Role of Munc18c in SNARE-mediated exocytosis

Background: The SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein Receptors) and SM (Sec1/Munc18) family of proteins form the core machinery that drives the fusion of vesicles in different membrane trafficking steps. They are highly conserved, implying a similar mode of binding and function. In vertebrates, Munc18a is essential for neuronal exocytosis. It binds to its partner syntaxin1a (Syx1a) at both its N-peptide and closed conformation, and thereby inhibits SNARE complex formation in vitro. By contrast, its close homolog Munc18c is thought to interact with only the N-peptide of its partner Syx4. Moreover, different effects of Munc18c on SNARE complex formation have been reported, suggesting that the two Munc18/Syx pairs act differently. Objective: The aim of the present study was to investigate whether the mechanism of action of Munc18c indeed deviates from that of Munc18a by using sensitive biochemical and biophysical methods. Results: I found that Munc18c does have a similar binding mode as Munc18a and interacts tightly with Syx4 at both the N-peptide and closed conformation. Moreover, I established, through a novel assay, that Munc18c inhibits SNARE complex assembly, with both the binding sites contributing to inhibition, similar to Munc18a. However, there were several subtle differences between the two Munc18/Syx pairs. Munc18a exerted stronger inhibition than Munc18c. Also their respective Syx partners were found to differ in the rate of binding to SNAP25, suggesting that the equilibrium of their open and closed conformations is different. Moreover, Munc18a was found to interact with Syx 1, 2, 3 but not 4, while Munc18c bound to Syx 2, 4 and 1 but not 3. By comparing the kinetics of interaction of Syx with either Munc18 or SNAP25, I found that the block of SNARE complex assembly by Munc18 is effective on a shorter time scale, but SNAP25 eventually binds to Syx resulting in SNARE complex formation. Nevertheless, these findings do not explain how Syx can escape the tight grip of Munc18, suggesting that other proteins or mechanisms are needed for this step. I also discovered that Munc18 is able to bind on the surface of the SNARE core complex; however, this observation needs to be tested more rigorously. Conclusion: Munc18c was found to be similar to Munc18a in its mode of binding to Syx and inhibition of SNARE complex assembly. However, differences in kinetics and interaction specificities were observed between the different Munc18/Syx pairs. -- Contexte : Les familles des protéines SNARE (Soluble N-ethylmaleimide-sensitive factor At- tachment protein Receptors) et SM (Sec1/Munc18) forment le coeur de la machinerie chargée de la fusion vésiculaire au cours des différentes étapes du trafic intracellulaire. Elles sont très conservées, suggérant un mode d'interaction et des fonctions semblables. Chez les Verté- brés, Munc18a est essentielle à l'exocytose neuronale. Elle se lie à sa partenaire d'interaction syntaxin1a (Syx1a) à la fois via un peptide N-terminal et la conformation fermée de celle-ci, inhibant ainsi la formation du complexe SNARE in vitro. Son homologue proche Munc18c au contraire, est supposée interagir seulement avec le peptide N-terminal de sa partenaire Syx4. En outre, différents effets de Munc18c sur la formation du complexe SNARE ont été décrits, suggérant que les deux paires Munc18/Syx fonctionnent différemment. Objectif : Le but de cette étude est de tester si les mécanismes de fonctionnement de Munc18c diffèrent vraiment de ceux de Munc18a par le biais de méthodes biochimiques et biophysiques très précises. Résultats : J'ai pu démontrer que Munc18c se comporte en effet de façon semblable à Munc18a, et interagit étroitement avec Syx4 à ses deux sites de liaison. J'ai pu de surcroît montrer par une nouvelle méthode que Munc18c inhibe l'assemblage du complexe SNARE en impliquant ces deux sites de liaison, comme le fait Munc18a. il existe cependant de subtiles différences entre les deux paires Munc18/Syx : Munc18a exerce une inhibition plus forte que Munc18c ; leurs Syx partenaires diffèrent également dans leur degré de liaison à SNAP25, ce qui suggère un équilibre different de leurs conformations ouverte et fermée. De plus, Munc18a interagit avec Syx 1, 2 et 3 mais pas Syx 4, alors que Munc18c se lie à Syx 2, 4 et 1 mais pas Syx 3. En comparant les cinétiques d'interaction de Syx avec Munc18 ou SNAP25, j'ai découvert que le blocage par Munc18 de l'assemblage du complexe SNARE est effectif de façon brève, bien que SNAP25 finisse par se lier à Syx et aboutir ainsi à la formation du complexe SNARE. Ces découvertes n'expliquent cependant pas comment Syx parvient à échapper à la solide emprise de Munc18, et suggèrent ainsi l'intervention nécessaire d'autres protéines ou mécanismes à cette étape. J'ai également découvert que Munc18 peut se lier à la surface de la partie centrale du complexe SNARE - cette observation reste à être testée de façon plus stringente. Conclusion : Il a pu être établi que Munc18c est semblable à Munc18a quant à son mode de liaison à Syx et d'inhibition de l'assemblage du complexe SNARE. Des différences de cinétique et de spécificité d'interaction entre les diverses paires Munc18/Syx ont cependant été identifiées.

Aquisição de tempos de vida de fosforescência com resolução de fase e sua aplicação ao estudo de relaxações em polímeros

We describe in this work a simple experimental set up to perform time dependent luminescence experiments in time scales from mili-seconds to seconds, based on the phase resolution of the emission signal. This system is composed by modulation of a continuous light source with an external chopper controlled by a lock-in amplifier. We exemplified the utility of the system in studies of phosphorecence emission using benzophenone dissolved in polystyrene and since the phosphorescence intensity and lifetime are temperature-dependent processes, we also studied polymer relaxation processes in the temperature range from 20 to 400K. A software that drives the spectrofluorimeter and controls both the cryosystem and the lock-in amplifier was developed.

Effects of modern variable frequency drive on the reliability of an old crane induction motor

The work aims to analyze the possibilities of utilizing old crane driving AC induction motors in modern pulse-width-modulated variable frequency drives. Bearing currents and voltage stresses are the two main problems associated with modern IGBT inverters, and they may cause premature failure of an old induction motor. The origins of these two problems are studied. An analysis of the mechanism of bearing failure is proposed. Certain types of bearing currents are considered in detail. The most effective and economical means are chosen for bearing currents mitigation. Transient phenomena of cables and mechanism of over voltages occurring at motor terminals are studied in the work. The weakest places of the stator winding insulation system are shown and recommendations are given considering the mitigation of voltage stresses. Only the most appropriate and cost effective preventative methods are chosen for old motor drives. Rewinding of old motors is also considered.

Considerações sobre a eletrogeração de peróxido de hidrogênio

This paper presents some results that may be used as previous considerations to a hydrogen peroxide electrogeneration process design. A kinetic study of oxygen dissolution in aqueous solution is carried out and rate constants for oxygen dissolution are calculated. Voltammetric experiments on vitreous carbon cathode shown that the low saturation concentration drives the oxygen reduction process to a mass transfer controlled process which exhibits low values of limiting currents. Results have shown that the hydrogen peroxide formation and its decomposition to water are separated by 400 mV on the vitreous carbon surface. Diffusion coefficients for oxygen and hydrogen peroxide are calculated using data taken from Levich and Tafel plots. In a series of bulk electrolysis experiments hydrogen peroxide was electrogenerated at several potential values, and concentration profiles as a function of the electrical charged passed were obtained. Data shown that, since limiting current plateaus are poorly defined onto reticulated vitreous carbon, cathodic efficiency may be a good criterion for choosing the potential value in which hydrogen peroxide electrogeneration should be carried out.

Taajuusmuuttajaohjatun pumppukäytön simulointimallin kehittäminen ja testaus

Pumppukäytöt vastaavat noin neljänneksestä Euroopan sähkömoottoreissa kulutettavasta sähkötehosta. Energian kallistumisen vuoksi energian säästämisestä ja energiatehokkuudesta on tullut yhä tärkeämpää. Taajuusmuuttajat ovat tuoneet yksinkertaisen säätötavan, jolla suurimmassa osassa pumppukäyttöjä voidaan saada huomattavia energiasäästöjä. Koko energiansäästöpotentiaalin hyödyntämiseksi täytyy kuitenkin pumppujen toimintaa tarkkailla. Tarkkailu voidaan toteuttaa käyttämällä taajuusmuuttajan sisäisiä mittauksia pumpun toimintapisteen määrittämiseen. Tässä työssä muodostetaan kaksi simulointimallia, joilla voidaan tutkia pumppukäytön toimintapistettä ja energiatehokkuutta. Pumpattavan nesteen ominaisuuksien vaikutusta malleihin tutkitaan ja tapoja korjata virhettä selvitetään. Mallien tuottamia estimaatteja verrataan laboratorio ja pilot-kohteiden mittauksiin. Pilot-kohteiden avulla tutkitaan voidaanko simulointimalleja käyttää teollisuuden energiansäästöpotentiaalia omaavien pumppukäyttöjen havaitsemiseen.

Mashup-alustan soveltuvuus tilannekuvan luontiin verkostopuolustusympäristössä

Verkostosodankäynti on suuren huomion kohteena useiden maiden puolustusvoimien järjestelmäkehityshankkeissa. Verkostosodankäynnin tavoitteena on kytkeä kaikki taistelukentän komponentit yhteen nopean tiedonsiirtoverkon avulla. Tällä pyritään tehokkaampaan tiedonjakoon ja edelleen resurssien tehokkaampaan käyttöön. Keskeisessä osassa verkostosodankäynnin tavoitteiden saavuttamisessa on palvelukeskeinen arkkitehtuuri (SOA). Tarve yhä monimutkaisemmille tietojärjestelmille pakottaa myös sotilasympäristön toimijat etsimään ratkaisuja valmiista kaupallisista toteutuksista. Verkottunut toiminta tuottaa valtavasti erilaista tilannetietoa. Tilannetiedon pohjalta muodostetaan erilaisia tilannekuvia, joita johtajat käyttävät päätöksentekonsa tukena. Työssä tutkitaan kaupallisen mashup-alustan käyttöä tilannekuvan luomiseen. Mashup-alusta on tietojärjestelmä, jolla voidaan helposti ja nopeasti integroida useista lähteistä saatavaa informaatiota. Mashup-alusta mahdollistaa niin kutsuttujen käyttäjämääriteltyjen tilannekuvien luomisen. Työn tuloksena mashup-alustan soveltuvuus tähän käyttöön on hyvä ja se soveltuu hyvin erityisesti tilanteisiin, joissa vaaditaan nopeita ratkaisuja. Jatkotutkimusta aiheesta tarvitaan, koska mashupalustan käyttöä sotilaallisissa tietojärjestelmissä ei ole juurikaan tutkittu ja aihe on suhteellisen uusi myös tiedeyhteisössä.

Position sensorless electrically excited synchronous motor drive for industrial use based on direct flux linkage and torque control

Synchronous machines with an AC converter are used mainly in large drives, for example in ship propulsion drives as well as in rolling mill drives in steel industry. These motors are used because of their high efficiency, high overload capacity and good performance in the field weakening area. Present day drives for electrically excited synchronous motors are equipped with position sensors. Most drives for electrically excited synchronous motors will be equipped with position sensors also in future. This kind of drives with good dynamics are mainly used in metal industry. Drives without a position sensor can be used e.g. in ship propulsion and in large pump and blower drives. Nowadays, these drives are equipped with a position sensor, too. The tendency is to avoid a position sensor if possible, since a sensor reduces the reliability of the drive and increases costs (latter is not very significant for large drives). A new control technique for a synchronous motor drive is a combination of the Direct Flux Linkage Control (DFLC) based on a voltage model and a supervising method (e.g. current model). This combination is called Direct Torque Control method (DTC). In the case of the position sensorless drive, the DTC can be implemented by using other supervising methods that keep the stator flux linkage origin centered. In this thesis, a method for the observation of the drift of the real stator flux linkage in the DTC drive is introduced. It is also shown how this method can be used as a supervising method that keeps the stator flux linkage origin centered in the case of the DTC. In the position sensorless case, a synchronous motor can be started up with the DTC control, when a method for the determination of the initial rotor position presented in this thesis is used. The load characteristics of such a drive are not very good at low rotational speeds. Furthermore, continuous operation at a zero speed and at a low rotational speed is not possible, which is partly due to the problems related to the flux linkage estimate. For operation in a low speed area, a stator current control method based on the DFLC modulator (DMCQ is presented. With the DMCC, it is possible to start up and operate a synchronous motor at a zero speed and at low rotational speeds in general. The DMCC is necessary in situations where high torque (e.g. nominal torque) is required at the starting moment, or if the motor runs several seconds at a zero speed or at a low speed range (up to 2 Hz). The behaviour of the described methods is shown with test results. The test results are presented for the direct flux linkage and torque controlled test drive system with a 14.5 kVA, four pole salient pole synchronous motor with a damper winding and electric excitation. The static accuracy of the drive is verified by measuring the torque in a static load operation, and the dynamics of the drive is proven in load transient tests. The performance of the drive concept presented in this work is sufficient e.g. for ship propulsion and for large pump drives. Furthermore, the developed methods are almost independent of the machine parameters.

Salient Pole Synchronous Machine Modelling in an Industrial Direct Torque Controlled Drive Application

Synchronous motors are used mainly in large drives, for example in ship propulsion systems and in steel factories' rolling mills because of their high efficiency, high overload capacity and good performance in the field weakening range. This, however, requires an extremely good torque control system. A fast torque response and a torque accuracy are basic requirements for such a drive. For large power, high dynamic performance drives the commonly known principle of field oriented vector control has been used solely hitherto, but nowadays it is not the only way to implement such a drive. A new control method Direct Torque Control (DTC) has also emerged. The performance of such a high quality torque control as DTC in dynamically demanding industrial applications is mainly based on the accurate estimate of the various flux linkages' space vectors. Nowadays industrial motor control systems are real time applications with restricted calculation capacity. At the same time the control system requires a simple, fast calculable and reasonably accurate motor model. In this work a method to handle these problems in a Direct Torque Controlled (DTC) salient pole synchronous motor drive is proposed. A motor model which combines the induction law based "voltage model" and motor inductance parameters based "current model" is presented. The voltage model operates as a main model and is calculated at a very fast sampling rate (for example 40 kHz). The stator flux linkage calculated via integration from the stator voltages is corrected using the stator flux linkage computed from the current model. The current model acts as a supervisor that prevents only the motor stator flux linkage from drifting erroneous during longer time intervals. At very low speeds the role of the current model is emphasised but, nevertheless, the voltage model always stays the main model. At higher speeds the function of the current model correction is to act as a stabiliser of the control system. The current model contains a set of inductance parameters which must be known. The validation of the current model in steady state is not self evident. It depends on the accuracy of the saturated value of the inductances. Parameter measurement of the motor model where the supply inverter is used as a measurement signal generator is presented. This so called identification run can be performed prior to delivery or during drive commissioning. A derivation method for the inductance models used for the representation of the saturation effects is proposed. The performance of the electrically excited synchronous motor supplied with the DTC inverter is proven with experimental results. It is shown that it is possible to obtain a good static accuracy of the DTC's torque controller for an electrically excited synchronous motor. The dynamic response is fast and a new operation point is achieved without oscillation. The operation is stable throughout the speed range. The modelling of the magnetising inductance saturation is essential and cross saturation has to be considered as well. The effect of cross saturation is very significant. A DTC inverter can be used as a measuring equipment and the parameters needed for the motor model can be defined by the inverter itself. The main advantage is that the parameters defined are measured in similar magnetic operation conditions and no disagreement between the parameters will exist. The inductance models generated are adequate to meet the requirements of dynamically demanding drives.

Multichannel distribution strategy for electrical equipment marketing

The thesis examines System Integration and original equipment manufacturer (OEM) channel in the St. Petersburg drives market. The aim of the study was to increase understanding the relationship between OEM and SI and producers, problems and ongoing trends. The collected data was analyzed in order to find out which features of a power electronic drive product exercise a significant influence for the Russian companies decision. An essential part of this study was interviews as primary information sources, organized with SI and OEM companies which represented the basic SPb industry segments. The wholesalers and end users are left out from the analysis. The collected data was analyzed in order to find out which features of a power electronic drive product exercise a significant influence for the Russian companies decision.

Conceitos de química dos ingressantes nos cursos de graduação do Instituto de Química da Universidade de São Paulo

A diagnostic instrument was developed to evaluate the basic chemistry concepts held by freshmen students of the three Chemistry undergraduate courses offered by the University of São Paulo. The instrument minimizes the use of algorithms or memorization by students and values high-order cognitive skills. Analysis of the students' performances reveals systematic use of "displacement reaction" as an algorithm and a mechanical use of Le Chatelier's Principle. Failure in comprehending the chemical equation and chemical language drives students to alternative models for chemical reactions in aqueous solution. For instance, reaction would occur between "ionic pairs" and/or between species situated in separate compartments.

Creación de unidades de análisis forense en bibliotecas

Las técnicas de análisis forense digital, de aplicación en investigación criminal, también se pueden usar en las bibliotecas para acceder a información digital almacenada en soportes o formatos obsoletos. Se analizan distintos ejemplos de departamentos de análisis forense creados por bibliotecas y se describen los elementos de hardware y software mínimos con los que se podría montar una unidad de análisis forense en cualquier biblioteca. Con este fin se presentan dos posibles configuraciones de equipamiento y se dan recomendaciones sobre organización del flujo de trabajo para la recuperación de antiguos discos duros y disquetes. Forensic analysis techniques, usually applied in criminal research, could also be used in libraries to access digital information stored in obsolete formats or storage devices. This article analyses some examples of forensic research departments created by libraries, and describes the minimal hardware and software elements required to set up a library unit specialized in forensic analysis. Two possible equipment settings are introduced and recommendations are given on how to organize a workflow to recover information stored in floppy disks, diskettes and old hard drives.

Autoamostrador de baixo custo para análise por injeção em fluxo

This paper describes a homemade autosampler for flow injection analysis (FIA), constructed with two step motors from old floppy disk drives (5¼-inch). The autosampler was connected to a computer through the parallel port and the sampling process was controlled by software in Quick Basic. The performance of the system was assessed by the determination of ammonium, based on the gaseous diffusion into a bromocresol purple solution, following the spectrophotometric determination of change in absorbance. The easy and simple construction is the main characteristics of this equipment and analytical results with RSD lower than 1% were obtained.

Solution thermodynamics of methocarbamol in some ethanol + water mixtures

Apparent thermodynamic functions, Gibbs energy, enthalpy and entropy of solution and mixing, for methocarbamol in ethanol + water mixtures, were evaluated from solubility data determined at temperatures from 293.15 K to 313.15 K and from calorimetric values of drug fusion. The drug solubility was greatest in the mixtures with 0.70 or 0.80 mass fraction of ethanol and lowest in neat water across all temperatures studied. Non-linear enthalpy-entropy compensation was found for the dissolution processes. Accordingly, solution enthalpy drives the respective processes in almost all the solvent systems analyzed.

Painediffusöörin suunnitteluperusteiden kehittäminen

Pesu on tärkeä osa sellun tuotantoprosessia. Eräs tapa toteuttaa sellun pesu on käyttää painediffusööriä. Painediffusööri toimii syrjäytyspesuperiaatteella, eli poistaa sellu-massasta keittolipeää paineistetun pesuveden avulla. Työssä on kehitetty painediffusöörin suunnittelun lähtökohtia keräämällä tietoa laitteen toiminnasta, rakenteesta, valmistuksesta sekä nykyisistä epäkohdista, joihin esitetään parannusmahdollisuuksia. Tärkeimmät kehitysalueet laitteessa ovat valmistus-toleranssien väljentäminen sekä sihdin pystysuuntaisen liikkeen tuottaminen. Laitteen valmistustoleranssit on analysoitu perusteellisesti, ja niiden väljentämis-mahdollisuuksia on tutkittu. Väljentämiseen ehdotetaan erilaisia keinoja. Sihdin liike tuotetaan tällä hetkellä hydrauliikalla. Hydrauliikkakomponenteille on koottu mitoitusohjeita, joiden jälkeen esitellään keinoja hydrauliikkajärjestelmän kehittämiseen. Lopuksi esitellään muita lineaarisen liikkeen tuottamisvaihtoehtoja, joilla hydrauliikan voisi korvata. Rakenteessa käytetyille valmistusmateriaaleille on etsitty olemassa olevat yleisimpien materiaalistandardien mukaiset nimikkeet materiaalinvalinnan helpottamiseksi jatkossa. Pääasiallisten valmistusmateriaalien lisäksi on kerätty tietoa myös vaihtoehtoisista konstruktiomateriaaleista sekä materiaalinvalinnasta ja tuotesuunnittelusta yleensä.

Reaaliaikainen Clarken muunnos

Työssä suunniteltiin ja rakennettiin laite, joka suorittaa reaaliaikaisen Clarken muunnoksen kolmivaiheiselle mittaussignaalille. Kolmivaiheisella mittaussignaalilla tarkoitetaan tässä kolmivaiheisesta järjestelmästä mitattuja vaihevirtoja tai -jännitteitä. Lähtö tuottaa kaksivaiheisen mittaussignaalin, jota voidaan tutkia oskilloskooppia. Käyttämällä oskilloskooppia xy-moodissa näytölle piirtyy virta- tai jänniteympyrä. Laitetta voidaan käyttää esimerkiksi taajuusmuuttajien testauksen apuvälineenä, jolloin parametrien muuttamisen vaikutus laitteen tuottaman sähkön laatuun nähdään välittömästi.