Vanadium oxide-porphyrin nanocomposites as gas sensor interfaces for probing low water content in ethanol

Vanadium pentoxide xerogels (VXG) incorporating meso(3- and 4-pyridyl)porphyrin cobalt(III) species coordinated to four [Ru(bipy)(2)Cl](+) complexes were employed as gas sensing materials capable of detecting small amounts of water in commercial ethanol and fuel supplies. According to their X-ray diffraction data, the original VXG lamellar framework was maintained in the nanocomposite material, but the interlamellar distance increased from 11.7 to 15.2 angstrom, reflecting the intercalation of the porphyrin species into the vanadium pentoxide matrix. The films generated by direct deposition of the nanocomposite aqueous suspensions exhibited good electrical and electrochemical performance for application in resistive sensors. The analysis of water in ethanol and fuels was carried out successfully using an especially designed electric setup incorporating a laminar gas flow chamber and interdigitated gold electrodes coated with the nanocomposites. (C) 2010 Elsevier B.V. All rights reserved.

Uso de antibióticos para o controle de bactérias endógenas visando à micropropagação da figueira

A descontaminação dos explantes é um dos princípios básicos para o sucesso da cultura de tecidos. Um dos problemas diagnosticados na propagação in vitro da figueira, através de gemas apicais, é a contaminação endógena dos explantes por bactérias. Este trabalho teve como objetivo avaliar a eficiência de alguns antibióticos em meio de cultura para o controle de bactérias endógenas em gemas apicais de figueira. Foram avaliados os seguintes tratamentos: T1(sem adição de antibiótico); T2 (30 mg L-1 de cloranfenicol); T3 (250 mg L-1 de ampicilina sódica); T4 (500 mg L-1 de ácido nalidícico); T5 (150 mg L-1 de cefalotina sódica); T6 (500 mg L-1 de tetraciclina), e T7 (400 mg L-1 de norfloxacina). Após coletados em campo, os segmentos de ramos contendo as gemas foram colocados em recipiente com água corrente. Posteriormente, as gemas apicais foram imersas em álcool etílico a 70% e hipoclorito de sódio a 2,5%. Todo procedimento de desinfestação externa dos explantes foi realizado em câmara de fluxo laminar. Os explantes foram inoculados em tubos de ensaio contendo 15 mL de meio básico MS suplementado, após a autoclavagem, com as doses de antibióticos de acordo com os tratamentos estabelecidos. Após a inoculação, os explantes foram mantidos em sala de crescimento por quatro dias no escuro e, em seguida, sob fotoperíodo de 16 horas de luz branca fria e irradiância de 25 µmol m s-1, na temperatura de 22 ± 3ºC. A assepsia realizada externamente nos explantes foi suficiente para o controle de contaminação fúngica, e a adição de antibióticos ao meio, após autoclavagem, foi eficiente para o controle de bactérias endógenas, cujo antibiótico ampicilina sódica proporcionou mais de 90% de explantes sobreviventes.

Produtividade de pleurotus ostreatus em resíduos da amazônia

The viability of the utilization of wood and agroindustrial residues available in the Amazon region in the formulation of alternative substrates for the cultivation of Pleurotus ostreatus was tested. Thus, two wood residues: marupa sawdust (SIAMP) and pau-de-balsa sawdust (SAPB), and two substrates derived from agroindustrial residues: sugar-cane bagasse (SIACN) and pupunheira stem (SIAPP), were used. These were supplemented with a mixture of rice bran, wheat and corn as protein source, with addition of 2-3% of CaCO(3) for pH correction (6.5). The substrates were placed in polyethylene (HDPE) bags, sterilized at 121 degrees C for 1h and inoculated in a laminar flow chamber. The cultivation was carried out in an axenic way, in a modified atmosphere. The productivity of the substrates was evaluated in relation to the biological efficiency, with the following mean results: 125.60, 99.80%, 94.00 and 64.60% for SIAPP, SIACN, SIAMP and SIAPB, respectively. The high biological efficiency of the substrates and the cultivation process clearly showed the viability of the utilization of the residues, suggesting the commercial cultivation of this mushroom, which may contribute for improving the social and economical conditions and sustainability of the regional biodiversity resources of Amazonia.

Incerteza espacial da emissão de 'CO IND.2' do solo em dois sistemas de manejo da palhada da cana-de-açúcar

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Dinâmica de carbono e nitrogênio em pastos de capim-xaraés submetidos a diferentes resíduos pós-pastejo

Pós-graduação em Zootecnia - FCAV

Estudo da prevalência de fungos em travesseiros de crianças com rinite e, ou, asma

Pós-graduação em Biologia Geral e Aplicada - IBB

Avaliação in vitro do crescimento micelial de cinco linhagens de Agaricus blazei em duas temperaturas

The objective of the work was to evaluate the in vitro mycelial growth of five A. blazei strains (ABL-05/53, ABL-04/49, ABL-03/44, ABL-99/30 and ABL-02/51) when submitted to the temperatures of 20 and 25 ºC. In a laminar flow chamber, discs of the strains were inoculated in the middle of Petri’s plates containing CA (compost-agar) medium and incubated in BOD. After 48 hours, measurements of the mycelial growth began, with the help of a ruler with scale in millimeters, by means of four equidistant measurements, until the moment when the fungal colony reached near the edges of the Petri’s plate in one of the treatments. The experimental design was totally randomized, in 5 x 2 factorial design. Each treatment consisted of seven repetitions, corresponding to one Petri’s plate, totalizing seventy experimental units. We verified that A. blazei growth is influenced by incubation temperature, being that the temperature of 25 ºC was more favorable for the mycelial growth of all A. blazei strains tested, with attention for ABL-04/49 and ABL-03/44 strains, which obtained the highest averages for mycelial growth under this temperature condition at the end of the cultivation cycle.

Resíduos de bananeira como substrato base para o cultivo in vitro de Coprinus comatus

Coprinus comatus is an edible and lignolitic fungus which has presented great potential for commercial use due to its easy development in the different residues, such as banana tree leave. Thus, the mycelial growth of Coprinus comatus in culture media based on leaves of Thap-Maeo, Prata-Anã, Pelipita and Caipira banana tree cultivars, supplemented with 20% of wheat, soy and rice brans, was evaluated. 7 mm-wide discs of CCO 01/01 strain of C. comatus were inoculated in the middle of Petri dishes containing culture medium, inside a laminar flow chamber. Next, the dishes were arranged totally at random inside an incubator at 25 ºC. The daily measurements of the mycelial growth began after 24 hours, until one of the treatments reached the borders of the Petri dish. According to the results obtained, we verified that there was not effect of the kind of supplementation for culture media based on Thap-Maeo, Prata-Anã and Pelipita; the best growth averages for culture media based on Caipira were provided by wheat and rice brans. Therefore, banana residues may be a viable and ecologically correct choice for the cultivation of C. comatus, especially for Thap-Maeo and Prata Anã sorts, which provided the best growth averages, regardless of the supplementation used.

Untersuchungen zu Migration, Homing und Effektor-Funktionen humaner T-Lymphozyten in NOD.Cg-Prkdc scid Il2r gtm1Wjl, SzJ-Mäusen im Rahmen der allogenen hämatopoetischen Stammzelltransplantation

Die akute myeloische Leukämie (AML) zählt zu den aggressivsten neoplastischen Erkrankungenrnder Hämatopoese. Die Mehrheit der Patienten mit AML erreicht nach Induktions-rnChemotherapie den Zustand der kompletten Remission, jedoch erleiden mehr als die Hälfterndieser Patienten anschließend einen Rückfall und versterben an den Folgen der Erkrankungrn[1]. Die allogene hämatopoetische Stammzelltransplantation (engl.: hematopoietic stem cellrntransplantation, HSCT) stellt die einzig putativ kurative Behandlungsform für rezidierendernPatienten und solche mit schlechter Prognose dar. Jedoch birgt diese Form der Therapiernauch eine Vielzahl an Risiken. Insbesondere das Auftreten einer akuten Transplantat-gegen-rnWirt-Erkrankung (engl.: graft-versus-host disease, GvHD) stellt die Hauptursache für transplantationsassoziierternMortalität und Morbidität dar [2]. Die Depletion von alloreaktiven zytotoxischenrnT Lymphozyten (CTL) aus dem Transplantat ermöglicht zwar die Prävention derrnEntstehung einer GvH-Erkrankung, jedoch häufig unter gleichzeitigem Verlust des förderlichen,rnanti-leukämischen Transplantat-gegen-Leukämie-Effekts (engl.: graft-versus-leukemia,rnGvL) [3]. Um den GvL-Effekt unter Vermeidung einer GvH-Erkrankung zu erhalten, bietetrnsich der gezielte adoptive Transfer von Leukämie-spezifischen, nicht alloreaktiven CTL alsrnattraktive Strategie der Immuntherapie für AML-Patienten nach allogener HSCT an. In derrnvorliegenden Arbeit konnte erfolgreich ein prä-klinisches murines AML-Modell unter Einsatzrndes stark immundefizienten NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ- (NSG-) Mausstamms und primärenrnAML-Blasten durch die Optimierung bereits publizierter Protokolle etabliert werden.rnBei zehn von 17 transplantierten primären AML-Proben konnte ein erfolgreiches Engraftmentrnder humanen Zellen und eine Rekonstitution der humanen Neoplasie in den NSG-Mäusenrnerzielt werden. Die Engraftment-Rate betrug somit 58,82% und lag etwas unter dem aus derrnLiteratur bekannten Wert von 65-70% [4, 5]. Es ließen sich gut, intermediär und schlecht anwachsendernAML-Proben anhand der Engraftment-Stärke und -Reproduzierbarkeit voneinanderrnunterscheiden. Anhand der Analyse von für das Engraftment kritischer Parameter konnternein Zusammenhang zwischen Engraftment-Rate in der Maus und Flt3-Mutationsstatus sowiernFAB-Klassifikation des Patienten hergestellt und somit Angaben aus der Literatur bestätigtrnwerden. Für zwei Patienten-spezifische AML-Modelle, MZ580 und MZ308, konnten in vitrornerfolgreich AML-reaktive, über einzelne bzw. duale HLA-Diskrepanzen restringierte CTLPopulationenrngeneriert und über einen Zeitraum von bis zu 70 Tagen expandiert werden.rnDeren adoptiver Transfer in zuvor mit humanen AML-Blasten inokulierte NSG-Mäuse führternzu einer nahezu vollständigen Eradikation der AML-Blasten und Remission der Versuchstiere.rnAnhand unterschiedlich langer in vitro Kultur-Zeiträume konnte ein für die in vivo ausgeübtenrnEffektor-Funktionen optimaler Reifungszustand der CTL-Populationen von maximalrn28 Tagen bestimmt werden. Die kinetische Analyse der lytischen Aktivität in vivo deutete auf eine relativ schnelle Ausübung der Effektor-Funktionen durch die CTL-Populationen innerhalbrnvon zwei bis 24 Stunden nach adoptivem Transfer hin. Durch die Verwendung von inrnvitro generierten EBV-reaktiven CTL aus einem irrelevanten Spender konnte zudem die Spezifitätrnder in vivo ausgeübten Effektor-Funktionen nachgewiesen werden. Die ex vivo Re-rnIsolation adoptiv transferierter CTL und deren in vitro Analyse in einem IFNγ ELISpot wiesrneine konstante Reaktivität der Zellen ohne Induktion einer Xeno-Reaktivität nach. Die zurrnVerbesserung der Persistenz humaner CTL-Populationen eingesetzten autologen CD4+ TrnZellen zeigten nur im AML MZ308-System eine positive Wirkung. Generell konnte die Persistenzrnin vivo jedoch trotz initialer Substitution mit den Zytokinen IL-2 und IL-7 nicht über einenrnZeitraum von sieben Tagen hinaus aufrechterhalten werden.rnZur Untersuchung des Extravasations-Mechanismus humaner T Zellen über murines Endothelrnwurden sowohl Flusskammer- als auch Transwell-Studien durchgeführt, um die molekularenrnGrundlagen des Adhäsions- und Transmigrationsprozesses aufzuklären. Durch denrnparallelen Einsatz humaner und muriner T Zellen auf murinen Endothelzellen unter Zusatzrnfunktionsblockierender monoklonaler Antikörper konnte gezeigt werden, dass derrnExtravasations-Mechanismus beider Spezies auf Interaktionen homologer Adhäsionsmolekül-rnPaare, nämlich VLA-4–VCAM-1 und LFA-1–ICAM-1, beruht. Für einzelne Moleküle konntenrnin Abhängigkeit der eingesetzten Endothelzellen Unterschiede in der Funktionalität zwischenrnden Spezies identifiziert werden. Der Adhäsionsprozess war durch die Blockade derrnVLA-4–VCAM-1-Interaktion stärker inhibierbar als durch die Blockade von LFA-1–ICAM-1.rnDie Transmigration hingegen war durch die Blockade beider Adhäsionsmolekül-Paare vergleichbarrnstark inhibierbar.

Differential requirement for ROCK in dendritic cell migration within lymphatic capillaries in steady-state and inflammation

Dendritic cell (DC) migration via lymphatic vessels to draining lymph nodes (dLNs) is crucial for the initiation of adaptive immunity. We imaged this process by intravital microscopy (IVM) in the ear skin of transgenic mice bearing red-fluorescent vasculature and yellow-fluorescent DCs. DCs within lymphatic capillaries were rarely transported by flow, but actively migrated within lymphatics and were significantly faster than in the interstitium. Pharmacologic blockade of the Rho-associated protein kinase (ROCK), which mediates nuclear contraction and de-adhesion from integrin ligands, significantly reduced DC migration from skin to dLNs in steady-state. IVM revealed that ROCK blockade strongly reduced the velocity of interstitial DC migration, but only marginally affected intralymphatic DC migration. By contrast, during tissue inflammation, ROCK blockade profoundly decreased both interstitial and intralymphatic DC migration. Inhibition of intralymphatic migration was paralleled by a strong up-regulation of ICAM-1 in lymphatic endothelium, suggesting that during inflammation ROCK mediates de-adhesion of DC-expressed integrins from lymphatic-expressed ICAM-1. Flow chamber assays confirmed an involvement of lymphatic-expressed ICAM-1 and DC-expressed ROCK in DC crawling on lymphatic endothelium. Overall, our findings further define the role of ROCK in DC migration to dLNs and reveal a differential requirement for ROCK in intralymphatic DC crawling during steady-state and inflammation.

DOCK2 is required for chemokine-promoted human T lymphocyte adhesion under shear stress mediated by the integrin alpha4beta1

The alpha4beta1 integrin is an essential adhesion molecule for recruitment of circulating lymphocytes into lymphoid organs and peripheral sites of inflammation. Chemokines stimulate alpha4beta1 adhesive activity allowing lymphocyte arrest on endothelium and subsequent diapedesis. Activation of the GTPase Rac by the guanine-nucleotide exchange factor Vav1 promoted by CXCL12 controls T lymphocyte adhesion mediated by alpha4beta1. In this study, we investigated the role of DOCK2, a lymphocyte guanine-nucleotide exchange factor also involved in Rac activation, in CXCL12-stimulated human T lymphocyte adhesion mediated by alpha4beta1. Using T cells transfected with DOCK2 mutant forms defective in Rac activation or with DOCK2 small interfering RNA, we demonstrate that DOCK2 is needed for efficient chemokine-stimulated lymphocyte attachment to VCAM-1 under shear stress. Flow chamber, soluble binding, and cell spreading assays identified the strengthening of alpha4beta1-VCAM-1 interaction, involving high affinity alpha4beta1 conformations, as the adhesion step mainly controlled by DOCK2 activity. The comparison of DOCK2 and Vav1 involvement in CXCL12-promoted Rac activation and alpha4beta1-dependent human T cell adhesion indicated a more prominent role of Vav1 than DOCK2. These results suggest that DOCK2-mediated signaling regulates chemokine-stimulated human T lymphocyte alpha4beta1 adhesive activity, and that cooperation with Vav1 might be required to induce sufficient Rac activation for efficient adhesion. In contrast, flow chamber experiments using lymph node and spleen T cells from DOCK2(-/-) mice revealed no significant alterations in CXCL12-promoted adhesion mediated by alpha4beta1, indicating that DOCK2 activity is dispensable for triggering of this adhesion in mouse T cells, and suggesting that Rac activation plays minor roles in this process.

Kindlin-3 regulates integrin activation and adhesion reinforcement of effector T cells

Activated T cells use very late antigen-4/α4β1 integrin for capture, rolling on, and firm adhesion to endothelial cells, and use leukocyte function-associated antigen-1/αLβ2 integrin for subsequent crawling and extravasation. Inhibition of α4β1 is sufficient to prevent extravasation of activated T cells and is successfully used to combat autoimmune diseases, such as multiple sclerosis. Here we show that effector T cells lacking the integrin activator Kindlin-3 extravasate and induce experimental autoimmune encephalomyelitis in mice immunized with autoantigen. In sharp contrast, adoptively transferred autoreactive T cells from Kindlin-3-deficient mice fail to extravasate into the naïve CNS. Mechanistically, autoreactive Kindlin-3-null T cells extravasate when the CNS is inflamed and the brain microvasculature expresses high levels of integrin ligands. Flow chamber assays under physiological shear conditions confirmed that Kindlin-3-null effector T cells adhere to high concentrations of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, albeit less efficiently than WT T cells. Although these arrested T cells polarize and start crawling, only few remain firmly adherent over time. Our data demonstrate that the requirement of Kindlin-3 for effector T cells to induce α4β1 and αLβ2 integrin ligand binding and stabilization of integrin-ligand bonds is critical when integrin ligand levels are low, but of less importance when integrin ligand levels are high.

siRNA mediated knockdown of tissue factor expression in pigs for xenotransplantation.

Acute vascular rejection (AVR), in particular microvascular thrombosis, is an important barrier to successful pig-to-primate xenotransplantation. Here, we report the generation of pigs with decreased tissue factor (TF) levels induced by small interfering (si)RNA-mediated gene silencing. Porcine fibroblasts were transfected with TF-targeting small hairpin (sh)RNA and used for somatic cell nuclear transfer. Offspring were analyzed for siRNA, TF mRNA and TF protein level. Functionality of TF downregulation was investigated by a whole blood clotting test and a flow chamber assay. TF siRNA was expressed in all twelve liveborn piglets. TF mRNA expression was reduced by 94.1 ± 4.7% in TF knockdown (TFkd) fibroblasts compared to wild-type (WT). TF protein expression in PAEC stimulated with 50 ng/mL TNF-α was significantly lower in TFkd pigs (mean fluorescence intensity TFkd: 7136 ± 136 vs. WT: 13 038 ± 1672). TF downregulation significantly increased clotting time (TFkd: 73.3 ± 8.8 min, WT: 45.8 ± 7.7 min, p < 0.0001) and significantly decreased thrombus formation compared to WT (mean thrombus coverage per viewing field in %; WT: 23.5 ± 13.0, TFkd: 2.6 ± 3.7, p < 0.0001). Our data show that a functional knockdown of TF is compatible with normal development and survival of pigs. TF knockdown could be a valuable component in the generation of multi-transgenic pigs for xenotransplantation.

Functional and physical associations of beta1 integrins in the activation of human T lymphocytes

Adhesion involves interactions between cells or cells with extracellular matrix components and is a fundamental process for all multicellular organisms as well as many pathogenic microbes. Integrins are heterodimeric transmembrane proteins that function as adhesion molecules and transduce signals between the extracellular environment and the intracellular cytoskeletal machinery. β1 integrin subfamily is highly expressed on T lymphocytes and mediates cell spreading, adhesion and coactivation. T lymphocytes have an important role in the regulation and homeostasis of the immune system therefore, the goals of this study were to first to investigate β1 integrin interaction with fibronectin binding protein A (FnbpA), a surface protein expressed on gram-negative bacteria Staphylococcus aureus. Second, characterize the association and function of a non-integrin surface protein, CD98, with β1 integrins on T lymphocytes. ^ FnbpA binds to fibronectin (FN), also a ligand for α5β1 and α4β1 integrins on T lymphocytes. Since both bacterial proteins FnbpA and T cell integrins utilize FN, it was of interest to determine the effects FnbpA on T cell activation. Results demonstrated that recombinant FnbpA (rFnbpA) coimmobilized with OKT3 mediated T cell coactivation in a soluble FN-dependent manner. Integrin α5β1 was identified as the main integrin utilized by Staphylococcus aureus FnbpA from studies using soluble antibodies to inhibit T cell proliferation and parallel plate flow chamber assays. The mechanism of rFnbpA-mediated coactivation was one that used soluble FN as a bridge between rFnbpA and integrin α5β1 on the T lymphocyte. ^ Since integrins are utilized by T lymphocytes and bacterial proteins, it was of interest to identify proteins involved in integrin regulation. Anti-CD98 mAb 80A10 was identified and characterized from a screen to identify surface proteins involved in integrin signaling and functions. CD98 is a non-integrin protein that was sensitive to integrin inhibition in human T lymphocyte aggregation and activation, thus suggested that CD98 shared a common signaling pathway with integrins. These results led to the question of whether CD98 physically associates with β1 integrins. Fluorescence microscopy and biochemical analysis determined that CD98 is specifically associated with β1 integrin on human T lymphocytes and may be part of a larger multimolecular signaling complex. ^

Determination of the lifetime and force dependence of interactions of single bonds between surface-attached CD2 and CD48 adhesion molecules

We studied single molecular interactions between surface-attached rat CD2, a T-lymphocyte adhesion receptor, and CD48, a CD2 ligand found on antigen-presenting cells. Spherical particles were coated with decreasing densities of CD48–CD4 chimeric molecules then driven along CD2-derivatized glass surfaces under a low hydrodynamic shear rate. Particles exhibited multiple arrests of varying duration. By analyzing the dependence of arrest frequency and duration on the surface density of CD48 sites, it was concluded that (i) arrests were generated by single molecular bonds and (ii) the initial bond dissociation rate was about 7.8 s−1. The force exerted on bonds was increased from about 11 to 22 pN; the detachment rate exhibited a twofold increase. These results agree with and extend studies on the CD2–CD48 interaction by surface plasmon resonance technology, which yielded an affinity constant of ≈104 M−1 and a dissociation rate of ≥6 s−1. It is concluded that the flow chamber technology can be an useful complement to atomic force microscopy for studying interactions between isolated biomolecules, with a resolution of about 20 ms and sensitivity of a few piconewtons. Further, this technology might be extended to actual cells.