Characterization of the mechanisms underlying the inflammatory response to Polistes lanio lanio (paper wasp) venom in mouse dorsal skin

Stings by Polistes wasps can cause life-threatening allergic reactions, pain and inflammation. We examined the changes in microvascular permeability and neutrophil influx caused by the venom of Polistes lanio a paper wasp found in southeastern Brazil. The intradermal injection of wasp venom caused long-lasting paw oedema and dose-dependently increased microvascular permeability in mouse dorsal skin. SR140333, an NK(1) receptor antagonist, markedly inhibited the response, but the NK(2) receptor antagonist SR48968 was ineffective. The oedema was reduced in capsaicin-treated rats, indicating a direct activation of sensory fibres. Dialysis of the venom partially reduced the oedema and the remaining response was further inhibited by SR140333. Mass spectrometric analysis of the venom revealed two peptides (QPPTPPEHRFPGLM and ASEPTALGLPRIFPGLM) with sequence similarities to the C-terminal region of tachykinin-like peptides found in Phoneutria nigniventer spider venom and vertebrates. Wasp venom failed to release histamine from mast cells in vitro and spectrofluorometric assay of the venom revealed a negligible content of histamine in the usual dose of P.l. lanio venom (1 nmol of histamine/7 mu g of venom)that was removed by dialysis. The histamine H(1) receptor antagonist pyrilamine, but not bradykinin B(1) or B(2) receptor antagonists, inhibited venom-induced oedema. In conclusion, P. l. lanio venom induces potent oedema and increases vascular permeability in mice, primarily through activation of tachykinin NK(1) receptors by substance P released from sensory C fibres, which in turn releases histamine from dermal mast cells. This is the first description of a neurovascular mechanism for P. l. lanio venom-mediated inflammation. The extent to which the two tachykinin-like peptides identified here contribute to this neurogenic inflammatory response remains to be elucidated. (c) 2008 Elsevier Ltd. All rights reserved.

Peptide gomesin triggers cell death through L-type channel calcium influx, MAPK/ERK, PKC and PI3K signaling and generation of reactive oxygen species

Gomesin is an antimicrobial peptide isolated from hemocytes of a common Brazilian tarantula spider named Acanthoscurriagomesiana. This peptide exerts antitumor activity in vitro and in vivo by an unknown mechanism. In this study, the cytotoxic mechanism of gomesin in human neuroblastoma SH-SY5Y and rat pheochromocytoma PC12 cells was investigated. Gomesin induced necrotic cell death and was cytotoxic to SH-SY5Y and PC12 cells. The peptide evoked a rapid and transient elevation of intracellular calcium levels in Fluo-4-AM loaded PC12 cells, which was inhibited by nimodipine, an L-type calcium channel blocker. Preincubation with nimodipine also inhibited cell death induced by gomesin in SH-SY5Y and PC12 cells. Gomesin-induced cell death was prevented by the pretreatment with MAPK/ERK, PKC or PI3K inhibitors, but not with PKA inhibitor. In addition, gomesin generated reactive oxygen species (ROS) in SH-SY5Y cells, which were blocked with nimodipine and MAPK/ERK, PKC or PI3K inhibitors. Taken together, these results suggest that gomesin could be a useful anticancer agent, which mechanism of cytotoxicity implicates calcium entry through L-type calcium channels, activation of MAPK/ERK, PKC and PI3K signaling as well as the generation of reactive oxygen species. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Differential effects of alpha-helical and beta-hairpin antimicrobial peptides against Acanthamoeba castellanii

In this work we evaluated the ability of different types of antimicrobial peptides to promote permeabilization and growth inhibition of Acanthamoeba castellanii trophozoites, which cause eye keratitis. We used cationic alpha-helical peptides P5 and a beta-hairpin amphipathic molecule (gomesin), of the spider Acanthoscurria gomesiana haemocytes. A. castellanii permeabilization was obtained after 1 h incubation with micromolar concentrations of both types of peptides. While permeabilization induced by gomesin increased with longer incubations, P5 permeabilization did not increase with time and occurred at doses that are more toxic for SIRC cells, P5, however, at doses below the critical dose used to kill rabbit corneal cells was quite effective in promoting growth inhibition. Similarly, P5 was more effective when serine protease inhibitor was added simultaneously to the permeabilization assay. High performance chromatography followed by mass spectrometry analysis confirmed that, in contrast to gomesin, P5 is hydrolysed by A. castellanii culture supernatants. We conclude that the use of antimicrobial peptides to treat A. castellanii infections requires the search of more specific peptides that are resistant to proteolysis.

Interaction of membrane mimetic vesicles with the antimicrobial peptide gomesin and its analogues

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

Expression and characterization of LTx2, a neurotoxin from Lasiodora sp effecting on calcium channels

Here, we described the expression and characterization of the recombinant toxin LTx2, which was previously isolated from the venomous cDNA library of a Brazilian spider, Lasiodora sp. (Mygalomorphae, Theraphosidae). The recombinant toxin found in the soluble and insoluble fractions was purified by reverse phase high-performance liquid chromatography (HPLC). Ca2+ imaging analysis revealed that the recombinant LTx2 acts on calcium channels of BC3H1 cells, blocking L-type calcium channels. (C) 2008 Elsevier Inc. All rights reserved.

Acanthoscurrin Fragment 101-132: Total Synthesis at 60 degrees C of a Novel Difficult Sequence

Glycine-rich proteins (GRP), serve a variety of biological functions. Acanthoscurrin is an antimicrobial GRP isolated front hemocytes-of the Brazilian spider Acanthoscurria gomesiana. Aiming to contribute to the knowledge of the secondary structure and stepwise solid-phase synthesis of GRPs` glycine-rich domains, we attempted to prepare G(101)GGLGGGRGGGYG(113) GGGGYGGGYG(123)GGy(126)GGGKYK(132)-NH(2), acanthoscurrin C-terminal amidated fragment. Although a theoretical prediction did not indicate high aggregation potential for this peptide, repetitive incomplete aminoacylations were observed after incorporating Tyr(126) to the growing peptide-MBHA resin (Boc chemistry) at 60 degrees C. The problem was not solved by varying the coupling reagents or solvents, adding chaotropic salts to the reaction media or changing the resin/chemistry (Rink amide resin/Fmoc chemistry). Some improvement was mode when CLEAR amide resin (Fmoc chemistry) was 32 used, as it allowed for obtaining fragment (G(113)-K(132) NIR-FT-Raman spectra collected for samples of the growing peptide-MBHA, -Rink amide resin and -CLEAR amide resin revealed the presence of beta-sheet structures. Only the combination of CLEAR-amide resin, 60 degrees C, Fmoc-(Fmoc-Hmb)Gly-OH and LiCl (the last two used alternately) was able to inhibit the phenomenon, as proven by NIR-FT-Raman analysis of the growing peptide-resin, allowing the total synthesis of desired 132 fragment Gly(101)-K(132). In summary, this work describes a new difficult sequence, contributes to understanding stepwise solid-phase synthesis of this type of peptide and shows that, at least while protected and linked to a resin, this GRPs glycine-rich motif presents all early tendency to assume beta-sheet structures. (c) 2008 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 92: 65-75, 2009.

Effects of the antimicrobial peptide gomesin on the global gene expression profile, virulence and biofilm formation of Xylella fastidiosa

In the xylem vessels of susceptible hosts, such as citrus trees, Xylella fastidiosa forms biofilm-like colonies that can block water transport, which appears to correlate to disease symptoms. Besides aiding host colonization, bacterial biofilms play an important role in resistance against antimicrobial agents, for instance antimicrobial peptides (AMPs). Here, we show that gomesin, a potent AMP from a tarantula spider, modulates X. fastidiosa gene expression profile upon 60 min of treatment with a sublethal concentration. DNA microarray hybridizations revealed that among the upregulated coding sequences, some are related to biofilm production. In addition, we show that the biofilm formed by gomesin-treated bacteria is thicker than that formed by nontreated cells or cells exposed to streptomycin. We have also observed that the treatment of X. fastidiosa with a sublethal concentration of gomesin before inoculation in tobacco plants correlates with a reduction in foliar symptoms, an effect possibly due to the trapping of bacterial cells to fewer xylem vessels, given the enhancement in biofilm production. These results warrant further investigation of how X. fastidiosa would respond to the AMPs produced by citrus endophytes and by the insect vector, leading to a better understanding of the mechanism of action of these molecules on bacterial virulence.

ICC-profilers användbarhetvid gråbalansjusteringav arkoffsettryck

I detta examensarbete har gråbalansstyrning och ICC-profilers duglighet undersökts. ICC-profilernahar utvärderats utifrån den tryckkvalitet de genererat tillsammans med olika papper.Gråbalansstyrning har använts som gemensamma likare för samtliga testtryckningar. Detta tillfördeen variabel som var lika för samtliga papper i utredningen. Här med ökade sannolikheten att ICCprofilerskapade från olika papper, skulle ge en likvärdig kvalitet.I arbetet har många mätningar och beräkningar genomförts. De två främsta anledningarna till dettavar dels att utreda i vilken mån det gick att gråbalansjustera trycket och dels att inhämta jämförbaravärden på likheter och divergenser mellan olika kombinationer av ICC-profiler och papper. Mätdatahjälpte även till att ta hänsyn till de felkällor som fanns.Det var möjligt att gråbalansjustera trycket och det fanns likheter mellan en ICC-profil på olika papperoch även mellan fler ICC-profiler på samma papper. Samtliga resultat och slutsatser var beroendeav att neutral gråbalans upprätthölls under tryckningarna. Subjektiva och objektiva jämförelser visadebåda på att en ICC-profil inte kommer att påverkas av variationer mellan olika pappersleveranser,såvida dessa inte är större än skillnaden mellan de olika papperna.Med säkerhet kunde inte förutsägas från vilket papper en ICC-profil skulle skapas för att ge en högkvalitet på flera papper. Mest troligt var att likvärdigt resultat skulle kunna uppnås på G-Print, MultiArt Matt och Multi Art Silk.

Utvärdering av ICC-profiler och profil-inställningar för HP Indigo Press 1000

Det nystartade digitaltryckeriet Digaloo planerar att inom en snar framtid införa ICC-hantering i sittarbetsflöde. Med anledning av detta har en studie utförts där antalet erfordrade ICC-profiler undersöktssamt vilka akromatiska inställningar som bäst lämpar sig för tryckeriets HP Indigo Press 1000.Testtryckning utfördes på sex av Digaloos mest använda papperskvaliteter. Genom inläsning av testkartorhar de olika papperskvaliteternas reproducerbara färgrymder åskådliggjorts. Grundat på resultatetfrån denna testtryckning framställdes ICC-profiler för valda papperskvaliteter. Dessa genereradesmed olika inställningar för akromatisk repro och TIC (Total Ink Coverage). Vid en andra testtryckninganvändes dessa ICC-profiler för konvertering av mellanton-, natt- och snöbilder, vilka trycktespå olika papperskvaliteter.Resultatet från den första testtryckningen visar att HP Indigo Press 1000 återger störst förgrymd påpapperskvaliteten Silverblade Silk, tätt följd av Silverblade Art. De minsta färgrymderna reproduceraspå Lessebo Linné Gultonat och Lessebo Linné Naturvit.Vid perceptuell bedömning av de olika bilderna blev resultatet att bäst tryckresultat uppnås medpappersspecifika ICC-profiler. Detta gäller dock endast i två av tre fall. Vid bedömning av bäst lämpadTIC ansågs den idag på Digaloo använda nivån som mycket dålig. Genom att antingen sänka eller höjadenna nivå bedömdes tryckresultatet öka i kvalitet.Slutligen visar studien även att akromatik i form av UCR ger bäst bildåtergivning i mellanton- ochnattbilder på de valda papperskvaliteterna. De olika graderna av GCR i snöbilder anses dock passaolika bra beroende på vilken papperskvalitet som används.

ICC-profilers duglighet vid gråbalansjustering av arkoffsettryck

Adjustment to a neutral colour balance when printing was the starting point in this degree project. Onthis base ICC-profiles were evaluated and assessed. The survey was concentrated on the similarity betweenreproductions on different papers with various ICC-profiles. The colour balancing introduced aconsistent variable between four graphic papers. With raised uniformity the probability increased toreceive similarities between the various combinations of ICC-profiles and papers.Numerous of measurements and calculations have been carried out, mainly to confirm whether or notadjustment to a neutral colour balance is possible and repeatable. Further more this gained in comparablevalues corresponding to similarities and divergences between combinations of ICC-profiles andpaper.The research concluded in the fact that it was possible to reach a neutral colour balance. One ICCprofileused together with various papers and also one paper with several ICC-profiles resulted in similaritiesand formed groups of samples. Since equivalent results could be reached on different samples,it implicated that variations within one paper shouldn’t affect the ICC-profiles. This was valid when thevariations were less than the divergence between the papers.Certain predictions on the question of from which paper the ICC-profiles should be generated to createhighest quality in print hasn’t been able to assemble. It’s likely that equivalent results could begenerated on G-Print, Multi Art Matt and Multi Art Silk.

"Vos sos la mujer araña, que atrapa a los hombres en su tela" : La construcción de la identidad feminina en El beso de la Mujer araña, de Manuel Puig.

La novela El beso de la mujer araña del escritor argentino Manuel Puig es una novela que trata dedos presos en una cárcel bonaerense durante la dictadura militar argentina. Esta tesina se centrará enla identidad femenina de Molina. El objetivo de esta tesina es examinar la celebración de lafemineidad de Molina, y su construcción de una identidad femenina. Esta identidad se conectarácon la idea de la identidad femenina como socialmente construida, opuesto a la noción de génerocomo algo innato. Es decir, la identidad femenina de Molina y sus ideas de lo femeninocorresponden a la idea de la identidad femenina como socialmente construida. Apoyándonos enteoría feminista y teoría queer, estableceremos la conexión que existe entre la identidad femeninacomo la ve Molina, y la identidad femenina como construcción social.

Índices de avaliação de processos : abordagem univariada e multivariada

Esta dissertação versa sobre índices de avaliação de processo nas suas abordagens univariada e multivariada. Analisa-se a utilização da Análise de Componentes Principais (ACP) como ferramenta no desenvolvimento de índices capazes de avaliar processos multivariados. O trabalho apresenta uma revisão teórica sobre os índices univariados de aplicação mais comum na indústria (CP/PP , CPK/PPK , CPM/PPM e CPMK/PPMK), o índice multivariado MCpm e sobre os índices MCP , MCPK , MCPM e MCPMK , associados a ACP. Os índices de avaliação de processo são analisados quanto à sua adequação ao uso, através de um estudo de caso na indústria de componentes automotivos. Para tanto, examina-se o processo de fabricação de um componente do freio de veículos médios e pesados, denominado spider, em que doze variáveis de processo são controladas por meio de controle estatístico de processo. Através do estudo de caso, faz-se uma comparação entre os resultados dos índices univariados e multivariados; pressupõe-se que através da Análise de Componentes Principais poder-se-á concluir sobre a capacidade e o desempenho de processos multivariados. Por fim, a partir da análise univariada dos componentes principais, apresenta-se uma técnica complementar para quantificar a contribuição das variáveis controladas à variabilidade de processos multivariados.

Larval development of Notolopas brasiliensis Miers, 1886 (Brachyura: Majoidea: Pisidae) described from laboratory reared material and a reappraisal of the characters of Pisidae

O completo desenvolvimento larval de Notolopas brasiliensis é descrito, a partir de material criado em laboratório, com ênfase na morfologia externa de Majoidea e comparado aos demais gêneros de Pisidae. O desenvolvimento larval de N. brasiliensis consiste em dois estágios de zoea e um de megalopa. A duração media de cada estágio foi de 4.2 ± 1.0 dias para a Zoea I e 3.8 ± 0.7 dias para a Zoea II, a megalopa aparece entre 8.1 ± 0.4 dias após a eclosão. Os caracteres previamente utilizados para definir as formas larvais de Pisidae ou são simplesiomórficos ou altamente homoplásticos. Foi observado que não existe um conjunto de caracteres capazes de definir Pisidae até o presente.Contudo foi mostrado que uma combinação de caracteres pode ser utilizada para diferenciar Notolopas dos demais gêneros da família.

A field study to describe diel, tidal and semilunar rhythms of larval release in an assemblage of tropical rocky shore crabs

Available information on the larval release rhythms of brachyurans is biased to temperate estuarine species and outcomes resulting from some sort of artificial manipulation of ovigerous females. In this study we applied field methods to describe the larval release rhythms of an assemblage of tropical rocky shore crabs. Sampling the broods of ovigerous females of Pachygrapsus transversus at two different shores indicated a spatially consistent semilunar pattern, with larval release maxima around the full and new moon. Yet, synchronism between populations varied considerably, with the pattern obtained at the site exposed to a lower wave action far more apparent. Breeding cohorts at one of the sampled shores apparently belonged to actual age groups composing the ovigerous population. The data suggest that these breeding groups release their larvae in alternate syzygy periods, responding to a lunar cycle instead of the semilunar pattern observed for the whole population. For the description of shorter-term rhythms, temporal series at hour intervals were obtained by sampling the plankton and confinement boxes where ovigerous females were held. Unexpectedly, diurnal release activity prevailed over nocturnal hatching. Yet, only grapsids living higher on the shore exhibited strong preferences over the diel cycle, with P. transversus releasing their larvae during the day and Geograpsus lividus during the night. The pea crab Dissodactylus crinitichelis, the spider crab Epialtus brasiliensis and a suite of xanthoids undertook considerable releasing activity in both periods. Apart from the commensal pea crab D. crinitichelis, all other taxa revealed tide-related rhythms of larval release, with average estimates of the time of maximum hatching always around the time of high tides; usually during the flooding and slack, rather than the ebbing tide. Data obtained for P. transversus females held in confinement boxes indicated that early larval release is mostly due to nocturnal hatching, while zoeal release in diurnal groups took place at the time of high tide. Since nocturnal high tides at the study area occurred late, sometimes close to dusk, early release would allow more time for offshore transport of larvae when the action of potential predators is reduced.

Source populations in coastal crabs: parameters affecting egg production

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