The octadecanoic pathway: signal molecules for the regulation of secondary pathways.

Plant defense against microbial pathogens and herbivores relies heavily on the induction of defense proteins and low molecular weight antibiotics. The signals between perception of the aggression, gene activation, and the subsequent biosynthesis of secondary compounds are assumed to be pentacylic oxylipin derivatives. The rapid, but transient, synthesis of cis-jasmonic acid was demonstrated after insect attack on a food plant and by microbial elicitor addition to plant suspension cultures. This effect is highly specific and not caused by a number of environmental stresses such as light, heavy metals, or cold or heat shock. Elicitation of Eschscholtzia cell cultures also led to a rapid alkalinization of the growth medium prior to jasmonate formation. Inhibition of this alkalinization process by the protein kinase inhibitor staurosporine also inhibited jasmonate formation. The induction of specific enzymes in the benzo[c]phenanthridine alkaloid pathway leading to the antimicrobial sanguinarine was induced to a qualitatively and quantitatively similar extent by fungal elicitor, methyl jasmonate, and its linolenic acid-derived precursor 12-oxophytodienoic acid. It is herein proposed that a second oxylipid cascade may exist in plants starting from linoleic acid via 15,16-dihydro-12-oxophytodienoic acid to 9,10-dihydrojasmonate. Experiments with synthetic trihomojasmonate demonstrated that beta-oxidation is not a prerequisite for biological activity and that 12-oxophytodienoic acid and derivatives are most likely fully active as signal transducers. Octadecanoic acid-derived compounds are essential elements in modulating the synthesis of antibiotic compounds and are thus integral to plant defense.

Characterization of the stable L-arginine-derived relaxing factor released from cytokine-stimulated vascular smooth muscle cells as an NG-hydroxyl-L-arginine-nitric oxide adduct.

The nature of an L-arginine-derived relaxing factor released from vascular smooth muscle cells cultured on microcarrier beads and stimulated for 20 h with interleukin 1 beta was investigated. Unlike the unstable relaxation elicited by authentic nitric oxide (NO) in a cascade superfusion bioassay system, the effluate from vascular smooth muscle cells induced a stable relaxation that was susceptible to inhibition by oxyhemoglobin. Three putative endogenous NO carriers mimicked this stable relaxing effect: S-nitroso-L-cysteine, low molecular weight dinitrosyl-iron complexes (DNICs), and the adduct of NG-hydroxy-L-arginine (HOArg) with NO. Inactivation of S-nitroso-L-cysteine by Hg2+ ions or trapping of DNICs with agarose-bound bovine serum albumin abolished their relaxing effects, whereas that of the vascular smooth muscle cell effluate remained unaffected. In addition, neither S-nitrosothiols nor DNICs were detectable in the effluate from these cells, as judged by UV and electron spin resonance (ESR) spectroscopy. The HOArg-NO adduct was instantaneously generated upon reaction of HOArg with authentic NO under bioassay conditions. Its pharmacological profile was indistinguishable from that of the vascular smooth muscle cell effluate, as judged by comparative bioassay with different vascular and nonvascular smooth muscle preparations. Moreover, up to 100 nM HOArg was detected in the effluate from interleukin 1 beta-stimulated vascular smooth muscle cells, suggesting that sufficient amounts of HOArg are released from these cells to spontaneously generate the HOArg-NO adduct. This intercellular NO carrier probably accounts for the stable L-arginine-derived relaxing factor released from cytokine-stimulated vascular smooth muscle cells and also from other NO-producing cells, such as macrophages and neutrophils.

Dinâmica do fósforo no solo em função da adição de ácidos orgânicos de baixa massa molar

A baixa eficiência da adubação fosfatada em solos altamente intemperizados é devido, entre outros fatores, à adsorção do fósforo (P) à superfície das argilas silicatadas do tipo 1:1 e, principalmente, dos (hidr)óxidos de Fe e de Al. Manejos do solo que induzem a solubilização de formas de P indisponíveis para as plantas têm sido intensamente estudados nos últimos anos. Uma tentativa de aumentar a concentração de P disponível na solução do solo para sua absorção pelas plantas é a mobilização de P por ânions de ácidos orgânicos de baixa massa molar (AOBMM). Ânions derivados de AOBMM exsudados pelas raízes de plantas ou excretados por microrganismos são associados com algumas condições de rizosfera como deficiência de P e fitotoxidez de Al e interagem com o solo de forma a aumentar a biodisponibilidade de P. Dependendo dos atributos do solo, do grau de dissociação, das propriedades e do número de grupos carboxílicos dos ânions orgânicos, o P pode ser mobilizado do solo principalmente devido à dissolução complexométrica de minerais e à adsorção competitiva dos grupos funcionais carboxílicos e fosfato nos sítios de superfície coloidais. A capacidade dos ânions citrato, malato e oxalato em mobilizar P de amostras de um Neossolo Quartzarênico típico (RQ) e de um Latossolo Vermelho ácrico (LVwf) foi avaliada por meio de um estudo de lixiviação de ânions em colunas. Devido a não detecção de P nos efluentes das colunas com LVwf, foi realizado outro estudo em colunas, no qual somente citrato foi lixiviado, mas num volume maior, e as alterações das formas de P nas amostras desse solo induzidas pela lixiviação de citrato foram identificadas por espectroscopia de absorção de raios-X na borda K do fósforo (X-ray absorption near edge structure -XANES - spectroscopy). A capacidade dos ânions de AOBMM em solubilizar P foi mais dependente do teor de P disponível e de outros atributos do solo que do número de grupos funcionais carboxílicos dos ânions orgânicos. Somente o oxalato mobilizou P do RQ, enquanto todos os ânions de AOBMM foram capazes de mobilizar P do LVwf. Quando baixos volumes de solução contendo ânions de AOBMM foram lixiviados no solo, além do aumento do pH, a mobilização de P foi acompanhada pela mobilização de Al no RQ (pH água = 5), e pela mobilização de Ca no LVwf (pH água = 5.6), o que indica solubilização de P pela complexação de Al, Ca, ou Fe, de fosfatos insolúveis, ou pela inibição da precipitação de P com esses metais. Ao lixiviar um volume maior de citrato no LVwf, o P também não foi detectado nos efluentes das colunas, mas houve lixiviação intensa de Al e Fe, bem como mudanças nas proporções de formas de P no solo caracterizadas pelos espectros XANES. Embora tenhamos encontrado indícios da ação dos principais mecanismos de solubilização de P (dissolução complexométrica de minerais e troca de ligantes entre grupos funcionais carboxílicos e P adsorvido ao solo), os ânions de AOBMM mostraram pouco potencial de efetivamente aumentar a biodisponibilidade de P.

Modelagem matemática do processo industrial de coqueamento retardado.

A unidade de coqueamento retardado é um processo térmico de conversão, utilizado pelas refinarias, para converter cargas residuais em produtos de baixo peso molecular e com alto valor agregado (gases, nafta e gasóleo) e coque verde de petróleo. Um pequeno aumento no rendimento líquido da unidade de coqueamento retardado proporciona benefícios económicos consideráveis, especialmente no destilado líquido. A concorrência no mercado, as restrições sobre as especificações do produto e gargalos operacionais exigem um melhor planejamento da produção. Portanto, o desenvolvimento de novas estratégias e modelos matemáticos, focados em melhores condições de operação do processo industrial e formulações de produtos, é essencial para alcançar melhores rendimentos e um acompanhamento mais preciso da qualidade do produto. Este trabalho tem como objetivo o desenvolvimento de modelo matemático do conjunto forno-reator do processo de coqueamento, a partir de informações obtidas em uma planta industrial. O modelo proposto é baseado na caracterização da carga e dos produtos em pseudocomponentes, modelos cinéticos de grupos e condições de equilíbrio liquido-vapor. Além disso, são discutidos os principais desafios para o desenvolver o modelo matemático do forno e do reator, bem como a caracterização rigorosa do resíduo de vácuo e dos produtos para determinar os parâmetros que afetam a morfologia do coque e a zona de reação no interior do reator de coque.

Propriedades físico-químicas e mecânicas de membranas porosas de carboximetilquitosana e hidrogéis de quitosana para aplicação em engenharia de tecidos

Este trabalho teve como principal objetivo produzir membranas porosas de carboximetilquitosana e hidrogéis de quitosana com propriedades físico-químicas e mecânicas adequadas para aplicações em Engenharia de Tecidos. Para isso, quitosanas com diferentes graus de acetilação (4,0%<GA<40%) e de elevada massa molar média viscosimétrica (Mv>750.000 g.mol-1) foram produzidas através da aplicação de processos consecutivos de desacetilação assistida por irradiação de ultrassom de alta intensidade (DAIUS) à beta-quitina extraída de gládios de lulas Doryteuthis spp. A carboximetilação de quitosana extensivamente desacetilada (Qs-3; GA=4%) foi realizada pela reação com ácido monocloroacético em meio isopropanol/solução aquosa de NaOH, gerando a amostra CMQs-0 (GS≈0,98; Mv≈190.000 g.mol-1). A irradiação de ultrassom de alta intensidade foi empregada para tratar solução aquosa de CMQs-0 durante 1 h e 3 h, resultando nas amostras CMQs-1 (Mv≈94.000 g.mol-1) e CMQs-3 (Mv≈43.000 g.mol-1), respectivamente. Para a produção de membranas reticuladas, genipina foi adicionada em diferentes concentrações (1,0x10-4 mol.L-1, 3,0x10-4 mol.L-1 ou 5,0x10-4 mol.L-1) às soluções aquosas das CMQs, que foram vertidas em placas de Petri e a reação de reticulação procedeu por 24 h. Em seguida, as membranas reticuladas (M-CMQs) foram liofilizadas, neutralizadas, lavadas e liofilizadas novamente, resultando em nove amostras, que foram caracterizadas quanto ao grau médio de reticulação (GR), grau médio de hidratação (GH), morfologia, propriedades mecânicas e quanto à susceptibilidade à degradação por lisozima. O grau médio de reticulação (GR) foi tanto maior quanto maior a concentração de genipina empregada na reação, variando de GR≈3,3% (M-CMQs-01) a GR≈17,8% (M-CMQs-35). As análises de MEV revelaram que as membranas reticuladas M-CMQs são estruturas porosas que apresentam maior densidade de poros aparentes quanto maiores os valores de Mve GR. Entretanto, as membranas preparadas a partir de CMQs de elevada massa molar (Mv>94.000 g.mol-1) e pouco reticuladas (GR<10%), apresentaram propriedades mecânicas superiores em termos de resistência máxima à tração (>170 kPa) e elongação máxima à ruptura (>40%). Por outro lado, as membranas mais susceptíveis à degradação enzimática foram aquelas preparadas a partir de CMQs de baixa massa molar (Mv≈43.000 g.mol-1) e que exibiram baixos graus de reticulação (GR<11%). Hidrogéis estáveis de quitosana sem o uso de qualquer agente de reticulação externo foram produzidos a partir da gelificação de soluções aquosas de quitosana com solução de NaOH ou vapor de NH3. Os hidrogéis produzidos a partir de soluções de quitosana de elevada massa molar média ponderal (Mw≈640.000 g.mol-1) e extensivamente desacetilada (DA≈2,8%) em concentrações poliméricas acima 2,0%, exibiram melhores propriedades mecânicas com o aumento da concentração polimérica, devido à formação de numerosos emaranhamentos físicos das cadeias poliméricas em solução. Os resultados mostram que as propriedades físico-químicas e mecânicas dos hidrogéis de quitosana podem ser controladas variando a concentração do polímero e o processo de gelificação. A avaliação biológica de tais hidrogéis para a regeneração de miocárdio infartado de ratos revelou que os hidrogéis de quitosana preparados a partir de soluções de polímero a 1,5% foram perfeitamente incorporados sobre a superfície do epicárdio do coração e apresentaram degradação parcial acompanhada por infiltração de células mononucleares.

Marcadores de lesão renal na nefropatia diabética

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Marcadores de lesão renal na nefropatia diabética

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Nanostructures, Semicrytalline Morphology, and Nanoscale Confinement Effect on the Crystallization Kinetics in Self-Organized Block Copolymer/Thermoset Blends

This work reports the first instance of self-organized thermoset blends containing diblock copolymers with a crystallizable thermoset-immiscible block. Nanostructured thermoset blends of bisphenol A-type epoxy resin (ER) and a low-molecular-weight (M-n = 1400) amphiphilic polyethylene-block-poly(ethylene oxide) (EEO) symmetric diblock copolymer were prepared using 4,4'-methylenedianiline (MDA) as curing agent and were characterized by transmission electron microscopy (TEM), atomic force microscopy (AFM), small-angle X-ray scattering (SAXS), and differential scanning calorimetry (DSC). All the MDA-cured ER/EEO blends do not show macroscopic phase separation but exhibit microstructures. The ER selectively mixes with the epoxy-miscible PEO block in the EEO diblock copolymer whereas the crystallizable PE blocks that are immiscible with ER form separate microdomains at nanoscales in the blends. The PE crystals with size on nanoscales are formed and restricted within the individual spherical micelles in the nanostructured ER/EEO blends with EEO content up to 30 wt %. The spherical micelles are highly aggregated in the blends containing 40 and 50 wt % EEO. The PE dentritic crystallites exist in the blend containing 50 wt % EEO whereas the blends with even higher EEO content are completely volume-filled with PE spherulites. The semicrystalline microphase-separated lamellae in the symmetric EEO diblock copolymer are swollen in the blend with decreasing EEO content, followed by a structural transition to aggregated spherical micellar phase morphology and, eventually, spherical micellar phase morphology at the lowest EEO contents. Three morphological regimes are identified, corresponding precisely to the three regimes of crystallization kinetics of the PE blocks. The nanoscale confinement effect on the crystallization kinetics in nanostructured thermoset blends is revealed for the first time. This new phenomenon is explained on the basis of homogeneous nucleation controlled crystallization within nanoscale confined environments in the block copolymer/thermoset blends.

Glass transition behaviour of fructose

The glass transition temperature and the second transition (the endothermic change between the glass transition and melting temperatures) of fructose were studied. The thermal history strongly affected both transitions of fructose. Storage for 10 days at 22degreesC increased the dynamic glass transition temperature from 16 to 25degreesC and decreased the second transition of fructose from 110 to 98degreesC in the first differential scanning calorimetric (DSC) scan. The amplitude of the second transition increased slightly with storage time and reached 260% of the first transition for vacuum oven dried samples. The effect of thermal history on the glass transition temperature of fructose can be removed by scanning the sample in a DSC to 130degreesC. The effects of water content, glucose and sucrose on the two transitions were also investigated.

Direct observation of covalent adducts with Cys34 of human serum albumin using mass spectrometry

The interactions of the unpaired thiol residue (Cys34) of human serum albumin (HSA) with low-molecular-weight thiols and an Au(I)-based antiarthritic drug have been examined using electrospray ionization mass spectrometry. Early measurements of the amount of HSA containing Cys34 as the free thiol suggested that up to 30% of circulating HSA bound cysteine as a mixed disulfide. It has also been suggested that reaction of HSA with cysteine, occurs only on handling and storage of plasma. In our experiments, there were three components of HSA in freshly collected plasma from normal volunteers, HSA, HSA + cysteine, and HSA + glucose in the ratio similar to50:25:25. We addressed this controversy by using iodoacetamide to block the free thiol of HSA in fresh plasma, preventing its reaction with plasma cysteine. When iodoacetamide was injected into a vacutaner tube as blood was collected, the HSA was modified by iodoacetamide, with 20-30% present as the mixed disulfide with cysteine (HSA + cys). These data provide strong evidence that 20-30% of HSA in normal plasma contains one bound cysteine. Reaction of HSA with [Au(S2O3)(2)](3-) resulted in formation of the adducts HSA + Au(S2O3) and HSA + Au. Reaction of HSA with iodoacetamide prior to treatment with [Au(S2O3)(2)](3-) blocked the formation of gold adducts. (C) 2003 Elsevier Inc. All rights reserved.

Conjugation of an antibody to cross-linked fibrin for targeted delivery of anti-restenotic drugs

There is an urgent need to treat restenosis, a major complication of the treatment of arteries blocked by atherosclerotic plaque, using local delivery techniques. We observed that cross-linked fibrin (XLF) is deposited at the site of surgical injury of arteries. An antibody to XLF, conjugated to anti-restenotic agents, should deliver the drugs directly and only to the site of injury. An anti-XLF antibody (H93.7C.1D2/48; 1D2) was conjugated to heparin (using N-succinimidyl 3-(2-pyridyldithio)-propionate), low molecular weight heparin (LMWH) (adipic acid dihydrazide) and rapamycin (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide), and the conjugates purified and tested for activity before use in vivo. Rabbits had their right carotid arteries de-endothelialised and then given a bolus of 1D2-heparin, 1D2-LMWH or 1D2-rapamycin conjugate or controls of saline, heparin, LMWH, rapamycin or 1D2 (+/-heparin bolus) and sacrificed after 2 or 4 weeks (12 groups, n=6/group). Rabbits given any of the conjugates had minimal neointimal development in injured arteries, with up to 59% fewer neointimal cells than those given control drugs. Rabbits given 1D2-heparin or 1D2-LMWH had an increased or insignificant reduction in luminal area, with positive remodelling, while the medial and total arterial areas of rabbits given 1D2-rapamycin were not affected by injury. Arteries exposed to 1D2-heparin or 1D2-rapamycin had more endothelial cells than rabbits given control drugs. Thus, XLF-antibodies can site-deliver anti-restenotic agents to injured areas of the artery wall, where the conjugates can influence remodelling, re-endothelialisation and neointimal cell density, with reduced neointimal formation. (C) 2004 Elsevier B.V. All rights reserved.

Targeted delivery of heparin and LMWH using a fibrin antibody prevents restenosis

This study investigates a stent-less local delivery system for anti-restenotic agents utilizing antibodies to cross-linked fibrin (XLF). Heparin and low molecular weight heparin (LMWH) were conjugated to an antibody to cross-linked fibrin D-dinner (1D2). Rabbit right carotid arteries were injured with a balloon catheter, then the animals were given a bolus injection of 40 mug/k,g 1D2-heparin (26-70 mug/kg heparin) or 1D2-LMWH (29-80 mug/kg LMWH) conjugates or controls of saline (0.5 ml/kg), heparin (150 U/kg), LMWH (2 mg), or 1D2 (40 mug/kg), with or without a heparin bolus and sacrificed after 2 weeks (8 groups, n = 6/group). The injured artery of rabbits given 1D2-heparin or 1D2-LMWH conjugates had reduced neointimal development, with decreased luminal narrowing and positive remodelling compared with animals given control drugs. Animals given 1D2-heparin conjugate (with a heparin bolus) had three to five times more endothelial cells than the rabbits given saline or unconjugated heparin, while rabbits given 1D2-LMWH conjugate had up to 59% fewer neointimal cells than those given unconjugated drugs. There was little difference in extracellular matrix organization or composition. Thus cross-linked fibrin-antibodies can site-deliver anti-restenotic agents to injured areas of the artery wall where they influence wall remodelling and endothelial and neointimal cell number, reducing neointimal formation without systemic complications. Local delivery of anti-restenotic agents should minimise systemic effects, bleeding complications and potentially the cost of treatment due to a single, lower dose. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Crystallization and preliminary x-ray diffraction analysis of the unliganded human growth hormone receptor

The crystal structure of the extracellular domain of growth hormone receptor complexed to its ligand, growth hormone, has been known since 1992. However, no information exists for the unliganded form of the receptor. The human growth hormone receptor's extracellular ligand-binding domain, encompassing amino-acid residues 1 - 238, has been expressed in Escherichia coli, purified by anion ion-exchange chromatography and crystallized in its unliganded state by the hanging-drop vapour-diffusion method in 100 mM HEPES pH 7.0 containing 27.5%(w/v) PEG 5000 monomethyl ether and 200 mM ammonium sulfate as the co-precipitants. The crystals belong to the othorhombic space group C222(1), have unit-cell parameters a = 99.7, b = 112.2, c = 93.2 Angstrom and diffract to 2.5 Angstrom resolution using synchrotron radiation. The crystal structure will shed light on the nature of any conformation changes that occur upon ligand binding and will provide information to develop potential low-molecular-weight agonists/antagonists to treat clinical diseases in which the growth hormone receptor is implicated.

Regulation of antigen processing and presentation molecules in West Nile virus-infected human skin fibroblasts

Infection of humans with the West Nile flavivirus principally occurs via tick and mosquito bites. Here, we document the expression of antigen processing and presentation molecules in West Nile virus (WNV)-infected human skin fibroblast (HFF) cells. Using a new Flavivirus-specific antibody, 4G4, we have analyzed cell surface human leukocyte antigen (HLA) expression on virus-infected cells at a single cell level. Using this approach, we show that West Nile Virus infection alters surface HLA expression on both infected HFF and neighboring uninfected HFF cells. Interestingly, increased surface HLA evident on infected HFF cultures is almost entirely due to virus-induced interferon (IFN)alpha/beta because IFNalpha/beta-neutralizing antibodies completely prevent increased surface HLA expression. In contrast, RT-PCR analysis indicates that WNV infection results in increased mRNAs for HLA-A, -B, and -C genes, and HLA-associated molecules low molecular weight polypeptide-2 (LMP-2) and transporter associated with antigen presentation-1 (TAP-1), but induction of these mRNAs is not diminished in HFF cells cultured with IFNalpha/beta-neutralizing antibodies. Taken together, these data support the idea that that both cytokine-dependent and cytokine-independent mechanisms account for WNV-induced HLA expression in human skin fibroblasts. (C) 2004 Elsevier Inc. All rights reserved.