Nedd4-2 functionally interacts with ClC-5 - Involvement in constitutive albumin endocytosis in proximal tubule cells

Constitutive albumin uptake by the proximal tubule is achieved by a receptor-mediated process in which the Cl- channel, ClC-5, plays an obligate role. Here we investigated the functional interaction between ClC-5 and ubiquitin ligases Nedd4 and Nedd4-2 and their role in albumin uptake in opossum kidney proximal tubule (OK) cells. In vivo immunoprecipitation using an anti-HECT antibody demonstrated that ClC-5 bound to ubiquitin ligases, whereas glutathione S-transferase pull-downs confirmed that the C terminus of ClC-5 bound both Nedd4 and Nedd4-2. Nedd4-2 alone was able to alter ClC-5 currents in Xenopus oocytes by decreasing cell surface expression of ClC-5. In OK cells, a physiological concentration of albumin (10 mug/ml) rapidly increased cell surface expression of ClC-5, which was also accompanied by the ubiquitination of ClC-5. Albumin uptake was reduced by inhibiting either the lysosome or proteasome. Total levels of Nedd4-2 and proteasome activity also increased rapidly in response to albumin. Overexpression of ligase defective Nedd4-2 or knockdown of endogenous Nedd4-2 with small interfering RNA resulted in significant decreases in albumin uptake. In contrast, pathophysiological concentrations of albumin (100 and 1000 mug/ml) reduced the levels of ClC-5 and Nedd4-2 and the activity of the proteasome to the levels seen in the absence of albumin. These data demonstrate that normal constitutive uptake of albumin by the proximal tubule requires Nedd4-2, which may act via ubiquitination to shunt ClC-5 into the endocytic pathway.

Recombinant human activated protein C improves pulmonary function in ovine acute lung injury resulting from smoke inhalation and sepsis

Objective: To investigate the effects of recombinant human activated protein C (rhAPC) on pulmonary function in acute lung injury (ALI) resulting from smoke inhalation in association with a bacterial challenge. Design: Prospective, randomized, controlled, experimental animal study with repeated measurements. Setting: Investigational intensive care unit at a university hospital. Subjects: Eighteen sheep (37.2 +/- 1.0 kg) were operatively prepared and randomly allocated to either the sham, control, or rhAPC group (n = 6 each). After a tracheotomy had been performed, ALI was produced in the control and rhAPC group by insufflation of 4 sets of 12 breaths of cotton smoke. Then, a 30 mL suspension of live Pseudomonas aeruginosa bacteria (containing 2-5 x 10(11) colony forming units) was instilled into the lungs according to an established protocol. The sham group received only the vehicle, i.e., 4 sets of 12 breaths of room air and instillation of 30 mL normal saline. The sheep were studied in the awake state for 24 hrs and were ventilated with 100% oxygen. RhAPC (24 mu g/kg/hr) was intravenously administered. The infusion was initiated 1 hr post-injury and lasted until the end of the experiment. The animals were resuscitated with Ringer's lactate solution to maintain constant pulmonary artery occlusion pressure. Measurements and Main Results., In comparison with nontreatment in controls, the infusion of rhAPC significantly attenuated the fall in PaO2/FiO(2) ratio (control group values were 521 +/- 22 at baseline [BL], 72 +/- 5 at 12 hrs, and 74 +/- 7 at 24 hrs, vs. rhAPC group values of 541 +/- 12 at BL, 151 +/- 29 at 12 hours [p < .05 vs. control], and 118 +/- 20 at 24 hrs), and significantly reduced the increase in pulmonary microvascular shunt fraction (Qs/Qt; control group at BL, 0.14 +/- 0.02, and at 24 hrs, 0.65 +/- 0.08; rhAPC group at BL, 0.24 +/- 0.04, and at 24 hrs, 0.45 +/- 0.02 [p < .05 vs. control]) and the increase in peak airway pressure (mbar; control group at BL, 20 +/- 1, and at 24 hrs, 36 +/- 4; rhAPC group at BL, 21 +/- 1, and at 24 hrs, 28 +/- 2 [p < .05 vs. control]). In addition, rhAPC limited the increase in lung 3-nitrotyrosine (after 24 hrs [%]: sham, 7 +/- 2; control, 17 +/- 1; rhAPC, 12 +/- 1 [p < .05 vs. control]), a reliable indicator of tissue injury. However, rhAPC failed to prevent lung edema formation. RhAPC-treated sheep showed no difference in activated clotting time or platelet count but exhibited less fibrin degradation products (1/6 animals) than did controls (4/6 animals). Conclusions. Recombinant human activated protein C attenuated ALI after smoke inhalation and bacterial challenge in sheep, without bleeding complications.

Successful use of recombinant factor VII in massive hemoptysis due to community-acquired pneumonia

Recombinant activated factor VII (rFVIIa) is a powerful hemostatic agent developed for use in hemophilia. It has been used increasingly in life-threatening hemorrhage in a variety of other settings in which conventional medical or surgical therapy is unsuccessful. This report describes the successful use of rFVIIa for pulmonary hemorrhage due to a focal bleeding source in a regional hospital where bronchial artery embolization or surgery were not available. rFVIIa may be a useful temporizing measure in the unstable patient with pulmonary hemorrhage without coagulopathic bleeding when conventional treatment. is not immediately available.

A study of the effect of harmonics in the system on the performance of residual current-operated circuit-breakers and electromechanical overcurrent relays

Residual current-operated circuit-breakers (RCCBs) have proved useful devices for the protection of both human beings against ventricular fibrillation and installations against fire. Although they work well with sinusoidal waveforms, there is little published information on their characteristics. Due to shunt connected non-linear devices, not the least of which is the use of power electronic equipment, the supply is distorted. Consequently, RCCBs as well as other protection relays are subject to non-sinusoidal current waveforms. Recent studies showed that RCCBs are greatly affected by harmonics, however the reasons for this are not clear. A literature search has also shown that there are inconsistencies in the analysis of the effect of harmonics on protection relays. In this work, the way RCCBs operate is examined, then a model is built with the aim of assessing the effect of non-sinusoidal current on RCCBs. Tests are then carried out on a number of RCCBs and these, when compared with the results from the model showed good correlation. In addition, the model also enables us to explain the RCCBs characteristics for pure sinusoidal current. In the model developed, various parameters are evaluated but special attention is paid to the instantaneous value of the current and the tripping mechanism movement. A similar assessment method is then used to assess the effect of harmonics on two types of protection relay, the electromechanical instantaneous relay and time overcurrent relay. A model is built for each of them which is then simulated on the computer. Tests results compare well with the simulation results, and thus the model developed can be used to explain the relays behaviour in a harmonics environment. The author's models, analysis and tests show that RCCBs and protection relays are affected by harmonics in a way determined by the waveform and the relay constants. The method developed provides a useful tool and the basic methodology to analyse the behaviour of RCCBs and protection relays in a harmonics environment. These results have many implications, especially the way RCCBs and relays should be tested if harmonics are taken into account.

A converter compensator for power factor control in 3-phase induction machines

The advent of the harmonic neutralised shunt Converter Compensator as a practical means of reactive power compensation in power transmission systems has cleared ground for wider application of this type of equipment. An experimental 24-pulse voltage sourced convector has been successfully applied in controlling the terminal power factor of a 1.5kW, 240V three phase cage rotor induction motor, whose winding has been used in place of the usual phase shifting transformers. To achieve this, modifications have been made to the conventional stator winding of the induction machine. These include an unconventional phase spread and facilitation of compensator connections to selected tapping points between stator coils to give a three phase winding with a twelve phase connection to the twenty four pulse converter. Theoretical and experimental assessments of the impact of these modifications and attachment of the compensator have shown that there is a slight reduction in the torque developed at a given slip and in the combined system efficiency. There is also an increase in the noise level, also a consequence of the harmonics. The stator leakage inductance gave inadequate coupling reactance between the converter and the effective voltage source, necessitating the use of external inductors in each of the twelve phases. The terminal power factor is fully controllable when the induction machine is used either as a motor or as a generator.

Fluid dynamics and platelet deposition in a model arterial stenosis

The objective of this study was to gain further understanding and elucidation of the fluid dynamic factors and flow-induced mechanisms of the thrombogenic process of platelet deposition onto, and possible subsequent embolization from, the walls of an arterial stenosis. This has been accomplished by measurement of the axial dependence of platelet deposition within a modeled arterial stenosis for a transitional flow and a completely laminar flow field. The stenotic region of the model was collagen-coated to simulate a damaged endothelial lining of an artery. Fluid dynamics within a stenosis was studied using qualitative flow visualization, and was further compared to the in vitro platelet deposition studies. Normalized platelet density (NPD) measurements indicate decreased levels of NPD in the high shear throat region of the stenosis for a Reynolds number of 300 and a drastic increase in NPD at the throat for a Reynolds number of 175. This study provides further understanding of the flow dynamic effects on thrombus development within a stenosis. ^

Elastic modulus study of gold thin film for use as an actuated membrane in a superconductive radio frequency (RF) microelectromechanical (MEM) switch

The objective of this research was to find Young's elastic modulus for thin gold films at room and cryogenic temperatures based on the flexional model which has not been previously attempted. Electrical Sonnet simulations and numerical methods using Abacus for the mechanical responses were employed for this purpose. A RF MEM shunt switch was designed and a fabrication process developed in house. The switch is composed of a superconducting YBa2 Cu3O7 coplanar waveguide structure with an Au bridge membrane suspended above an area of the center conductor covered with BaTiO3 dielectric. The Au membrane is actuated by the electrostatic attractive force acting between the transmission line and the membrane when voltage is applied. The value of the actuation force will greatly depend on the switch pull-down voltage and on the geometry and mechanical properties of the bridge material. Results show that the elastic modulus for Au thin film can be 484 times higher at cryogenic temperature than it is at room temperature. ^

Temperature dependence and characterization of gold thin film actuated membranes for radio frequency microelectromechanical switches

Over the last 10 years, the development and the understanding of the mechanical properties of thin film material have been essential for improving the reliability and lifetime in operation of microelectromechanical systems (MEMS). Although the properties of a bulk material might be well characterized, thin-film properties are considerably different from those of the bulk and it cannot be assumed that mechanical properties measured using bulk specimens will apply to the same materials when used as a thin film in MEMS. For many microelectronic thin films, the material properties depend strongly on the details of the deposition process and the growth conditions on its substrate. ^ The purpose of this dissertation is to determine the temperature dependence of a gold thin film membrane on the pull down voltage of a MEMS switch as the temperature is varied from room temperature (300 K) to cryogenic temperature (10 K). For this purpose, an RF MEMS shunt switch was designed and fabricated. The switch is composed of a gold coplanar waveguide structure with a gold bridge membrane suspended above an area of the center conductor which is covered by a dielectric (BaTiO3). The gold membrane is actuated by an electrostatic force acting between the transmission line and the membrane when voltage is applied. ^ Material characterization of the gold evaporated thin film membrane was obtained via AFM, SEM, TEM and X-ray diffraction analyses. A mathematical relation was used to estimate the pull down voltage of the switch at cryogenic temperature and results showed that the mathematical theory match the experimental values of the tested MEMS switches. ^

Tailoring heme -thiolate proteins into efficient biocatalysts with high specificity and selectivity

Cytochrome P450 monooxygenases, one of the most important classes of heme-thiolate proteins, have attracted considerable interest in the biochemical community because of its catalytic versatility, substrate diversity and great number in the superfamily. Although P450s are capable of catalyzing numerous difficult oxidation reactions, the relatively low stability, low turnover rates and the need of electron-donating cofactors have limited their practical biotechnological and pharmaceutical applications as isolated enzymes. The goal of this study is to tailor such heme-thiolate proteins into efficient biocatalysts with high specificity and selectivity by protein engineering and to better understand the structure-function relationship in cytochromes P450. In the effort to engineer P450cam, the prototype member of the P450 superfamily, into an efficient peroxygenase that utilizes hydrogen peroxide via the “peroxide-shunt” pathway, site-directed mutagenesis has been used to elucidate the critical roles of hydrophobic residues in the active site. Various biophysical, biochemical and spectroscopic techniques have been utilized to investigate the wild-type and mutant proteins. Three important P450cam variants were obtained showing distinct structural and functional features. In P450camV247H mutant, which exhibited almost identical spectral properties with the wild-type, it is demonstrated that a single amino acid switch turned the monooxygenase into an efficient preoxidase by increasing the peroxidase activity nearly one thousand folds. In order to tune the distal pocket of P450cam with polar residues, Leu 246 was replaced with a basic residue, lysine, resulting in a mutant with spectral features identical to P420, the inactive species of P450. But this inactive-species-like mutant showed catalytic activities without the facilitation of any cofactors. By substituting Gly 248 with a histidine, a novel Cys-Fe-His ligation set was obtained in P450cam which represented the very rare case of His ligation in heme-thiolate proteins. In addition to serving as a convenient model for hemoprotein structural studies, the G248H mutant also provided evidence about the nature of the axial ligand in cytochrome P420 and other engineered hemoproteins with thiolate ligations. Furthermore, attempts have been made to replace the proximal ligand in sperm whale myoglobin to construct a heme-thiolate protein model by mimicking the protein environment of cytochrome P450cam and chloroperoxidase.

Tailoring Heme-Thiolate Proteins into Efficient Biocatalysts with High Specificity and Selectivity

Cytochrome P450 monooxygenases, one of the most important classes of heme-thiolate proteins, have attracted considerable interest in the biochemical community because of its catalytic versatility, substrate diversity and great number in the superfamily. Although P450s are capable of catalyzing numerous difficult oxidation reactions, the relatively low stability, low turnover rates and the need of electron-donating cofactors have limited their practical biotechnological and pharmaceutical applications as isolated enzymes. The goal of this study is to tailor such heme-thiolate proteins into efficient biocatalysts with high specificity and selectivity by protein engineering and to better understand the structure-function relationship in cytochromes P450. In the effort to engineer P450cam, the prototype member of the P450 superfamily, into an efficient peroxygenase that utilizes hydrogen peroxide via the “peroxide-shunt” pathway, site-directed mutagenesis has been used to elucidate the critical roles of hydrophobic residues in the active site. Various biophysical, biochemical and spectroscopic techniques have been utilized to investigate the wild-type and mutant proteins. Three important P450cam variants were obtained showing distinct structural and functional features. In P450camV247H mutant, which exhibited almost identical spectral properties with the wild-type, it is demonstrated that a single amino acid switch turned the monooxygenase into an efficient preoxidase by increasing the peroxidase activity nearly one thousand folds. In order to tune the distal pocket of P450cam with polar residues, Leu 246 was replaced with a basic residue, lysine, resulting in a mutant with spectral features identical to P420, the inactive species of P450. But this inactive-species-like mutant showed catalytic activities without the facilitation of any cofactors. By substituting Gly 248 with a histidine, a novel Cys-Fe-His ligation set was obtained in P450cam which represented the very rare case of His ligation in heme-thiolate proteins. In addition to serving as a convenient model for hemoprotein structural studies, the G248H mutant also provided evidence about the nature of the axial ligand in cytochrome P420 and other engineered hemoproteins with thiolate ligations. Furthermore, attempts have been made to replace the proximal ligand in sperm whale myoglobin to construct a heme-thiolate protein model by mimicking the protein environment of cytochrome P450cam and chloroperoxidase.

Elastic modulus study of gold thin film for use as an actuated membrane in a superconductive radio frequency (RF) MicroElectroMechanical (MEM) switch

The objective of this research was to find Young's elastic modulus for thin gold films at room and cryogenic temperatures based on the flexional model which has not been previously attempted. Electrical Sonnet simulations and numerical methods using Abacus for the mechanical responses were employed for this purpose. A RF MEM shunt switch was designed and a fabrication process developed in house. The switch is composed of a superconducting YBa2Cu3O7 coplanar waveguide structure with an Au bridge membrane suspended above an area of the center conductor covered with BaTiO3 dielectric. The Au membrane is actuated by the electrostatic attractive force acting between the transmission line and the membrane when voltage is applied. The value of the actuation force will greatly depend on the switch pull-down voltage and on the geometry and mechanical properties of the bridge material. Results show that the elastic modulus for Au thin film can be 484 times higher at cryogenic temperature than it is at room temperature.

Contribuições à estratégia de controle sem detecção de harmônicos aplicada a um filtro ativo de potência paralelo

The conventional control schemes applied to Shunt Active Power Filters (SAPF) are Harmonic extractor-based strategies (HEBSs) because their effectiveness depends on how quickly and accurately the harmonic components of the nonlinear loads are identified. The SAPF can be also implemented without the use of the load harmonic extractors. In this case, the harmonic compensating term is obtained from the system active power balance. These systems can be considered as balanced-energy-based schemes (BEBSs) and their performance depends on how fast the system reaches the equilibrium state. In this case, the phase currents of the power grid are indirectly regulated by double sequence controllers with two degrees of freedom, where the internal model principle is employed to avoid reference frame transformation. Additionally the DSC controller presents robustness when the SAPF is operating under unbalanced conditions. Furthermore, SAPF implemented without harmonic detection schemes compensate simultaneously harmonic distortion and reactive power of the load. Their compensation capabilities, however, are limited by the SAPF power converter rating. Such a restriction can be minimized if the level of the reactive power correction is managed. In this work an estimation scheme for determining the filter currents is introduced to manage the compensation of reactive power. Experimental results are shown for demonstrating the performance of the proposed SAPF system.

Systematic Review of Surgical Management of Nonmetastatic Renal Cell Carcinoma with Vena Caval Thrombus

Copyright © 2015 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Étude du cross-talk entre la voie de synthèse des prostaglandines et des leucotriènes dans les membranes synoviales arthrosiques humaines

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Contributors to and impact of residual shunting after device closure of atrial septal defects.

BACKGROUND: The prevalence of residual shunt in patients after device closure of atrial septal defect and its impact on long-term outcome has not been previously defined. METHODS: From a prospective, single-institution registry of 408 patients, we selected individuals with agitated saline studies performed 1 year after closure. Baseline echocardiographic, invasive hemodynamic, and comorbidity data were compared to identify contributors to residual shunt. Survival was determined by review of the medical records and the Social Security Death Index. Survival analysis according to shunt included construction of Kaplan-Meier curves and Cox proportional hazards modeling. RESULTS: Among 213 analyzed patients, 27% were men and age at repair was 47 ± 17 years. Thirty patients (14%) had residual shunt at 1 year. Residual shunt was more common with Helex (22%) and CardioSEAL/STARFlex (40%) occluder devices than Amplatzer devices (9%; P = .005). Residual shunts were more common in whites (79% vs 46%, P = .004). At 7.3 ± 3.3 years of follow-up, 13 (6%) of patients had died, including 8 (5%) with Amplatzer, 5 (25%) with CardioSEAL/STARFlex, and 0 with Helex devices. Patients with residual shunting had a higher hazard of death (20% vs 4%, P = .001; hazard ratio 4.95 [1.59-14.90]). In an exploratory multivariable analysis, residual shunting, age, hypertension, coronary artery disease, and diastolic dysfunction were associated with death. CONCLUSIONS: Residual shunt after atrial septal defect device closure is common and adversely impacts long-term survival.