles