Stroke causes CNS injury associated with strong fast microglial activation as part of the inflammatory response. Fast activation of microglia in response to neuronal damage requires the rapid availability of a large amount of energy to trigger diverse cytotoxic or neuroprotective signals. ATP-dependent potassium (K(ATP)) channels play important roles in many cellular functions by coupling cell metabolism to electrical activity. K(ATP) channels were first detected in cardiac myocytes and later found in beta-cells of the pancreas, skeletal muscle, neurons, smooth muscle, heart, pituitary, and tubular cells of the kidney. Our group and others have also demonstrated its expression in reactive microglia after brain injury. In rat models of stroke, blockade of the sulfonylurea receptor (SUR), with glibenclamide (Gbc) reduced cerebral edema and infarct volume. Furthermore, clinical data suggest the effectiveness of Gbc to treat stroke. Gbc close the K(ATP) channel by interaction with two drug-binding sites on SUR subunits, as well as, the astroglial NC(Ca-ATP) channel, which mediates the Gbc-induced prevention of edema after cerebral ischemia. In these studies however, the function of the K(ATP) channel remained unclear. Therefore, as Gbc may bind to constitute functional K(ATP) channels after ischemic stroke, other possible effects of Gbc might explain the effectiveness of this drug in the treatment of stroke. Giving the fact that, SUR1-regulated channels are exquisitely sensitive to changes in the metabolic state of the cell, and that microglia are sensing the environment, the expression of K(ATP) channels in activated microglia, will couple cell energy to membrane potential. We herein postulate, that the effectiveness of Gbc to treat stoke, at least in part, is caused by the KATP channel closure expressed by activated microglia, which may then be critical in determining, their participation in the pathogenic process. Given the analogy with beta-cells, K(ATP) channel blockade in microglia would response faster and more efficiently to the external signals released after brain injury. If true, blockade of microglial K(ATP) channel with low doses of Gbc during the early stages of stroke might foster neuroprotective microglial activity, could enhance ischemia-induced neurogenesis in the SVZ, and consequently will lead to an improved functional outcome. The work presented in this thesis demonstrates that, Gbc improves functional neurological outcome in stroke, accompanied by neuron preservation in the core of the ischemic brain. In this region, reactive microglia from tMCAO rats upregulate the K(ATP) channel, which makes microglia a target to Gbc actions in the early stages of stroke. Furthermore, Gbc also strengthens the neuroprotective role of microglia in the acute phase after focal cerebral ischemia, enhance long-term neurogenesis and brain repair processes. As such, identify microglial K(ATP) channels as a key target for stroke treatment. Overall, these results provide new therapeutic avenues for the treatment of other neurological disorders that involve microglia.

Adenosine triphosphate (ATP) is the energy currency of all living cells and its production is a key reaction in the energy metabolism of living organisms. Cells produce most of the ATP they require through ATP synthase, a unique molecular rotary motor driven by the movement of protons across the lipid membrane. In E.coli, ATP synthase is composed of a soluble domain called F1, which houses the catalytic sites, and a transmembrane domain called F0 that shuttles protons across the membrane to drive ATP production in the F1 sector. The F0 domain is built of three subunit types: subunit a and a dimer of subunit b form the stator of the motor, while a decameric c ring forms the rotor. The dynamic interface between a and c10 forms the proton channel. The ultimate goal of this work is to determine the structure of the proton transport machinery and understand the molecular mechanism of proton translocation in ATP synthase. We have characterized some of the key events in the stepwise assembly of the F0--complex. We have designed and validated a model protein, consisting of genetically fused subunits a and c, for structural studies. We have made progress towards determining the structure of the proton channel, including the development of a novel procedure for purification of subunit a and the a/c fusion protein, and crystallization of subunit a. Medical applications of this work include the potential development of novel antibiotic compounds, as well as the characterization and potential treatment of three human diseases caused by disruptions in proton transport through F0.

Hemocytes are important as storage and producers of proteins of the innate immune defence, as well as actors of the cellular immune response. Therefore the hematopoietic process is critical for survival of most invertebrates. In order to search for molecules of importance for hemocyte development in crayfish we investigated proteins in crayfish plasma, which were increased after microbial challenge. As a result we were able to identify, purify and characterize a new invertebrate cytokine named astakine, and could clearly show that this protein is important for hematopoietic development in vivo as well as in an in vitro cell culture system. Astakine contains a prokineticin (PK) domain shown for the first time in an invertebrate, however, unlike the vertebrate PKs, astakine binds to a cell surface F1 ATP synthase β subunit located on the hematopoietic tissue (hpt) cell membranes. Extracellular ATP synthases as receptors have earlier been reported in different vertebrate cells and here we show that extracellular ATP synthase β subunit acts as a receptor for an invertebrate cytokine and is involved in hematopoiesis. We also found two other groups of proteins, which were increased in plasma after microbial challenge and they were further characterized. A great number of different Kazal type proteinase inhibitors were produced by the hemocytes and this type of proteinase inhibitors have variable reactive sites determining the specificity of their inhibition. In crayfish Kazal inhibitors with similar reactive sites were found as a response to specific microorganisms suggesting that the crayfish Kazal proteinase inhibitors may provide enough variability to participate in diverse innate immune reactions against different pathogens. Antimicrobial peptides were synthesized by the hemocytes and were likewise released in high amount upon microbial infection and we have characterized the main group of cystein-rich crustin-like antimicrobial peptides and investigated their tissue distribution and expression pattern.

The task for this Master thesis was to evaluate different black-box arc models for circuit-breakers with the purpose of finding criteria for the breaking ability. A black-box model is a model that requires no knowledge from the user of the underlying physical processes. Black-box arc models have been used in circuit-breaker development for many years. Arc voltages from tests made in the High Power Laboratory in Ludvika were used for validation, along with the resistance calculated at current zero, R0, and 500 ns before current zero, R500. Three different arc models were evaluated: Cassie-Mayr, KEMA and an arc model based on power calculations. The third model gave very good results and if the model is developed further, the breaking ability could easily be estimated. The arc model based on power calculations could be improved by using better approximations of the quantities in the model, and by representing the current better. A further suggestion for the following work is to combine the second arc model tested, the KEMA model, with the model based on power calculations in order to estimate the KEMA model parameters. The R0 and R500 values should also be calculated from more tests, in order to find a clear limit of the breaking ability.

oday, globalization has become a phenomenon of all the industry in the world, business need to quickly response to the supply chain changes, or could obiously harm to iself. Artificial industry in Taiwan, orders from downstream members are huge and with large diversity, it Is important to build an alignment to the sales department and the production department. To decrease the information gaps between sales department and the production department, there are mechanisms whichh could be apply into this industry In Taiwan. This study shows the difference of apply two kinds of sale / production alignment machanism, and compare the performance of those two to the processes nowaday through simulation tools.

An individual who has sustained either irreversible cessation of circulatory and respiratory functions, or irreversible cessation of all functions of the entire brain, including the brain stem is dead. Brain death is currently the legal definition of death in many countries. Many people confuse brain death with vegetative states. Patients in a vegetative state are unaware of themselves or their environment. Both patients with brain death and those in a vegetative state are unconscious following severe brain injury. Unlike the brain death, vegetative patient¡¦s vital vegetative functions, such as cardiac action, respiration, and maintenance of blood pressure are preserved. The rostral ventrolateral medulla (RVLM) is the origin of a ¡§life-and-death¡¨ signal identified from systemic arterial blood pressure spectrum and intimately related to brain death. Based on the animal models of brain death, the observations that the power density of the vasomotor components of SAP signals undergoes both augmentation and reduction during the progression towards death strongly suggest that both ¡¥¡¥pro-life¡¦¡¦ and ¡¥¡¥pro-death¡¦¡¦ programs are present in the RVLM. A number of those ¡¥¡¥pro-life¡¦¡¦ and ¡¥¡¥pro-death¡¦¡¦ programs in the RVLM has now been identified along with their cellular and molecular mechanisms. As the neural substrate that is intimately related to brain death, one unresolved question is whether the proteome expressed in RVLM is unique. To address the issue, we used the cerebral cortex, which is defunct under persistent vegetative state for comparison. 2-DE electrophoresis, MALTI-TOF MS and peptide mass fingerprinting were used for investigation the proteomic difference between the rat RVLM and cerebral cortex. Quantitative analysis on silver-stained 2-DE electrophoresis gels revealed highly comparable distribution patterns of these protein spots for both brain regions, with 85.9 ¡Ó 2.3 % of protein spots from RVLM matched those from cerebral cortex. According to the protein function, these proteins were classed into binding activity, chaperone, antioxidant, oxidoreductase, ubiquitin- proteasome system, cell cycle, catalytic activity, glycolysis, tricarboxylic acid cycle, electron transport chain, endocytosis and exocytosis, structural molecular function, apoptosis, transport, differentiation and neurogenesis, protein biosynthesis, cell junction, and others. We found that a group of antioxidant proteins, including members of the peroxiredoxin (Prx) family (Prx-1, Prx-2, Prx-5, and Prx-6), thioredoxin and mitochondrial manganese superoxide dismutase exhibited significantly higher protein and mRNA expression levels in RVLM when compared to cerebral cortex. Tissue oxygen, ATP contents and ATP synthase subunits alpha and beta in RVLM were also significantly elevated. On the other hand, protein and mRNA levels of members of the ubiquitin-proteasome system, including proteasome subunit alpha type-1, ubiquitin, uniquitin-conjugating enzyme E2 N, ubiquitin carboxyl-terminal hydrolase isozyme L1 and L3, were comparable in both brain regions. The presence of higher levels of tissue oxygen and ATP synthase subunits in RVLM, leading to augmented ATP production, provides a cellular safeguard mechanism to reduce the possibility of irreversible reduction in intracellular ATP contents that precipitate brain death. By manifesting an augmented tissue oxygen and metabolic energy production, RVLM is more prone to oxidative stress. We conclude that a significantly elevated level of antioxidant proteins and mRNA in RVLM is consistent with the exhibition of higher tissue oxygen tension and metabolic energy production in this neural substrate, which together constitute a safeguard mechanism against brain death.

Face the change of globalization business, business opportunities far and wide. In contrast, corporate challengers, is no longer just a regional rival, but the strong competitors from different countries. Now, although the market is large, but also a substantial growth of the overall market supply, the supply quickly exceeded demand. In saturated markets, companies how to overcome or co-competitors to grab a larger market share, profit? For enterprises, existence of the business mission is to provide products, services, customers. Without customers, there is no demand, companies will not be able to survive. Therefore, in addition to providing standard, high quality products, we must be able to respond more quickly to customer needs, get the trust and improve customer satisfaction. Enterprise subject to different tangible and intangible constraints, How to play the best of ability, flexibility and rapid response to customer demand among the existing constraints? Improve operational efficiency, reduce inventory costs, efficient delivery and with the best service in the competition for maximum profit. This study base on many constraints in the continuous industry using thought tool of theory of constraints to break the bottleneck and response customer's demand. Through the ERP combined with ATP / CTP mechanism to improve the order promising process. Effectively improve service efficiency, shorten customer product delivery, while enhancing the overall supply chain efficiency, enhance corporate competitiveness and profitability.

Thèse de doctorat : Médecine : Nancy 1 : 2005. / Titre provenant de l'écran-titre.

The ATP-PRTase enzyme catalyses the first committed step of histidine biosynthesis in archaea, bacteria, fungi and plants.1 As the catalyst of an energetically expensive pathway, ATP-PRTase is subject to a sophisticated, multilevel regulatory system.2 There are two families of this enzyme, the long form (HisGL) and the short form (HisGS) that differ in their molecular architecture. A single HisGL chain comprises three domains. Domains I and II house the active site of HisGL while domain III, a regulatory domain, forms the binding site for histidine as an allosteric inhibitor. The long form ATP-PRTase adopts a homo-hexameric quaternary structure.3,4 HisGS comprises a similar catalytic core to HisGL but is devoid of the regulatory domain and associates with a second protein, HisZ, to form a hetero-octameric assembly.5 This thesis explores the allosteric regulation of the short form ATP-PRTase, as well as the functional and evolutionary relationship between the two families. New insight into the mode allosteric inhibition of the short form ATP-PRTase from Lactococcus lactis is reported in chapter two. A conformational change upon histidine binding was revealed by small angle X-ray scattering, illuminating a potential mechanism for the allosteric inhibition of the enzyme. Additionally, characterisation of histidine binding to HisZ by isothermal titration calorimetry, in the presence and absence of HisGS, provided evidence toward the location of the functional allosteric binding site within the HisZ subunit. Chapter three details the extensive effort towards the purification of the short form ATP-PRTase from Neisseria menigitidis, the causative agent of bacterial meningitis. This enzyme is of particular interest as a potential target for novel, potent inhibitors to combat this disease. The attempts to purify the long form ATP-PRTase from E. coli, in order to clarify earlier research on the functional multimeric state of the enzyme, are also discussed. Chapter four reports the investigation of a third ATP-PRTase sequence architecture, in which hisZ and hisGS comprise a single open reading frame, forming a putative fusion enzyme. The engineering of two covalent linkers between HisZ and HisGS from L. lactis and the transfer of the regulatory domain from HisGL to HisGS, is also discussed, in an attempt to delineate the evolutionary pathway of the ATP-PRTase enzymes. Finally, the in vivo activity of each functional and putative ATP-PRTase was assessed by E. coli BW25113∆hisG complementation assays.

The maintenance of a functional energy balance in ectothermic fauna could be challenging in a thermally disparate environment. Biochemical adaptations at the enzyme and membrane levels allows for a set compensatory mechanism that allow the individual to maintain an energetic surplus, thus allocating energy for growth and reproduction. The present work describes how the energetic machinery in the cell, particularly the mitochondrion, could be affected by temperature changes. More specifically, this work aimed to determine how environmental temperature affects the mitochondria energetic performance of fishes from disparate thermal regimes. Mitochondrial ATP production efficiency was evaluated in fishes from polar, tropical and subtropical regions. In polar fishes, mitochondria remained functional at temperatures well beyond whole organismal critical temperatures. On the other hand, tropical and subtropical teleosts exhibited a decrease in mitochondrial efficiency at temperatures commonly found during summer seasons. This remarkable variability of mitochondrial thermal sensitivity may restrict the energy allocated for growth and reproduction during the summer months in tropical and subtropical regions. The observed variation in the thermal window of tolerance of mitochondrial function in fishes provides further insight into how the energetic machinery responds to thermal changes, like those associated with warming trends in marine ecosystems. In addition, the reduced efficiency in mitochondrial function among teleosts from latitudinal gradients suggest that warm-adapted species are close to their upper tolerance range, and further warming trends could severely impact the energy budget of fishes.