Role of mutations in the phosphoprotein on RNA polymerase activity of canine distemper virus following cell culture adaptation

Abstract: The canine distemper virus A75/17 wild-type strain, which is unable to replicate in cell lines, was adapted to growth in Vero cells. Sequence comparison between the A75/17 and the Vero cell-adapted A75/17-V virus revealed 7 amino acid differences between the 2 viruses. Three of these were located in the matrix protein, three in the phosphoprotein also changing the V protein but not the C protein and one in the large protein. The phosphoprotein and the large protein constituted the viral RNA polymerase whose activity was studied by transfection experiments using a reverse genetic system with a plasmid encoding a minireplicon and expression plasmids encoding the nucleocapsid protein and the viral RNA polymerase subunits. Surprinsingly, the enzyme of A75/17 CDV was significantly more active in cell lines compared to the polymerase of A75/17-V CDV. The decrease in overall enzyme activity was found to be due to both decreased replication and transcription activity. This polymerase attenuation was confirmed in CHO cells infection stably expressing the dog SLAM receptor mainly found in dog's lymphoid organs and allowing both virus strains to enter these cells at the same efficiency. A75/17-V CDV replicated more slowly in CHODogSLAM cells than A75/17 CDV and syncytium formation was significantly decreased compared to A75/17 infected CHODogSLAM cells.. Cell culture adaptation lead to an attenuated virus strain both in vitro and in vivo with decreased polymerase activity and syncytium forming capability showing an important role of the polymerase in determining the phenoytpe of the virus. In addition, this reduced phenotype of A75/17-V CDV was shown to be due to the P mutations in the P protein only, showing an important function of the polycistronic P gene in the adaptation process. The role of the matrix protein was found not to have any effect on polymerase activity, however its participation in the adaptation process still needs to be elucidated. The accessory proteins V and C were shown to act on polymerase activity, but their functions in virus pathogenicity and in inhibiting the interferon system have not been studied in this thesis. The V proteins have an activating effect on the polymerase of both the A75/17 and the A75/17-V CDV strains. Although the C protein amino acid sequence was not changed during adaptation of wild-type canine distemper virus in Vero cells, the C protein was demonstrated to have opposite effects on polymerase activity of both virus strains suggesting a different interaction of the C protein with the proteins forming the polymerase complex, which could modulate polymeras activity. These effects were demonstrated by transfection experiments and studying recombinant viruses not expressing the C protein. Thus, the abrogation of the C protein decrease the activity of the wild-type polymerase. In contrast, the polymerase activity of the Vero cell- adapted virus is enhanced in the absence of the C protein and this has also been demonstrated with a recombinant virus, which grew faster in the first 48 hours of infection. Future studies will focus on the generation of recombinant wild-type viruses, which should be very helpful in understanding the molecular mechanisms underlying the adaptation process and the loss of pathogenicity.

La Comissió Jurídica Assessora i l'autonomia de Catalunya durant la Segona República: la creació del sistema polític i institucional d'autogovern

[cat] La tesi que es defensa en aquest article és la posició de centralitat que va tenir la Comissió Jurídica Assessora durant el període de la Generalitat republicana en la creació i la configuració del sistema polític i institucional d'autogovern a Catalunya. Per a demostrar aquesta tesi s'analitzen els avantprojectes de llei que va elaborar durant aquest període la Comissió Jurídica Assessora: la Constitució o Estatut interior, la Llei municipal, el Tribunal de Cassació de Catalunya i els recursos en les vies governativa i contenciosa administrativa. L'anàlisi d'aquests avantprojectes posa en relleu com durant aquest període es van forjar les institucions d'autogovern, es van posar les bases de la democràcia i de l'autonomia local, com també es va crear una jurisdicció contenciosa administrativa pròpia. L'avantprojecte de llei de recursos en les vies governativa i contenciosa administrativa serà un precedent rellevant, però oblidat, de les lleis estatals sobre procediment administratiu i jurisdicció contenciosa administrativa. En definitiva, el sistema polític i institucional català que hi ha en l'actualitat no podria entendre's sense els antecedents de la Generalitat republicana que, recollint la legitimació històrica de les institucions medievals, van saber configurar les bases d'un sistema adaptat a les necessitats de la societat contemporània.

Identification of new transformation products during enzymatic treatment of tetracycline and erythromycin antibiotics at laboratory scale by an on-line turbulent flow liquid-chromatography coupled to a high resolution mass spectrometer LTQ-Orbitrap

This work describes the formation of transformation products (TPs) by the enzymatic degradation at laboratory scale of two highly consumed antibiotics: tetracycline (Tc) and erythromycin (ERY). The analysis of the samples was carried out by a fast and simple method based on the novel configuration of the on-line turbulent flow system coupled to a hybrid linear ion trap – high resolution mass spectrometer. The method was optimized and validated for the complete analysis of ERY, Tc and their transformation products within 10 min without any other sample manipulation. Furthermore, the applicability of the on-line procedure was evaluated for 25 additional antibiotics, covering a wide range of chemical classes in different environmental waters with satisfactory quality parameters. Degradation rates obtained for Tc by laccase enzyme and ERY by EreB esterase enzyme without the presence of mediators were ∼78% and ∼50%, respectively. Concerning the identification of TPs, three suspected compounds for Tc and five of ERY have been proposed. In the case of Tc, the tentative molecular formulas with errors mass within 2 ppm have been based on the hypothesis of dehydroxylation, (bi)demethylation and oxidation of the rings A and C as major reactions. In contrast, the major TP detected for ERY has been identified as the “dehydration ERY-A”, with the same molecular formula of its parent compound. In addition, the evaluation of the antibiotic activity of the samples along the enzymatic treatments showed a decrease around 100% in both cases

Sähköinen huutokauppa ja dynaaminen hankintajärjestelmä

Opinnäytetyöni tavoitteena on esitellä olennaiset kohdat julkisten hankintojen sähköisessä menettelyssä. Arvioinnin kohteena on lokakuussa 2011 voimaan astunut laki sähköisestä huutokaupasta ja dynaamisesta hankintajärjestelmästä. Opinnäytetyöni on kirjoituspöytätutkimus, joka perustuu julkisia hankintoja koskevaan lainsäädäntöaineistoon sekä julkisia hankintoja tutkivien sidosryhmien tuottamaan kirjallisuuteen. Näkökulmana on sähköisen menettelyn sääntelyn tarkoituksenmukaisuus hankintayksiköiden sekä toimittajien näkökulmista. Sääntely julkisten hankintojen sähköisestä menettelystä tarkoittaa julkisilla varoilla hankittavien palvelujen sekä tavaroiden hankintaa sähköisessä verkkoympäristössä. Hankintayksikkö, kuten valtion tai kunnan viranomainen, voi tämän lain myötä suorittaa hankintaprosessin sähköisesti käyttöliittymän kautta haluamastaan tuotteesta tai palvelusta.

Effect of Hevea brasiliensis latex sap gel on healing of acute skin wounds induced on the back of rats

Objective : to evaluate the effect of topical delivery of latex cream-gel in acute cutaneous wounds induced on the back of rats. Methods : we subjected sixteen rats to dermo-epidermal excision of a round dorsal skin flap, with 2.5cm diameter. We divided the animals into two groups: Latex Group: application of cream-gel-based latex throughout the wound bed on postoperative days zero, three, six and nine; Control group: no treatment on the wound. Photographs of the lesions were taken on the procedure day and on the 6th and 14th postoperative days, for analyzing the area and the larger diameter of the wound. We carried out euthanasia of all animals on the 14th postoperative day, when we resected he dorsal skin and the underlying muscle layer supporting the wound for histopathological study. Results : there was no statistically significant difference in the percentage of wound closure, in the histopathological findings or in the reduction of the area and of the largest diameter of the wounds among the groups studied on the 14th postoperative day. Conclusion : according to the experimental conditions in which the study was conducted, latex cream-gel did not interfere in the healing of acute cutaneous wounds in rats.

Simulation of structural stress history based on dynamic analysis

Modern machine structures are often fabricated by welding. From a fatigue point of view, the structural details and especially, the welded details are the most prone to fatigue damage and failure. Design against fatigue requires information on the fatigue resistance of a structure’s critical details and the stress loads that act on each detail. Even though, dynamic simulation of flexible bodies is already current method for analyzing structures, obtaining the stress history of a structural detail during dynamic simulation is a challenging task; especially when the detail has a complex geometry. In particular, analyzing the stress history of every structural detail within a single finite element model can be overwhelming since the amount of nodal degrees of freedom needed in the model may require an impractical amount of computational effort. The purpose of computer simulation is to reduce amount of prototypes and speed up the product development process. Also, to take operator influence into account, real time models, i.e. simplified and computationally efficient models are required. This in turn, requires stress computation to be efficient if it will be performed during dynamic simulation. The research looks back at the theoretical background of multibody dynamic simulation and finite element method to find suitable parts to form a new approach for efficient stress calculation. This study proposes that, the problem of stress calculation during dynamic simulation can be greatly simplified by using a combination of floating frame of reference formulation with modal superposition and a sub-modeling approach. In practice, the proposed approach can be used to efficiently generate the relevant fatigue assessment stress history for a structural detail during or after dynamic simulation. In this work numerical examples are presented to demonstrate the proposed approach in practice. The results show that approach is applicable and can be used as proposed.

The Environmental Status of the Port of HaminaKotka

This study is a part of the Ecologically Friendly Port Ust-Luga (EFP) project. The purpose of this study is to examine the environmental status of the Finnish ports and, more specifically, the Port of HaminaKotka. An analysis of the environmental status is performed mainly as a literature review, because the Finnish ports must comply with Finnish and EU legislation and with the binding international regulations and conventions created by different organizations. The International Maritime Organisation (IMO) has done groundbreaking work in the field of maritime safety and maritime environmental protection. The MARPOL convention has a great impact on decreasing pollution from international shipping and it applies to 99% of the world’s merchant tonnage. Pollution prevention covers: Oil pollution, Chemical pollution, Air pollution and GHG Emissions, Dumping of Wasted and Other Matters, Garbage, Sewage, Port Reception Facilities, Special Areas under MARPOL and Particularly Sensitive Sea Areas. There is also Pollution Prevention for other treaties like anti-fouling systems used on ships, the transfer of alien species by ships’ ballast water and the environmentally sound recycling of ships. There are more than twenty different EU and international regulations that influence ports and port operations in Finland. In addition, there is also national legislation that has an effect on Finnish ports. For the most part, the legislation for ports is common in the EU area, but the biggest and most important difference between the legislation in Finland and other EU countries is due to the Act on Environmental Impact Assessment Procedure. The Act states that the environmental impact assessment procedure shall be applied to projects that may have significant adverse environmental impacts, due to the special features of Finland`s nature and environment. In this Act, the term environmental impact refers to the direct and indirect effects inside and outside Finnish territory of a project or operations on human health, living conditions and amenity; soil, water, air, climate, organisms, interaction between them and biodiversity; community structure, buildings, landscape, townscape and cultural heritage; utilization of natural resources. In Finland, the Environmental Permit requires that ports collect all necessary information concerning environmental effects and make required reports to the Finnish authorities, stakeholders and the public. Commonly, environmental reporting is public and environmental achievements are emphasized in reporting and in media. At the moment, the problem in environmental reporting is that it’s difficult to compare data from different ports. There is enough data concerning the environmental effects and performance, but the manner of reporting and the quality of the data varies between ports. There are differences in the units and codes used, in some cases the information is not sufficient and it can even be rather unreliable. There are also differences regarding the subjects that are emphasized in reporting.

Chronic effect of antidopaminergic drugs or estrogen on male Wistar rat lactotrophs and somatotrophs

The aim of the present study was to evaluate the effect of antidopaminergic agents on the somatotrophs in the presence of hyperprolactinemia. Adult male Wistar rats were divided into 6 groups: a control group and five groups chronically treated (60 days) with haloperidol, fluphenazine, sulpiride, metoclopramide or estrogen. Somatotrophs and lactotrophs were identified by immunohistochemistry and the data are reported as percent of total anterior pituitary cells counted. The drugs significantly increased the percentage of lactotrophs: control (mean ± SD) 21.3 ± 4.4, haloperidol 27.8 ± 2.2, fluphenazine 34.5 ± 3.6, sulpiride 32.7 ± 3.5, metoclopramide 33.4 ± 5.5 and estrogen 42.4 ± 2.8. A significant reduction in somatotrophs was observed in animals treated with haloperidol (23.1 ± 3.0), fluphenazine (22.1 ± 1.1) and metoclopramide (24.2 ± 3.0) compared to control (27.3 ± 3.8), whereas no difference was observed in the groups treated with sulpiride (25.0 ± 2.2) and estrogen (27.1 ± 2.8). In the groups in which a reduction occurred, this may have simply been due to dilution, secondary to lactotroph hyperplasia. In view of the duplication of the percentage of prolactin-secreting cells, when estrogen was applied, the absence of a reduction in the percent of somatotrophs suggests a replication effect on this cell population. These data provide additional information about the direct or indirect effect of drugs which, in addition to interfering with the dopaminergic system, may act on other pituitary cells as well as on the lactotrophs.

Effects of melatonin on the ovarian response to pinealectomy or continuous light in female rats: similarity with polycystic ovary syndrome

The current study was conducted to investigate the relationship between melatonin and chronic anovulation. Adult (3-4 months old) female Wistar rats were submitted to pinealectomy: group I: pinealectomized ovariectomized melatonin-treated (N = 10); group II: pinealectomized ovariectomized placebo-treated (N = 12); group III: pinealectomized light-treated placebo-treated(N = 10) or maintained under continuous light; group IV: maintained under continuous light, ovariectomized melatonin-treated (N = 22); group V: maintained under continuous light, ovariectomized placebo-treated (N = 10); group VI: maintained under continuous light placebo-treated (N = 10). In order to assess ovarian modifications, unilateral ovariectomy was performed during the fourth month in groups I, II, IV, V and the other ovary was removed after 8 months. Ovariectomy was performed in groups III and VI only after eight months. Melatonin (200 µg/100 g body weight) dissolved in 0.02 ml absolute ethanol was injected intramuscularly daily during the last 4 months into groups I and IV. The other groups were treated with placebo (NaCl). The ovarian cysts were analyzed and their area, perimeter and maximum diameter, as well as the thickness of the ovarian capsule were measured. Daily colpocytological smears were performed throughout the study. Persistent estrous condition and ovarian cysts were observed in all groups. In pinealectomized rats the ovarian and vaginal alterations disappeared at the end of the study and in rats maintained under continuous light the vaginal and ovarian polycystic aspect was reversed only in those treated with melatonin. We conclude that melatonin may act on the ovarian response reverting chronic anovulation induced by pinealectomy or continuous light.

Negative inotropic and chronotropic effects on the guinea pig atrium of extracts obtained from Averrhoa carambola L. leaves

It has been reported that star fruit can lead to a fatal outcome in uremic patients. The intoxication syndrome consists of hiccups, mental confusion, dizziness, and vomiting. On the other hand, folk medicine uses teas and infusions of carambola leaves to treat headache, vomiting, cough, insomnia, and diabetes. This motivated us to determine if Averrhoa carambola can act on the contractility and automaticity of the guinea pig heart. We measured the atrial isometric force in stimulated left atria and determined the chronotropic changes in spontaneously beating right atria. The carambola leaf extracts (1.5 mg/ml) abolished the contractile force in a concentration-dependent manner. Among the crude, methanolic, ethanolic, aqueous, and acetic extracts, the aqueous one was the most potent (EC50 = 520 ± 94 µg/ml; flavonoids and tannins are the main constituents; Na+ and K+ contents in 1.0 mg/ml of aqueous extract were 0.12 ± 0.016 and 1.19 ± 0.15 mM, respectively). The aqueous extract abolished the positive Bowditch staircase phenomenon and reduced the inotropic response to CaCl2 (0.17-8.22 mM), events that are dependent on the cellular Ca2+ inward current. The adrenergic, muscarinic or opioid membrane receptors do not seem to participate in the mechanism of action of the cardioactive substance(s). In spontaneously beating atria, the aqueous extract promoted a negative chronotropic effect that was antagonized by 0.1 µM isoproterenol bitartrate. With this agonist, the EC50 of the aqueous extract increased from 133 ± 58 to 650 ± 100 µg/ml. These data regarding the effect of A. carambola on guinea pig atrial contractility and automaticity indicate an L-type Ca2+ channel blockade.

Collective redundancies in the State of California of the United States and in the Republic of Finland. Procedures and Provisions

Video games industry has recently bonded California and Finland in a new way and where the employers are recruiting they also need to be aware of the provisions and procedures related to terminations. In general, collective dismissals are on a relatively high level both in Finland and in California. In California, collective redundancies are regulated under the WARN law. The WARN obligates employers with 75 or more employees to give a 60-day notice prior to a mass lay off and some other similar events. Employers with less than 75 employees are free to administer the terminations without the WARN notice period. Generally, the California at-will presumption allows employment relationship to be terminated any day with or without reason and without notice period if conditions of collective agreements or employment contract do not limit this right. Termination cannot anyhow be in violation of the anti-discrimination law. In Finland the termination related provisions are part of the Employment Contracts Act and the Act on Co-operation within Undertakings. Collective redundancies are allowed under financial and production related grounds. Small employers with less than 20 employees follow the termination provisions of the Employment Contracts Act and are obligated to inform the employee to be terminated on the details of the termination itself and also the services of the Employment and Economic Development Office. Employers with 20 or more employees are to initiate co-operation procedure under the Act on Co-operation within Undertakings when reducing personnel. The co- operation negotiations are to inform employees on the employer’s plans and financial situation as well as to involve them in the decision making regarding the terminations. The employer’s duty to inform the employees of the services of Employment and Economic Development Office needs to be fulfilled also in terminations under the co-operation procedure. Discrimination is prohibited in Finland in terminations of employment. As an alternative for terminations, employees can for example be transferred to another position or be temporarily laid off. Employer’s duties related to search of alternatives for layoff are broader in Finland than in California. The recent development of the labor laws in Finland and in California suggests that the labor law is not static in either one of these environments but changes can be expected as the needs of the business life so require.

Reflections on the sociology of law: A rejection of law as 'socially marginal'

Rejecting the concept of law as subservient to social pathology, the principle aim of this article is to locatc law as a critical matter of social structure - and power - which requires to be considered as a central element in the construction of society and social institutions. As such, this article contends that wider jurisprudential notions such as legal procedure and procedural justice, and juridical power and discretion are cogent, robust normative social concerns (as much as they are legal concerns) that positively require consideration and representation in the ernpifical study of sociological phenomena. Reflecting upon scholarship and research evidence on legal procedure and decision-making, the article attempts to elucidate the inter-relationship between power, 'the social', and the operation of law. It concludes that law is not 'socially marginal' but socially, totally central. (c) 2009 Elsevier Ltd. All rights reserved.

The impact of fruit flavonoids on memory and cognition

There is intense interest in the studies related to the potential of phytochemical-rich foods to prevent age-related neurodegeneration and cognitive decline. Recent evidence has indicated that a group of plant-derived compounds known as flavonoids may exert particularly powerful actions on mammalian cognition and may reverse age-related declines in memory and learning. In particular, evidence suggests that foods rich in three specific flavonoid sub-groups, the flavanols, anthocyanins and/or flavanones, possess the greatest potential to act on the cognitive processes. This review will highlight the evidence for the actions of such flavonoids, found most commonly in fruits, such as apples, berries and citrus, on cognitive behaviour and the underlying cellular architecture. Although the precise mechanisms by which these flavonoids act within the brain remain unresolved, the present review focuses on their ability to protect vulnerable neurons and enhance the function of existing neuronal structures, two processes known to be influenced by flavonoids and also known to underpin neuro-cognitive function. Most notably, we discuss their selective interactions with protein kinase and lipid kinase signalling cascades (i.e. phosphoinositide-3 kinase/Akt and mitogen-activated protein kinase pathways), which regulate transcription factors and gene expression involved in both synaptic plasticity and cerebrovascular blood flow. Overall, the review attempts to provide an initial insight into the potential impact of regular flavonoid-rich fruit consumption on normal or abnormal deteriorations in cognitive performance.

Microcystins -LA, -YR, and -LR action on neutrophil migration

Microcystins (MCs) produced by some freshwater cyanobacterial species possess potent liver toxicity as evidenced by acute neutrophil infiltration. Here, we investigate the ability of three structurally distinct toxins (MC-LA, MC-LR, and MC-YR) to evoke neutrophil recruitment per se and their effects on migration pathways. Intravital Microscopic Studies showed that topical application of only MC-LR enhanced the numbers of rolling and adhered leukocytes in the endothelium of postcapillary mesenteric venules. The latter effects may be dependent upon induction of the synthesis and expression Of L-selectin and beta(2)-integrin in neutrophils, as assessed by flow cytometry and RT-PCR, respectively. Conversely, the three toxins promoted direct locomotion of neutrophils and enhanced their migration in response to NO, as measured by Boyden chamber assays, and increased intracellular calcium, a messenger in the chemotaxic process. In conclusion, our results show that MCs act on specific pathways of neutrophil recruitment, indicating their potential effect on neutrophils activation. (C) 2009 Elsevier Inc. All rights reserved.

Essays in taxation and savings

Esta tese é composta por três artigos. Dois deles investigam assuntos afeitos a tributação e o terceiro é um artigo sobre o tema “poupança”'. Embora os objetos de análise sejam distintos, os três possuem como característica comum a aplicação de técnicas de econometria de dados em painel a bases de dados inéditas. Em dois dos artigos, utiliza-se estimação por GMM em modelos dinâmicos. Por sua vez, o artigo remanescente é uma aplicação de modelos de variável dependente latente. Abaixo, apresenta-se um breve resumo de cada artigo, começando pelos dois artigos de tributação, que dividem uma seção comum sobre o ICMS (o imposto estadual sobre valor adicionado) e terminando com o artigo sobre poupança. O primeiro artigo analisa a importância da fiscalização como instrumento para deter a evasão de tributos e aumentar a receita tributária, no caso de um imposto sobre valor adicionado, no contexto de um país em desenvolvimento. O estudo é realizado com dados do estado de São Paulo. Para tratar questões relativas a endogeneidade e inércia na série de receita tributária, empregam-se técnicas de painel dinâmico. Utiliza-se como variáveis de controle o nível do PIB regional e duas proxies para esforço fiscal: a quantidade e o valor das multas tributárias. Os resultados apontam impacto significativo do esforço fiscal nas receitas tributárias. O artigo evidencia, indiretamente, a forma como a evasão fiscal é afetada pela penalidade aplicada aos casos de sonegação. Suas conclusões também são relevantes no contexto das discussões sobre o federalismo fiscal brasileiro, especialmente no caso de uma reforma tributária potencial. O segundo artigo examina uma das principais tarefas das administrações tributárias: a escolha periódica de contribuintes para auditoria. A melhora na eficiência dos mecanismos de seleção de empresas tem o potencial de impactar positivamente a probabilidade de detecção de fraudes fiscais, provendo melhor alocação dos escassos recursos fiscais. Neste artigo, tentamos desenvolver este mecanismo calculando a probabilidade de sonegação associada a cada contribuinte. Isto é feito, no universo restrito de empresas auditadas, por meio da combinação “ótima” de diversos indicadores fiscais existentes e de informações dos resultados dos procedimentos de auditoria, em modelos de variável dependente latente. Após calculados os coeficientes, a probabilidade de sonegação é calculada para todo o universo de contribuintes. O método foi empregado em um painel com micro-dados de empresas sujeitas ao recolhimento de ICMS no âmbito da Delegacia Tributária de Guarulhos, no estado de São Paulo. O terceiro artigo analisa as baixas taxas de poupança dos países latino-americanos nas últimas décadas. Utilizando técnicas de dados em painel, identificam-se os determinantes da taxa de poupança. Em seguida, faz-se uma análise contrafactual usando a China, que tem apresentado altas taxas de poupança no mesmo período, como parâmetro. Atenção especial é dispensada ao Brasil, que tem ficado muito atrás dos seus pares no grupo dos BRICs neste quesito. O artigo contribui para a literatura existente em vários sentidos: emprega duas amplas bases de dados para analisar a influência de uma grande variedade de determinantes da taxa de poupança, incluindo variáveis demográficas e de previdência social; confirma resultados previamente encontrados na literatura, com a robustez conferida por bases de dados mais ricas; para alguns países latino-americanos, revela que as suas taxas de poupança tenderiam a aumentar se eles tivessem um comportamento mais semelhante ao da China em outras áreas, mas o incremento não seria tão dramático.