Aplicació de la termoquimiolisi - cromatografia de gasos - espectrometria de masses a l'estudi de la matèria orgànica del sòl

Estudi realitzat a partir d’una estada a la Université de Poitiers, França, entre 2007 i 2009. El treball s'ha centrat en dues activitats bàsiques. El treball realitzat s’ha centrat en dues activitats bàsiques. D’una banda, la posada a punt d'un protocol de fraccionament de la matèria orgànica del sòl, per extraccions successives amb solvents alcalins després d'una seqüència de pretractaments al sòl: cap pretractament, atac amb àcid (per destruir els carbonats), atac amb ditionit (per reduir els òxids de Fe i Al i facilitar l'extracció de la matèria orgànica associada a aquests compostos). El protocol dóna una visió de conjunt de la situació de la matèria orgànica del sòl, combinant aspectes físics (protecció, precipitació, oclusió per carbonats) i químics (grau d'humificació). D’altra banda, l'aprenentatge de la tècnica de termoquimiolisi-cromatografia de gasos-espectrometria de masses. Aquest era l'objectiu de l'estada a Poitiers, al qual hem donat prioritat. Ens hem centrat en l'estudi de fraccions físiques (densimètriques) obtingudes en estudis anteriors sobre sòls forestals. Les fraccions considerades són: fracció lleugera (FL), tres fraccions ocluïdes (OC1, OC2 i OC3) i fracció densa (FD). L’aplicació de la termoquimiolisi permet de caracteritzar diversos grups de substàncies, de les quals ens hem centrat en alguns indicadors bioquímics: àcids grasos, alcohols, diàcids, productes fenòlics i altres productes aromàtics, derivats de carbohidrats. L’estudi de conjunt d’aquests productes indica que és a les fraccions ocluïdes (que solen ser minoritàries a tots els horitzons) on la matèria orgànica d’origen microbià és dominant, mentre que a les fraccions lleugera (FL) i densa (FD) la matèria orgànica d’origen vegetal sembla dominant. Es preveu aplicar aquesta tècnica a l’estudi de les fraccions obtingudes a la primera part del treball, actualment congelades i a l’espera de ser processades.

Translocation of the yeast dolichol-phosphate-mannose synthase into microsomal membranes.

Dolichol-phosphate-mannose synthase catalyzes the formation of Dolichol-phosphate-mannose from Dolichol-phosphate and GDP-mannose. Analysis of the primary amino acid sequence of the yeast enzyme predicts a luminal orientation of the enzyme in the endoplasmic reticulum. We analysed the translocation of the Dolichol-phosphate-mannose synthase into dog pancreatic microsomal membranes: resistance to proteolytic attack provides evidence of its luminal orientation and asks for a reevaluation of the topology of the reaction.

PSMA-Specific CAR-Engineered T Cells Eradicate Disseminated Prostate Cancer in Preclinical Models.

Immunology-based interventions have been proposed as a promising curative chance to effectively attack postoperative minimal residual disease and distant metastatic localizations of prostate tumors. We developed a chimeric antigen receptor (CAR) construct targeting the human prostate-specific membrane antigen (hPSMA), based on a novel and high affinity specific mAb. As a transfer method, we employed last-generation lentiviral vectors (LV) carrying a synthetic bidirectional promoter capable of robust and coordinated expression of the CAR molecule, and a bioluminescent reporter gene to allow the tracking of transgenic T cells after in vivo adoptive transfer. Overall, we demonstrated that CAR-expressing LV efficiently transduced short-term activated PBMC, which in turn were readily stimulated to produce cytokines and to exert a relevant cytotoxic activity by engagement with PSMA+ prostate tumor cells. Upon in vivo transfer in tumor-bearing mice, CAR-transduced T cells were capable to completely eradicate a disseminated neoplasia in the majority of treated animals, thus supporting the translation of such approach in the clinical setting.

Fabricació i anàlisi del biodièsel

La producció de biodièsel a partir d'olis de cuina utilitzats, amb l'objectiu de transformar un residu en un producte amb valor comercial i ambiental, és més net que els combustibles fòssils i contribueix a disminuir el gran consum de petroli que estem fent. En una primera part teòrica s’ha realitzat un treball de recerca d' informació del biodièsel per saber si és un bon combustible i si podria ser un possible substitut dels combustibles fòssils. En quant a la part pràctica l'objectiu principal ha estat fabricar el biodièsel . Les matèries primeres són oli de gira-sol i oli de cuina utilitzat, per tal de comparar les seves qualitats. S’ha arribat a la conclusió que presenten característiques molt semblants. Després de fer les anàlisis físico-químics de qualitat vam fer una mescla dels dos biodièsels fabricats, i el vam provar en un motor Dièsel per comprovar si funciona correctament i fer una comparació dels gasos emesos pel nostre combustible amb els emesos pel gasoil comercial. El resultat va ser molt positiu ja que el motor va funcionar correctament, i la comparació de gasos va sortir tal i com esperàvem ja que les quantitats de diòxid i monòxid de carboni emeses eren menors que en el gasoil.

Development of an immunoenzymatic assay using a monoclonal antibody against a 50-kDa catabolite from the P126 Plasmodium falciparum protein to the diagnosis of malaria infection

The WHO criterion of defering any donation of blood by a confirmed case of malaria for three years after cessation of therapy can not be applied in areas where malaria in endemic. For this reason we developed an immunoenzymatic assay for the detection of plasmodial antigens for blood screening in malararial endemic areas. So, we tested sera from 191 individuals. Among patients with active disease 100% of the cases of Plasmodium falciparum or mixed infections and 91.7% of those with P. vivax were positive for the presence of plasmodial antigens. The lower parasitaemia detected was 0.0003% for P. vivax malária. When the frequency of positive circulating malarial antigens was evaluated among asymptomatic and symptomatic individuals with negative TBS, positive results were found in respectively 38.7% and 17.7% of the individuals studied in the 30 days after confirmed malaria attack. Data provide by these assays have shown that ELISA seemed to be more sensitive than parasitological examination for malaria diagnosis. This test by virtue of its high sensivity and the facilities in processing a large number of specimens, can prove to be useful in endemic areas for the recognition of asymptomatic malaria and screening of blood donors.

Antibodies in falciparum malaria: what matters most, quantity or quality?

In view of the recent demonstration that antibodies that are protective agains Plasmodium falciparum malaria may act in collaboration with blood monocytes, we have investigated the isotype content of sera from individuals with defined clinical states of resistance or susceptibility to malaria. Profound differences in the distribution of each Ig subclass and particulary in the ratio of cytophilic versus noncytophilic antibodies were found. In protected subjects, two cytophilic isotypes, IgG1 and IgG3 were found to predominate. In non-protected subjects, i.e. children and primary attack adults, three different situations were encountered: a) an imbalance in which IgG2, a non-cytophilic class, predominated (mostly seen in primary attacks); b) imbalance in which mostly IgM antibodies predominated (a frequent event in children) or c) less frequently, an overall low level of antimalarial antibodies. Of 33 non immune subjects studied all, except one, had one of the above defects. The function of total Ig presenting such an isotype imbalance was studied in vitro in Antibody-Dependent -Cellular-Inhibition assays. Not only did IgG from protected subjects cooperate efficiently with blood monocytes, whilst IgG from non-protected groups did not, but moreover the latter inhibit the in vitro effect of the former: in competition assays whole IgG from primary attack cases with increased IgG2 content, competed with IgG from immune adults, thus suggesting that non-protected subjects had antibodies to epitopes critical for protection, but that these antibodies are non functional.

Modern immunological approaches to assess malaria transmission and immunity and to diagnose plasmodial infection

The present paper reviews our recent data concerning the use of immunological methods employing monoclonal antibodies and synthetic peptides to study malaria transmission and immunity and to diagnose plasmodial infection. As concerns malaria transmission, we studied the main vectors of human malaria and the plasmodial species transmitted in endemic areas of Rondônia state, Brazil. The natural infection on anopheline was evaluated by immunoradiometric assay (IRMA) using monoclonal antibodies to an immunodominant sporozoite surface antigen (CS protein) demonstrated to be species specific. Our results showed that among six species of Anopheles found infected, An. darlingi was the main vector transmitting Plasmodium falciparum and P. vivax malaria in the immediate vicinity of houses. In order to assess the level of anti-CS antibodies we studied, by IRMA using the synthetic peptide corresponding to the repetitive epitope of the sporozoite CS protein, sera of individuals living in the same areas where the entomological survey has been performed. In this assay the prevalence of anti-CS antibodies was very low and did not reflect the malaria transmission rate in the studied areas. In relation to malaria diagnosis, a monoclonal antibody specific to an epitope of a 50 kDa exoantigen, the major component of supernatant collected at the time of schizont rupture, was used as a probe for the detection of P. falciparum antigens. This assay seemed to be more sensitive than parasitological examination for malaria diagnosis since it was able to detect plasmodial antigens in both symptomatic and asymtomatic individuals with negative thick blood smear at different intervals after a last parasitologically confirmed confirmed attack of malaria.

Aseptic meningoencephalitis mimicking transient ischaemic attacks.

PURPOSE: To highlight meningoencephalitis as a transient ischaemic attack (TIA) mimic and suggest clinical clues for differential diagnosis. METHODS: This was an observational study of consecutively admitted patients over a 9.75-year period presenting as TIAs at a stroke unit. RESULTS: A total of 790 patients with TIAs and seven with TIA-like symptoms but a final diagnosis of viral meningoencephalitis were recognised. The most frequent presentations of meningoencephalitis patients were acute sensory hemisyndrome (6) and cognitive deficits (5). Signs of meningeal irritation were minor or absent on presentation. Predominantly lymphocytic pleocytosis, hyperproteinorachia and a normal cerebrospinal fluid (CSF)/serum glucose index (in 5 out of 6 documented patients) were present. Meningeal thickening on a brain magnetic resonance imaging (MRI) scan was the only abnormal imaging finding. Six patients received initial vascular treatment; one thrombolysed. Finally, six patients were treated with antivirals and/or antibiotics. Although neither bacterial nor viral agents were identified on extensive testing, viral meningoencephalitis was the best explanation for all clinical and laboratory findings. CONCLUSIONS: Aseptic meningoencephalitis should be part of the differential diagnosis in patients presenting as TIA. The threshold for a lumbar puncture in such patients should be set individually and take into account the presence of mild meningeal symptoms, age and other risk factors for vascular disease, the results of brain imaging and the basic diagnostic work-up for a stroke source.

Prevalence and levels of IgG and IgM antibodies against Plasmodium falciparum and P. vivax in blood donors from Rondônia, Brazilian Amazon

Antibodies of IgG and IgM isotopes reacting with Plasmodium falciparum and P. vivax thick-smear antigens were searched for by the indirect fluorescent antibody test (IFAT) in a random sample of 230 blood donors at the transfusion centre of Porto Velho (HEMERON), Rondônia State, western Brazilian Amazon. A high prevalence of IgG seropositivity (32% against P. falciparum, 24% against P. vivax and 37% against either P. falciparum or P. vivax antigens) was detected among them, despite the fact that candidates reporting recent (<12 months) malaria attacks were not elegible. Only a small proportion of them had also detectable IgM antibodies to these antigens. These data suggest an intense, relatively recent exposure to malaria in such an urban population sample. However, parasitaemia (as detected by microscopical examination of Giemsa-stained thick smears) was patent in only one prospective donor. The antibody profile of blood donors was compared with that of healthy subjects of all age groups, living in a close endemic area (Candeias village, 21 km east of Porto Velho). The villagers were classified into two groups according to their history of a recent (<12 months) or a remote (>12 months) past malaria attack due to either P. falciparum or P. vivax. Extensive overlapping was observed when the distribution of antibody titres of healthy subjects from Candeias village with a recent and remote malaria history was compared. In conclusion, subjects with a recent or a remote malaria history could not be distinguished by sorological criteria alone.

Evidence for multiple B- and T-cell epitopes in Plasmodium falciparum liver-stage antigen 3.

Liver-stage antigen 3 (LSA-3) is a new vaccine candidate that can induce protection against Plasmodium falciparum sporozoite challenge. Using a series of long synthetic peptides (LSP) encompassing most of the 210-kDa LSA-3 protein, a study of the antigenicity of this protein was carried out in 203 inhabitants from the villages of Dielmo (n = 143) and Ndiop (n = 60) in Senegal (the level of malaria transmission differs in these two villages). Lymphocyte responses to each individual LSA-3 peptide were recorded, some at high prevalences (up to 43%). Antibodies were also detected to each of the 20 peptides, many at high prevalence (up to 84% of responders), and were directed to both nonrepeat and repeat regions. Immune responses to LSA-3 were detectable even in individuals of less than 5 years of age and increased with age and hence exposure to malaria, although they were not directly related to the level of malaria transmission. Thus, several valuable T- and B-cell epitopes were characterized all along the LSA-3 protein, supporting the antigenicity of this P. falciparum vaccine candidate. Finally, antibodies specific for peptide LSP10 located in a nonrepeat region of LSA-3 were found significantly associated with a lower risk of malaria attack over 1 year of daily clinical follow-up in children between the ages of 7 and 15 years, but not in older individuals.

Differential allocation and deployment of direct and indirect defences by Vicia sepium along elevation gradients

Dissecting drivers of plant defence investment remains central for understanding the assemblage of communities across different habitats. There is increasing evidence that direct defence strategies against herbivores, including secondary metabolites production, differ along ecological gradients in response to variation in biotic and abiotic conditions. In contrast, intraspecific variation in indirect defences remains unexplored. Here, we investigated variation in herbivory rate, resistance to herbivores, and indirect defences in ant-attracting Vicia species along the elevation gradient of the Alps. Specifically, we compared volatile organic compounds (VOCs) and ant attraction in high and low elevation ecotypes. Consistent with adaptation to the lower herbivory conditions that we detected at higher elevations in the field, high elevation plants were visited by fewer ants and were more susceptible to herbivore attack. In parallel, constitutive volatile organic compound production and subsequent ant attraction were lower in the high elevation ecotypes. We observed an elevation-driven trade-off between constitutive and inducible production of VOCs and ant attraction along the environmental cline. At higher elevations, inducible defences increased, while constitutive defence decreased, suggesting that the high elevation ecotypes compensate for lower indirect constitutive defences only after herbivore attack. Synthesis. Overall, direct and indirect defences of plants vary along elevation gradients. Our findings show that plant allocation to defences are subject to trade-offs depending on local conditions, and point to a feedback mechanism linking local herbivore pressure, predator abundance and the defence investment of plants.

Novel mechanisms of immune evasion by Schistosoma mansoni

The interaction of Schistosoma mansoni with its host's immune system is largely affected by multiple specific and non-specific evasion mechanisms employed by the parasite to reduce the host's immune reactivity. Only little is known about these mechanisms on the molecular level. The four molecules described below are intrinsic parasitic proteins recently identified and studied in our laboratory. 1. m28-A 28kDa membrane serine protease. m28 cleaves iC3b and can thus restrict attack by effector cells utilizing complement receptors (especially CR3). Treatment with protease inhibitors potentiates killing of schistosomula by complement plus neutrophils. 2. Smpi56-A 56kDa serine protease inhibitor. Smpi56 binds covalently to m28 and to neutrophil's elastase and blocks their proteolytic activity. 3. P70-A 70kDa C3b binding protein. The postulated activity of P70 includes binding to C3b and blocking of complement activation of the C3 step. 4. SCIP-1-A 94kDa schistosome complement inhibitor. SCIP-1 shows antigenic and functional similarities to the human 18kDa complement inhibitor CD59. Like CD59, SCIP-1 binds to C8 and C9 and blocks formation of the complement membrane attack complex. Antibodies directed to human CD59 bind to schistosomula and potentiate their killing by complement. The structure and function of these four proteins as well as their capacity to induce protection from infection with S. mansoni are under investigation.

The Security Council and the Legitimacy of the Use of Force: Legal, Normative, and Social Aspects.

Tal i com estableix la Carta de les Nacions Unides, el Consell de Seguretat determina l’existència d’una amenaça a la pau, un trencament de la pau o un acte d’agressió i decideix les mesures que han de fer-se servir per restaurar la pau i seguretat internacionals, també l’ús de la força. L’objectiu d’aquest article és explorar la legitimitat d’aquest ús de la força. Amb aquest objectiu, el text parteix d’una definició centrada en les seves dimensions legal, normativa i social. En segon lloc, s’analitzarà com aquestes dimensions estan representades als debats del Consell de Seguretat de la guerra d’Irak de 2003, un dels usos de la força més controvertits i que més debat ha generat als darrers anys. Finalment, l’anàlisi proposat permet treure algunes conclusions sobre les bases canviants de la legitimitat de l’ús de la força.

Els cultius in vitro i el desenvolupament vegetal

Biotecnologia Vegetal de materials de treball que incorporin les TICs. El material elaborat ha estat un llibre electrònic utilitzable tant on-line (web) com off-line (CD). Els materials generats com a resultes d’aquest projecte han estat situats en un servidor de la UdL accessible des de l’exterior de la Universitat. Aquest llibre electrònic és d’accés obert i es pot consultar on-line a l’URL (http://sakai.udl.es/cursos/76304/indexC.htm). El llibre electrònic conté un total de 98 Mb d’informació hipermèdia i hipertexual distribuïda en més de 380 arxius dels quals 50 són pàgines html, 10 arxius doc, 10 arxius pdf, 258 imatges fixes i 8 arxius de vídeo en format flash (swf). L’accés al llibre electrònic es realitza a través d’una pantalla inicial que dóna pas a un menú que distribueix els materials en 7 apartats. Els textos estan acompanyats de gran quantitat d’imatges fotogràfiques, gràfics, esquemes, imatges infogràfiques i videoclips generats de novo per aquest projecte. En dissenyar la web s’han tingut en compte criteris de confiabilitat, accessibilitat i usabilitat. El primer disseny de la web ha estat validat per un panel d’usuaris i utilitzat posteriorment amb alumnes durant el curs 2007-2008. Les observacions i suggeriments fets per aquests ja han estat incorporats en aquest document final. Una primera enquesta de satisfacció realitzada amb aquest alumnat permet concloure, a títol provisional donat lo reduït de la població enquestada, que l’alumnat mostra un grau de coneixement de les eines TIC suficient i que consideren positiva la incorporació de materials multimèdia com a recurs educatiu. A més a més, la realització d’aquest projecte i la seva aplicació a l’aula ha estat presentada en dues ponències al “Congreso Internacional de Docencia y Innovación Universitaria”.

Visualització dinàmica d'un procés metabòlic amb finalitats docents

La incorporació del Moodle com a eina de docència, i l’augment de hores no presencials a les diverses assignatures fa que calgui incorporar les noves tecnologies perquè els alumnes disposin de més material docent al seu abast. Però l’acumulació de material fa que només sigui útil aquell material que es guanyi a l’alumne. En aquest sentit, creiem que les animacions i les eines interactives poden ser materials atractius pels alumnes. En aquest projecte hem creat una eina d’animació interactiva perquè l’estudiant de Bioquímica practiqui i aprengui una de les rutes metabòliques principals: la glucòlisi. Aquesta eina consta d’una pantalla separada en 3 zones: 1) una zona lateral que inclou la ruta metabòlica completa, i en la que l’alumne pot prémer sobre cada un dels passos que estructuren la ruta (finestra del metabolisme); 2) una zona inferior que presenta la reacció individual de la ruta metabòlica, en la que s’observa l’estructura química de les molècules i informació de l’enzim implicat en la reacció (finestra de l’enzim); i 3) una zona central en la que a través d’una animació amb Macromedia Flash MX, l’alumne observa el mecanisme químic de la reacció (finestra del mecanisme químic). Les tres zones són interactives i en prémer sobre elles donen informació, respectivament sobre la ruta completa, l’enzim de cada pas en particular i el mecanisme d’acció. A més, la finestra del mecanisme químic permet aturar en qualsevol moment la animació, tornar enrere i veure els intermediaris. Durant el segon semestre del curs 2006-2007 hem avaluat l’eina amb alumnes de Bioquímica de la Llicenciatura de Química, incorporant-la als dossier electrònics. Creiem que hem assolit els objectius que es proposaven: aprendre una ruta metabòlica, de manera divertida; aprendre el mecanisme de cada un dels passos de la ruta i aprendre l’estructura química dels metabòlits intermediaris de la ruta.