Inverter-fed linear asynchronous machine

The behaviors of an arc-shaped stator induction machine (the sector-motor) and a disc-secondary linear induction motor are analyzed in this work for different values of the frequency. Variable frequency is produced by a voltage source controlled-current inverter which keeps constant the r.m.s. value of the phase current, also assuring a sinusoidal waveform. For the simulations of the machine developed thrust, an equivalent circuit is used. It is obtained through the application of the one-dimensional theory to the modeling. The circuit parameters take into account the end effects, always present is these kind of machines. The phase current waveforms are analyzed for their harmonic contents. Experimental measurements were carried out in laboratory and are presented with the simulations, for comparison.

Pathological and molecular analysis of Turkey coronavirus replication in captive wild Turkey embryos (broad-breasted bronze breeder)

The degree of genetic and pathologic variation exhibited by a turkey Coronavirus (TCoV) strain was investigated after nine serial passages in 25-day-old turkey embryos obtained from wild broad-breasted bronze breeders. In spite of spleen, liver, kidneys, cloacal bursa and thymus have been collected and analysed, the main histopathological changes were only documented in the intestine sections. Microscopic lesions were characterized as mild enteritis, low degree of enterocyte vacuolization and detachment of the intestinal villous after five consecutive passages and were considered absent in the last passages. Genealogic analysis based on S1 and S2 DNA sequences suggested that Brazilian isolate might be considered as originated from TCoV strains circulating in the United States, as 100% identity with TCoV-Gl strain. Although S1 S2 sequences from each passage revealed no significant point mutations, and no correlation could be speculate between S2 nucleotide changes and pathologic features in infected embryos. This is the first demonstration of wild turkey embryos as a model for TCoV isolation and propagation.

The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication

Background: Yellow fever virus (YFV) belongs to the Flavivirus genus and causes an important disease. An alarming resurgence of viral circulation and the expansion of YFV-endemic zones have been detected in Africa and South America in recent years. NS5 is a viral protein that contains methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, which are essential for viral replication, and the interactions between NS5 and cellular proteins have been studied to better understand viral replication. The aim of this study was to characterize the interaction of the NS5 protein with eukaryotic translation initiation factor 3 subunit L (eIF3L) and to evaluate the role of eIF3L in yellow fever replication. Methods. To identify interactions of YFV NS5 with cellular proteins, we performed a two-hybrid screen using the YFV NS5 RdRp domain as bait with a human cDNA library, and RNApol deletion mutants were generated and analyzed using the two-hybrid system for mapping the interactions. The RNApol region involved was segmented into three fragments and analyzed using an eIF3L-expressing yeast strain. To map the NS5 residues that are critical for the interactions, we performed site-direct mutagenesis in segment 3 of the interaction domain (ID) and confirmed the interaction using in vitro assays and in vivo coimmunoprecipitation. The significance of eIF3L for YFV replication was investigated using eIF3L overexpression and RNA interference. Results: In this work, we describe and characterize the interaction of NS5 with the translation factor eIF3L. The interaction between NS5 and eIF3L was confirmed using in vitro binding and in vivo coimmunoprecipitation assays. This interaction occurs at a region (the interaction domain of the RNApol domain) that is conserved in several flaviviruses and that is, therefore, likely to be relevant to the genus. eIF3L overexpression and plaque reduction assays showed a slight effect on YFV replication, indicating that the interaction of eIF3L with YFV NS5 may play a role in YFV replication. Conclusions: Although the precise function of eIF3L on interactions with viral proteins is not entirely understood, these results indicate an interaction of eIF3L with YF NS5 and that eIF3L overexpression facilitates translation, which has potential implications for virus replication. © 2013 Morais et al.; licensee BioMed Central Ltd.

Cultivation of PCV2 in swine testicle cells using the shell vial technique and monitoring of viral replication by qPCR and RT-qPCR

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Caffeine inhibits hepatitis C virus replication in vitro

Abstract Hepatitis C is considered the major cause of cirrhosis and hepatocellular carcinoma. Conventional treatment is not effective against some hepatitis C virus (HCV) genotypes; therefore, new treatments are needed. Coffee and, more recently, caffeine, have been found to have a beneficial effect in several disorders of the liver, including those manifesting abnormal liver biochemistry, cirrhosis and hepatocellular carcinoma. Caffeine acts directly by delaying fibrosis, thereby improving the function of liver cellular pathways and interfering with pathways used by the HCV replication cycle. In the current study, the direct relationship between caffeine and viral replication was evaluated. The Huh-7.5 cell line was used for transient infections with FL-J6/JFH-50 C19Rluc2AUbi and to establish a cell line stably expressing SGR-Feo JFH- 1. Caffeine efficiently inhibited HCV replication in a dosedependent manner at non-cytotoxic concentrations and demonstrated an IC50 value of 0.7263 mM after 48 h of incubation. These data demonstrate that caffeine may be an important new agent for anti-HCV therapies due to its efficient inhibition of HCV replication at non-toxic concentrations.

Natural compounds isolated from Brazilian plants are potent inhibitors of hepatitis C virus replication in vitro

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of canonical siRNA and dicer substrate RNA for inhibition of hepatitis C virus genome replication: a comparative study

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparative analysis of the replication of bovine herpesvirus 1 (BHV1) and BHV5 in bovine derived-neuron-like cells

Members of the subfamily Alphaherpesvirinae use the epithelium of the upper respiratory and/or genital tract as preferential sites for primary replication. However, bovine herpesvirus 5 (BoHV5) is neurotropic and neuroinvasive and responsible for meningoencephalitis in cattle and in animal models. A related virus, BoHV1 has also been occasionally implicated in natural cases of neurological infection and disease in cattle. The aim of the present study was to assess the in vitro effects of BoHV1 and BoHV5 replication in neuron-like cells. Overall, cytopathic effects, consisting of floating rounded cells, giant cells and monolayer lysis, induced by both viruses at 48 h postinfection (p.i.) resulted in a loss of cell viability and high virus titres (r = 0.978). The BoHV1 Cooper strain produced the lowest titres in neuron-like cells, although viral DNA was detected in infected cells during all experiments. Virus replication in infected cells was demonstrated by immunocytochemistry, flow cytometry and qPCR assays. BoHV antigens were better visualized at 48 h p.i. and flow cytometry analysis showed that SV56/90 and Los Angeles antigens were present at higher levels. In spite of the fact that BoHV titres dropped at 48 h p.i, viral DNA remained detectable until 120 h p.i. Sensitive TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) and annexin V assays were used to identify apoptosis. BoHV5 induced death in approximately 50 % of cells within 24 h p.i., similar to what has been observed for BoHV1 Los Angeles. Infection with the BoHV1 Cooper strain resulted in 26.37 % of cells being in the early stages of apoptosis; 63.69 % of infected cells were considered viable. Modulation of mitochondrial function, as measured by mitochondrial membrane depolarization, was synchronous with the virus replication cycle, cell viability and virus titres at 48 h p.i. Our results indicate that apoptosis plays an important role in preventing neuronal death and provides a bovine-derived in vitro system to study herpesvirus-neuron interactions.

INFLUENCE OF TYPE 2 BOVINE VIRAL DIARRHEA VIRUS NPRO ON ENHANCEMENT OF BOVINE RESPIRATORY SYNCYTIAL VIRUS REPLICATION MEDIATED BY ANTAGONISM OF HOST CELL INTERFERON TYPE I RESPONSES

Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus, Family Flaviviridae. The virus can infect many species of animals of the order Artiodactyla. The BVDV genome encodes an auto protease, Npro, that degrades interferon regulatory factor-3 (IRF-3) reducing type I interferon (IFN-I) production from host cells. Bovine respiratory syncytial virus (BRSV) is a member of the genus Pneumovirus, Family Paramyxoviridae. Concurrent infection with BVDV and BRSV causes more severe respiratory and enteric disease than infection with either virus alone. Our hypothesis was that Npro modulates the innate immune responses to BVDV infection and enhances replication of BVDV or BRSV co-infection. The noncytopathic BVDV2 viruses NY93/c N- Npro 18 EGFP (a mutant with modified Npro fused with enhanced green fluorescent protein), NY93 infectious clone (NY93/c), wild-type NY93-BVDV2 (NY93-wt), and BRSV were evaluated in this study. The objectives of this study were: (1) to characterize the replication kinetics and IFN-I induction in Madin-Darby bovine kidney (MDBK) cells following infection with each of the BVDV isolates, and (2) to characterize the influence of BVDV-mediated IFN-I antagonism on enhancement of BRSV replication in bovine turbinate (BT) cells. NY93/c N- Npro 18 EGFP replicated 0.4 – 1.6 TCID50 logs lower than NY93-wt in MDBK cells. NY93/c N- Npro 18 EGFP-infected MDBK cells synthesized IFN-I significantly higher than NY93/c- and NY93-wt-infected MDBK cells. BT cells co-infected with NY93/c N- Npro 18 EGFP/BRSV or NY93-wt/BRSV were evaluated to determine the effects of co-infection on BRSV replication and IFN-I induction in BT cells. BRSV RNA levels in NY93-wt/BRSV co-infected BT cells were 2.49, 2.79, and 2.89 copy number logs significantly greater than in NY93/c N- Npro 18 EGFP/BRSV co-infected BT cells on days 5, 7, and 9 post-infection, respectively. BVDV RNA levels in NY93/c N- Npro 18 EGFP-infected BT cells were 1.64 – 4.38 copy number logs lower than in NY93-wt-infected BT cells. NY93/c N- Npro 18 EGFP single and co-infected BT cells synthesized IFN-I significantly higher than NY93-wt single and co-infected BT cells. In summary, these findings suggest: (1) NY93/c N- Npro 18 EGFP BVDV2 induced higher levels of IFN-I than BVDV2-wt and may be useful as a safer, replicating BVDV vaccine, and (2) Enhancement of BRSV infection by BVDV co-infection is mediated by antagonism of IFN-I.

Asynchronous teams for joint lot-sizing and scheduling problem in flow shops

The integrated production scheduling and lot-sizing problem in a flow shop environment consists of establishing production lot sizes and allocating machines to process them within a planning horizon in a production line with machines arranged in series. The problem considers that demands must be met without backlogging, the capacity of the machines must be respected, and machine setups are sequence-dependent and preserved between periods of the planning horizon. The objective is to determine a production schedule to minimise the setup, production and inventory costs. A mathematical model from the literature is presented, as well as procedures for obtaining feasible solutions. However, some of the procedures have difficulty in obtaining feasible solutions for large-sized problem instances. In addition, we address the problem using different versions of the Asynchronous Team (A-Team) approach. The procedures were compared with literature heuristics based on Mixed Integer Programming. The proposed A-Team procedures outperformed the literature heuristics, especially for large instances. The developed methodologies and the results obtained are presented.

Effects of consequenses on patterns of interlocked contingencies; A replication of a metacontingency experiment

The concept of metacontingency was taught to undergraduate students of Psychology by using a "game" simulation proposed originally by Vichi, Andery and Glenn (2009). Twenty-five students, distributed into three groups were exposed to six experimental sessions in which they had to make bets and divide the amounts gained. The three groups competed against each other for photocopies quotas. Two contingencies shifted over the sessions. Under Contingency B, the group would win points only if in the previous round each member had received the same amount of points and under Contingency A, winning was contingent on an unequal distribution of the points. We observed that proportional divisions predominated independent of the contingency in course. The manipulation of cultural consequences (winning or losing points) produced consistent modifications in two response categories: 1) choices of the value bet in each round, and 2) divisions of the points among group members. Controlling relations between cultural consequences and the behavior of dividing were statistically significant in one of the groups, whereas in the other two groups controlling relations were observed only in Contingency B. A review of the reinforcement criteria used in the original experiment is suggested.

Chk1 phosphorylation of Metnase enhances DNA repair but inhibits replication fork restart

Chk1 both arrests replication forks and enhances repair of DNA damage by phosphorylating downstream effectors. Although there has been a concerted effort to identify effectors of Chk1 activity, underlying mechanisms of effector action are still being identified. Metnase (also called SETMAR) is a SET and transposase domain protein that promotes both DNA double-strand break (DSB) repair and restart of stalled replication forks. In this study, we show that Metnase is phosphorylated only on Ser495 (S495) in vivo in response to DNA damage by ionizing radiation. Chk1 is the major mediator of this phosphorylation event. We had previously shown that wild-type (wt) Metnase associates with chromatin near DSBs and methylates histone H3 Lys36. Here we show that a Ser495Ala (S495A) Metnase mutant, which is not phosphorylated by Chk1, is defective in DSB-induced chromatin association. The S495A mutant also fails to enhance repair of an induced DSB when compared with wt Metnase. Interestingly, the S495A mutant demonstrated increased restart of stalled replication forks compared with wt Metnase. Thus, phosphorylation of Metnase S495 differentiates between these two functions, enhancing DSB repair and repressing replication fork restart. In summary, these data lend insight into the mechanism by which Chk1 enhances repair of DNA damage while at the same time repressing stalled replication fork restart. Oncogene (2012) 31, 4245-4254; doi:10.1038/onc.2011.586; published online 9 January 2012

Expressiveness of Concurrent Languages

The aim of this thesis is to go through different approaches for proving expressiveness properties in several concurrent languages. We analyse four different calculi exploiting for each one a different technique. We begin with the analysis of a synchronous language, we explore the expressiveness of a fragment of CCS! (a variant of Milner's CCS where replication is considered instead of recursion) w.r.t. the existence of faithful encodings (i.e. encodings that respect the behaviour of the encoded model without introducing unnecessary computations) of models of computability strictly less expressive than Turing Machines. Namely, grammars of types 1,2 and 3 in the Chomsky Hierarchy. We then move to asynchronous languages and we study full abstraction for two Linda-like languages. Linda can be considered as the asynchronous version of CCS plus a shared memory (a multiset of elements) that is used for storing messages. After having defined a denotational semantics based on traces, we obtain fully abstract semantics for both languages by using suitable abstractions in order to identify different traces which do not correspond to different behaviours. Since the ability of one of the two variants considered of recognising multiple occurrences of messages in the store (which accounts for an increase of expressiveness) reflects in a less complex abstraction, we then study other languages where multiplicity plays a fundamental role. We consider the language CHR (Constraint Handling Rules) a language which uses multi-headed (guarded) rules. We prove that multiple heads augment the expressive power of the language. Indeed we show that if we restrict to rules where the head contains at most n atoms we could generate a hierarchy of languages with increasing expressiveness (i.e. the CHR language allowing at most n atoms in the heads is more expressive than the language allowing at most m atoms, with m

Real time monitoring of DNA hybridization and replication using optical and acoustic biosensors

Während in den letzten Jahren zahlreiche Biosensoren zum spezifischen Nachweis von DNA entwickelt wurden, ist die Anwendung oberflächen-sensitiver Methoden auf enzymatische Reaktionen ein vergleichsweise neues Forschungsgebiet. Trotz der hohen Empfindlichkeit und der Möglichkeit zur Echtzeit-Beobachtung molekularer Prozesse, ist die Anwendung dieser Methoden nicht etabliert, da die Enzymaktivität durch die Nähe zur Oberfläche beeinträchtigt sein kann. Im Rahmen dieser Arbeit wurde die enzymatische Verlängerung immobilisierter DNA durch eine DNA Polymerase mit Hilfe von Oberflächenplasmonen-Fluoreszenzspektroskopie (SPFS) und einer Quarzkristall-Mikrowaage (QCM) untersucht. Die Synthese von DNA wurde im Fall der QCM als Massenzuwachs detektiert, der sich im Abfall der Resonanzfrequenz des Schwingquarzes und einem Anstieg seiner Dissipationsenergie ausdrückte. Die viskoelastischen Eigenschaften der DNA-Schichten wurden bestimmt, indem die erhaltenen Daten mit einem auf Voigt basierenden Modell ausgewertet wurden. SPFS nutzt das evaneszente elektromagnetische Feld, das mit Oberflächenplasmonen einhergeht, zur oberflächen-sensitiven Anregung von Chromophoren. Auf diese Weise wurde der Einbau von Farbstoff-markierten Nukleotiden in die entstehende DNA-Sequenz als Indikator für das Voranschreiten der Reaktion ausgenutzt. Beide Meßtechniken konnten erfolgreich zum Nachweis der DNA-Synthese herangezogen werden, wobei die katalytische Aktivität des Enzyms vergleichbar zu der in Lösung gemessenen war.

Brain-Computer Interfaces for augmented communication: Asynchronous and adaptive algorithms and evaluation with end users

This thesis aimed at addressing some of the issues that, at the state of the art, avoid the P300-based brain computer interface (BCI) systems to move from research laboratories to end users’ home. An innovative asynchronous classifier has been defined and validated. It relies on the introduction of a set of thresholds in the classifier, and such thresholds have been assessed considering the distributions of score values relating to target, non-target stimuli and epochs of voluntary no-control. With the asynchronous classifier, a P300-based BCI system can adapt its speed to the current state of the user and can automatically suspend the control when the user diverts his attention from the stimulation interface. Since EEG signals are non-stationary and show inherent variability, in order to make long-term use of BCI possible, it is important to track changes in ongoing EEG activity and to adapt BCI model parameters accordingly. To this aim, the asynchronous classifier has been subsequently improved by introducing a self-calibration algorithm for the continuous and unsupervised recalibration of the subjective control parameters. Finally an index for the online monitoring of the EEG quality has been defined and validated in order to detect potential problems and system failures. This thesis ends with the description of a translational work involving end users (people with amyotrophic lateral sclerosis-ALS). Focusing on the concepts of the user centered design approach, the phases relating to the design, the development and the validation of an innovative assistive device have been described. The proposed assistive technology (AT) has been specifically designed to meet the needs of people with ALS during the different phases of the disease (i.e. the degree of motor abilities impairment). Indeed, the AT can be accessed with several input devices either conventional (mouse, touchscreen) or alterative (switches, headtracker) up to a P300-based BCI.