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NMDA receptors play a central role in shaping the strength of synaptic connections throughout development and in mediating synaptic plasticity mechanisms that underlie some forms of learning and memory formation in the CNS. In the hippocampus and the neocortex, GluN1 is combined primarily with GluN2A and GluN2B, which are differentially expressed during development and confer distinct molecular and physiological properties to NMDA receptors. The contribution of each subunit to the synaptic traffic of NMDA receptors and therefore to their role during development and in synaptic plasticity is still controversial. We report a critical role for the GluN2B subunit in regulating NMDA receptor synaptic targeting. In the absence of GluN2B, the synaptic levels of AMPA receptors are increased and accompanied by decreased constitutive endocytosis of GluA1-AMPA receptor. We used quantitative proteomic analysis to identify changes in the composition of postsynaptic densities from GluN2B−/−mouse primary neuronal cultures and found altered levels of several ubiquitin proteasome system components, in particular decreased levels of proteasome subunits. Enhancing the proteasome activity with a novel proteasome activator restored the synaptic levels of AMPA receptors in GluN2B−/−neurons and their endocytosis, revealing that GluN2B-mediated anchoring of the synaptic proteasome is responsible for fine tuning AMPA receptor synaptic levels under basal conditions.

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Intra-annual density fluctuations (IADFs) are anatomical features formed in response to changes in the environmental conditions within the growing season. These anatomical features are commonly observed in Mediterranean pines, being more frequent in younger and wider tree rings. However, the process behind IADF formation is still unknown. Weekly monitoring of cambial activity and wood formation would fill this void. Although studies describing cambial activity and wood formation have become frequent, this knowledge is still fragmentary in the Mediterranean region. Here we present data from the monitoring of cambial activity and wood formation in two diameter classes of maritime pine (Pinus pinaster Ait.), over two years, in order to test: (i) whether the differences in stem diameter in an even-aged stand were due to timings and/or rates of xylogenesis; (ii) if IADFs were more common in large trees; and (iii) if their formation is triggered by cambial resumption after the summer drought. Larger trees showed higher rates of cell production and longer growing seasons, due to an earlier start and later end of xylogenesis. When a drier winter occurs, larger trees were more affected, probably limiting xylogenesis in the summer months. In both diameter classes a latewood IADF was formed in 2012 in response to late-September precipitation, confirming that the timing of the precipitation event after the summer drought is crucial in determining the resumption of cambial activity and whether or not an IADF is formed. It was the first time that the formation of a latewood IADF was monitored at a weekly time scale in maritime pine. The capacity of maritime pine to adjust cambial activity to the current environmental conditions represents a valuable strategy under the future climate change conditions.

The plant cell wall degrading enzymes expressed by anaerobic microorganisms form large multienzyme complexes (cellulosomes). Cellulosomes assemble by the Type I dockerins on the catalytic subunits binding to the reiterated Type I cohesins in the molecular scaffold, while Type II dockerin-cohesin interactions anchor the complex onto the bacterial cell surface. Type I and Type II cohesin, dockerin pairs show no cross-specificity. Here we report the crystal structure of the Type II cohesin (CohII) from the Clostridium thermocellum cell surface anchoring protein SdbA. The protein domain contains nine beta-strands and a small alpha-helix. The beta-strands assemble into two elongated beta-sheets that display a typical jelly roll fold. The structure of CohII is very similar to Type I cohesins, and the dockerin binding site, which is centred at beta-strands 3, 5 and 6, is likely to be conserved in the two proteins. Subtle differences in the topology of the binding sites and a lack of sequence identity in the beta-strands that comprise the core of the dockerin binding site explain why Type I and Type II cohesins display such distinct specificities for their target dockerins.

African swine fever virus (ASFV), a large icosahedral deoxyvirus, is the causative agent of an economically relevant hemorrhagic disease that affects domestic pigs. The major purpose of the present study was to investigate the nuclear transport activities of the ASFV p37 and p14 proteins, which result from the proteolytic processing of a common precursor. Experiments were performed by using yeast-based nucleocytoplasmic transport assays and by analysis of the subcellular localization of different green fluorescent and Myc fusion proteins in mammalian cells. The results obtained both in yeast and mammalian cells clearly demonstrated that ASFV p14 protein is imported into the nucleus but not exported to the cytoplasm. The ability of p37 protein to be exported from the nucleus to the cytoplasm of both yeast and mammalian cells was also demonstrated, and the results clearly indicate that p37 nuclear export is dependent on the interaction of the protein with the CRM-1 receptor. In addition, p37 was shown to exhibit nuclear import activity in mammalian cells. The p37 protein nuclear import and export abilities described here constitute the first report of a nucleocytoplasmic shuttling protein encoded by the ASFV genome. Overall, the overlapping results obtained for green fluorescent protein fusions and Myc-tagged proteins undoubtedly demonstrate that ASFV p37 and p14 proteins exhibit nucleocytoplasmic transport activities. These findings are significant for understanding the role these proteins play in the replication cycle of ASFV.

The utilization of organized supramolecular assemblies to exploit the synergistic interactions afforded by close proximity, both for enzymatic synthesis and for the degradation of recalcitrant substrates, is an emerging theme in cellular biology. Anaerobic bacteria harness a multiprotein complex, termed the “cellulosome,” for efficient degradation of the plant cell wall. This megadalton catalytic machine organizes an enzymatic consortium on a multifaceted molecular scaffold whose “cohesin” domains interact with corresponding “dockerin” domains of the enzymes. Here we report the structure of the cohesin–dockerin complex from Clostridium thermocellum at 2.2-Å resolution. The data show that the β-sheet cohesin domain interacts predominantly with one of the helices of the dockerin. Whereas the structure of the cohesin remains essentially unchanged, the loop–helix–helix–loop–helix motif of the dockerin undergoes conformational change and ordering compared with its solution structure, although the classical 12-residue EF-hand coordination to two calcium ions is maintained. Significantly, internal sequence duplication within the dockerin is manifested in near-perfect internal twofold symmetry, suggesting that both “halves” of the dockerin may interact with cohesins in a similar manner, thus providing a higher level of structure to the cellulosome and possibly explaining the presence of “polycellulosomes.” The structure provides an explanation for the lack of cross-species recognition between cohesin–dockerin pairs and thus provides a blueprint for the rational design, construction, and exploitation of these catalytic assemblies.

Ionotropic glutamate receptors mediate the majority of excitatory synaptic transmission in the brain and are thought to be involved in learning and memory formation. The activity of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptors can be regulated by direct phosphorylation of their subunits, which affects the electrophysiological properties of the receptor, and the receptor association with numerous proteins that modulate membrane traffic and synaptic targeting of the receptor. In the present study we investigated the association of protein kinase C (PKC) γ isoform with the GluR4 AMPA receptor subunit. PKCγ was co-immunoprecipitated with GluR4 AMPA receptor subunit in rat cerebellum and in cultured chick retina cell extracts, and immunocytochemistry experiments showed co-localization of GluR4 and PKCγ in cultured chick retinal neurons. Pull-down assays showed that native PKCγ binds the GluR4 C-terminal membrane-proximal region, and recombinant PKCγ was retained by GST-GluR4 C-terminal fusion protein, suggesting that the kinase binds directly to GluR4. Furthermore, GST-GluR4 C-terminal protein was phosphorylated on GluR4 Ser-482 by bound kinases, retained by the fusion protein, including PKCγ. The GluR4 C-terminal segment that interacts with PKCγ, which lacks the PKC phosphorylation sites, inhibited histone H1 phosphorylation by PKC, to the same extent as the PKC pseudosubstrate peptide 19–31, indicating that PKCγ bound to GluR4 preferentially phosphorylates GluR4 to the detriment of other substrates. Additionally, PKCγ expression in GluR4 transfected human embryonic kidney 293T cells increased the amount of plasma membrane-associated GluR4. Our results suggest that PKCγ binds directly to GluR4, thereby modulating the function of GluR4-containing AMPA receptors.

The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors for the neurotransmitter glutamate are oligomeric structures responsible for most fast excitatory responses in the central nervous system. The activity of AMPA receptors can be directly regulated by protein phosphorylation, which may also affect the interaction with intracellular proteins and, consequently, their recycling and localization to defined postsynaptic sites. This review focuses on recent advances in understanding the dynamic regulation of AMPA receptors, on a short- and long-term basis, and its implications in synaptic plasticity.

We have previously reported that the activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors is potentiated by protein kinase C (PKC) in cultured chick retina amacrine neurons, and that constitutive PKC activity is necessary for basal AMPA receptor activity (Carvalho et al., 1998). In this study, we evaluated the phosphorylation of the GluR4 subunit, which is very abundant in cultured amacrine neurons, to correlate it with the effects of PKC on AMPA receptor activity in these cells. 32P-labelling of GluR4 increased upon AMPA receptor stimulation or cell treatment with phorbol 12-myristate 13-acetate (PMA) before stimulating with kainate. By contrast, phosphorylation of GluR4 was not changed when PKC was inhibited by treating the cells with the selective PKC inhibitor GF 109203X before stimulation with kainate. We conclude that GluR4 is phosphorylated upon PKC activation and/or stimulation of AMPA receptors in cultured amacrine cells. Additionally, AMPA receptor activation with kainate in cultured chick amacrine cells leads to translocation of conventional and novel PKC isoforms to the cell membrane, suggesting that PKC could be activated upon AMPA receptor stimulation in these cells.

AbtractCellulosomes are sophisticated multi-enzymatic nanomachines produced by anaerobes to effectively deconstruct plant structural carbohydrates. Cellulosome assembly involves the binding of enzyme-borne dockerins (Doc) to repeated cohesin (Coh) modules located in a non-catalytic scaffoldin. Docs appended to cellulosomal enzymes generally present two similar Coh-binding interfaces supporting a dual-binding mode, which may confer increased positional adjustment of the different complex components. Ruminococcus flavefaciens’ cellulosome is assembled from a repertoire of 223 Doc-containing proteins classified into 6 groups. Recent studies revealed that Docs of groups 3 and 6 are recruited to the cellulosome via a single-binding mode mechanism with an adaptor scaffoldin. To investigate the extent to which the single-binding mode contributes to the assembly of R. flavefaciens cellulosome, the structures of two group 1 Docs bound to Cohs of primary (ScaA) and adaptor (ScaB) scaffoldins were solved. The data revealed that group 1 Docs display a conserved mechanism of Coh recognition involving a single-binding mode. Therefore, in contrast to all cellulosomes described to date, the assembly of R. flavefaciens cellulosome involves single but not dual-binding mode Docs. Thus, this work reveals a novel mechanism of cellulosome assembly and challenges the ubiquitous implication of the dual-binding mode in the acquisition of cellulosome flexibility.

Early cognitive deficits in Alzheimer's disease (AD) have been related to deregulation of N-methyl-d-aspartate receptors (NMDARs) and synaptic dysfunction in response to amyloid-beta peptide. NMDAR anchorage to post-synaptic membrane depends in part on Src kinase, which is also implicated in NMDAR activation and actin cytoskeleton stabilization, two processes relevant for normal synaptic function. In this study we analyzed the changes in GluN2B subunit phosphorylation and the levels of proteins involved in Src related signaling pathways linking the Tyr kinase to actin cytoskeleton polymerization, namely reelin, disabled-1 (Dab1) and cortactin, in hippocampal and cortical homogenates obtained from the triple transgenic mouse model of AD (3xTg-AD) that shows progression of pathology as a function of age versus age-matched wild-type mice. Moreover, we evaluated regional post-synaptic actin polymerization using phalloidin labeling in hippocampal slices. Young (3month-old) 3xTg-AD male mice hippocampus exhibited decreased GluN2B Tyr1472 phosphorylation and reduced Src activity. In the cortex, decreased Src activity correlated with reduced levels of reelin and Dab1, implicating changes in the reelin pathway. We also observed diminished phosphorylated Dab1 and cortactin protein levels in the hippocampus and cortex of young 3xTg-AD male mice. Concordantly with the recognized role of these proteins in actin stabilization, we detected a significant decrease in post-synaptic F-actin in 3month-old 3xTg-AD male CA1 and CA3 hippocampal regions. These data suggest deregulated Src-dependent signaling pathways involving GluN2B-composed NMDARs and post-synaptic actin cytoskeleton depolymerization in the hippocampus in early stages of AD.

Ghrelin is a peptide mainly produced by the stomach and released into circulation, affecting energy balance and growth hormone release. These effects are guided largely by the expression of the ghrelin receptor growth hormone secretagogue type 1a (GHS-R1a) in the hypothalamus and pituitary. However, GHS-R1a is expressed in other brain regions, including the hippocampus, where its activation enhances memory retention. Herein we explore the molecular mechanism underlying the action of ghrelin on hippocampal- dependent memory. Our data show that GHS-R1a is localized in the vicinity of hippocampal excitatory synapses, and that its activation increases delivery of α-amino-3-hydroxy- 5- methyl-4-isoxazole propionic-type receptors (AMPARs) to synapses, producing functional modifications at excitatory synapses. Moreover, GHS-R1a activation enhances two different paradigms of long-term potentiation in the hippocampus, activates the phosphatidylinositol 3-kinase, and increases GluA1 AMPAR subunit and stargazin phosphorylation. We propose that GHS-R1a activation in the hippocampus enhances excitatory synaptic transmission and synaptic plasticity by regulating AMPAR trafficking. Our study provides insights into mechanisms that may mediate the cognitionenhancing effect of ghrelin, and suggests a possible link between the regulation of energy metabolism and learning., This work was supported by FCT and Fundo Europeu de Desenvolvimento Regional PTDC/BIA-BCM/113738/2009, PEst-C/SAU/LA0001/2013-2014, PTDC/NEUNMC/ 1098/2012, and PTDC/SAU-NEU/099440/2008 (to A.L.C.), and by the Spanish Ministry of Economy (SAF2011-24730 and CSD2010-00045) and Fundacion Ramon Areces (to J.A.E.).

The AMPA receptors for glutamate are oligomeric structures that mediate fast excitatory responses in the central nervous system. Phosphorylation of AMPA receptors is an important mechanism for short-term modulation of their function, and is thought to play an important role in synaptic plasticity in different brain regions. Recent studies have shown that phosphorylation of AMPA receptors by cAMP-dependent protein kinase (PKA) and Ca2+ - and calmodulin-dependent protein kinase II (CaMKII) potentiates their activity, but phosphorylation of the receptor subunits may also affect their interaction with intracellular proteins, and their expression at the plasma membrane. Phosphorylation of AMPA receptor subunits has also been investigated in relation to processes of synaptic plasticity. This review focuses on recent advances in understanding the molecular mechanisms of regulation of AMPA receptors, and their implications in synaptic plasticity.

Photoacoustic calorimetry was used to measure the quantum yields of singlet molecular oxygen production by the triplet states of tetraphenylporphyrin (TPP), ZnTPP and CuTPP in toluene, yielding values of 0.67 0.14, 0.68 0.19 and 0.03 0.01, respectively. We show that a novel dichlorophenyl derivative of ZnTPP is capable of singlet-oxygen production with a 0.90 0.07 quantum yield. The synthesis and characterisation of a new photostable chlorin with high absorptivity in the red that is capable of singlet-oxygen production with 0.54 0.06 quantum yield is described. Our results suggest that chlorinated chlorins may be interesting new sensitisers for photodynamic therapy.

In the present work we investigated the mechanisms controlling the release of acetylcholine (ACh) and of gamma-aminobutyric acid (GABA) from cultures of amacrine-like neurons, containing a subpopulation of cells which are simultaneously GABAergic and cholinergic. We found that 81.2 +/- 2.8% of the cells present in the culture were stained immunocytochemically with an antibody against choline acetyltransferase, and 38.5 +/- 4.8% of the cells were stained with an antibody against GABA. Most of the cells containing GABA (87.0 +/- 2.9%) were cholinergic. The release of acetylcholine and GABA was mostly Ca2+-dependent, although a significant release of [3H]GABA occurred by reversal of its transporter. Potassium evoked the Ca2+-dependent release of [3H]GABA and [3H]acetylcholine, with EC50 of 31.0 +/- 1.0 mm and 21.6 +/- 1.1 mm, respectively. The Ca2+-dependent release of [3H]acetylcholine was significantly inhibited by 1 micrometer tetrodotoxin and by low (30 nm) omega-conotoxin GVIA (omega-CgTx GVIA) concentrations, or by high (300 nm) nitrendipine (Nit) concentrations. On the contrary, the release of [14C]GABA was reduced by 30 nm nitrendipine, or by 500 nm omega-CgTx GVIA, but not by this toxin at 30 nm. The release of either transmitters was unaffected by 200 nm omega-Agatoxin IVA (omega-Aga IVA), a toxin that blocks P/Q-type voltage-sensitive Ca2+ channels (VSCC). The results show that Ca2+-influx through omega-CgTx GVIA-sensitive N-type VSCC and through Nit-sensitive L-type VSCC induce the release of ACh and GABA. However, the significant differences observed regarding the Ca2+ channels involved in the release of each neurotransmitter suggest that in amacrine-like neurons containing simultaneously GABA and acetylcholine the two neurotransmitters may be released in distinct regions of the cells, endowed with different populations of VSCC.

We report the three-dimensional crystal structure of acidic seminal fluid protein (aSFP), a 12.9 kDa polypeptide of the spermadhesin family isolated from bovine seminal plasma, solved by the multiple isomorphous replacement method and refined with data to 1.9 A resolution with a final R-factor of 17.3%. aSFP is built by a single CUB domain architecture, a 100 to 110 amino-acid-residue extracellular module found in 16 functionally diverse proteins. The structure of aSFP reveals that the CUB domain displays a beta-sandwich topology organised into two 5-stranded beta-sheets, each of which contain two parallel and four antiparallel strands. The structure of aSFP is almost identical to that of porcine spermadhesins PSP-I and PSP-II, which in turn show limited structural similarity with jellyroll topologies of certain virus capsid proteins. Essentially, topologically conserved residues in these proteins are those internal amino acids forming the hydrophobic core of the CUB and the jellyroll domains, suggesting their importance in maintaining the integrity of these protein folds. On the other hand, the structure of aSFP shows structural features that are unique to this protein and which may provide a structural ground for understanding the distinct biological properties of different members of the spermadhesin protein family.

The aim of the study was to determine whether betaxolol is a neuroprotective agent and can therefore slow down the changes seen in the retina following ischaemia/reperfusion. Ischaemia was induced in one rat eye by raising the intraocular pressure for 45 min. Three days later electroretinograms were recorded from both eyes and the retinas were examined immunohistochemically for the localisation of calretinin and choline acetyltransferase (ChAT) immunoreactivities. The effect of glutamate agonists, hypoxia or experimental ischaemia was examined on the GABA immunoreactivity, lactate dehydrogenase (LDH) and internal calcium levels ([Ca 2+ ] i ) of the isolated rabbit retina, rat cortical cultures and chick retinal cell cultures respectively. Betaxolol was tested to see whether it can attenuate the influence of the glutamate agonists, hypoxia or experimental ischaemia. Ischaemia for 45 min causes a change in the nature of the normal calretinin immunoreactivity, an obliteration of the ChAT immunoreactivity and a drastic reduction in the b-wave of the electroretinogram after 3 days of reperfusion. When betaxolol was injected i.p. into the rats before ischaemia and on the days of reperfusion the changes to the calretinin and ChAT immunoreactivities were reduced and the reduction of the b-wave was prevented. Rabbit retinas incubated in vitro in physiological solution lacking oxygen/glucose or containing the glutamate agonists kainate or NMDA caused a change in the nature of the GABA immunoreactivity. Inclusion of betaxolol partially prevented the changes caused by NMDA and lack of oxygen/glucose. Rat cortical cultures exposed to glutamate or hypoxia/reoxygenation resulted in a release of LDH. The release of the enzyme was almost completely attenuated when betaxolol was included in the culture medium. Kainate increased the [Ca 2+ ] i in chick retinal cultures, as measured with Indo-1. In a medium with sodium, this kainate-induced elevation of [Ca 2+ ] i was significantly reduced by betaxolol. The combined data show that betaxolol is a neuroprotective agent and attenuates the effects on the retina induced by raising the intraocular pressure to simulate an ischaemic insult as may occur in glaucoma. © 1997 Elsevier Science B.V. All rights reserved.

Doutoramento em Engenharia Química Os coeficientes de difusão (D 12) são propriedades fundamentais na investigação e na indústria, mas a falta de dados experimentais e a inexistência de equações que os estimem com precisão e confiança em fases comprimidas ou condensadas constituem limitações importantes. Os objetivos principais deste trabalho compreendem: i) a compilação de uma grande base de dados para valores de D 12 de sistemas gasosos, líquidos e supercríticos; ii) o desenvolvimento e validação de novos modelos de coeficientes de difusão a diluição infinita, aplicáveis em amplas gamas de temperatura e densidade, para sistemas contendo componentes muito distintos em termos de polaridade, tamanho e simetria; iii) a montagem e teste de uma instalação experimental para medir coeficientes de difusão em líquidos e fluidos supercríticos. Relativamente à modelação, uma nova expressão para coeficientes de difusão a diluição infinita de esferas rígidas foi desenvolvida e validada usando dados de dinâmica molecular (desvio relativo absoluto médio, AARD = 4.44%) Foram também estudados os coeficientes de difusão binários de sistemas reais. Para tal, foi compilada uma extensa base de dados de difusividades de sistemas reais em gases e solventes densos (622 sistemas binários num total de 9407 pontos experimentais e 358 moléculas) e a mesma foi usada na validação dos novos modelos desenvolvidos nesta tese. Um conjunto de novos modelos foi proposto para o cálculo de coeficientes de difusão a diluição infinita usando diferentes abordagens: i) dois modelos de base molecular com um parâmetro específico para cada sistema, aplicáveis em sistemas gasosos, líquidos e supercríticos, em que natureza do solvente se encontra limitada a apolar ou fracamente polar (AARDs globais na gama 4.26-4.40%); ii) dois modelos de base molecular biparamétricos, aplicáveis em todos os estados físicos, para qualquer tipo de soluto diluído em qualquer solvente (apolar, fracamente polar e polar). Ambos os modelos dão origem a erros globais entre 2.74% e 3.65%; iii) uma correlação com um parâmetro, específica para coeficientes de difusão em dióxido de carbono supercrítico (SC-CO2) e água líquida (AARD = 3.56%); iv) nove correlações empíricas e semi-empíricas que envolvem dois parâmetros, dependentes apenas da temperatura e/ou densidade do solvente e/ou viscosidade do solvente. Estes últimos modelos são muito simples e exibem excelentes resultados (AARDs entre 2.78% e 4.44%) em sistemas líquidos e supercríticos; e v) duas equações preditivas para difusividades de solutos em SC-CO2, em que os erros globais de ambas são inferiores a 6.80%. No global, deve realçar-se o facto de os novos modelos abrangerem a grande variedade de sistemas e moléculas geralmente encontrados. Os resultados obtidos são consistentemente melhores do que os obtidos com os modelos e abordagens encontrados na literatura. No caso das correlações com um ou dois parâmetros, mostrou-se que estes mesmos parâmetros podem ser ajustados usando um conjunto muito pequeno de dados, e posteriormente serem utilizados na previsão de valores de D 12 longe do conjunto original de pontos. Uma nova instalação experimental para medir coeficientes de difusão binários por técnicas cromatográficas foi montada e testada. O equipamento, o procedimento experimental e os cálculos analíticos necessários à obtenção dos valores de D 12 pelo método de abertura do pico cromatográfico, foram avaliados através da medição de difusividades de tolueno e acetona em SC-CO2. Seguidamente, foram medidos coeficientes de difusão de eucaliptol em SC-CO2 nas gamas de 202 – 252 bar e 313.15 – 333.15 K. Os resultados experimentais foram analisados através de correlações e modelos preditivos para D12. Diffusivities (D12) are fundamental properties both at research and industry levels, but the lack of experimental data and the non-existence of reliable and accurate equations to estimate them in compressed and condensed phases constitute important shortcomings. The main objectives of this work comprise: i) the compilation of a large database of D12 values in gas, liquid and supercritical systems; ii) the development and validation of new models for tracer diffusivities, applicable over wide ranges of temperature and density, for systems containing very distinct components in term of polarity, size and symmetry; iii) the installation and test of an experimental set-up to measure diffusion coefficients in liquids and supercritical fluids. Concerning modelling, a new accurate expression for tracer diffusion coefficients of hard sphere fluid was developed and validated using molecular dynamic data (average absolute relative deviation, AARD = 4.44%). The binary diffusion coefficients of real systems were also studied. An extensive database of diffusivities in gas and dense solvents was compiled (622 binary systems performing 9407 data points and comprehending 358 molecules) and used to validate the new models developed in this thesis. A set of new models were proposed for tracer diffusivities using different approaches: i) two molecularly-based models with one system-specific parameter that are applicable to gas, liquid, and supercritical systems, where the nature of solvent is limited to non-polar or weakly polar (global AARDs in the range 4.26-4.40%); ii) two molecularly-based models with two parameters, applicable in all physical states, for any solutes diluted in any type of solvent (non-polar, weakly-polar, and polar). Both models provide global errors between 2.74% and 3.65%; iii) one correlation with one parameter devoted to D 12 coefficients in supercritical carbon dioxide (SC-CO2) and liquid water (AARD = 3.56%); iv) nine empirical and semi-empirical correlations involving two parameters, dependent on temperature and/or solvent density and/or solvent viscosity. These models are very simple and provide accurate results (AARDs between 2.78% and 4.44%) in liquid and supercritical systems; and v) two predictive equations for diffusivities of solutes in SC-CO2 where the global deviations for both are inferior to 6.80%. In the whole, it may be emphasized that the new models cover the large variety of systems and molecules generally found. The results achieved are consistently better than those obtained by well known models and approaches taken from the literature. In the case of the 1- and 2-parameter correlations, it has been shown that such parameters can be fitted to a very small set of data, and subsequently used to predict D 12 values far from the original set of points. A new experimental set-up to measure binary diffusion coefficients by chromatographic techniques was designed and tested. The equipment, experimental procedure and analytical calculations to obtain the D 12 values by the chromatographic peak broadening technique were assessed by measuring diffusivities of toluene and acetone in SC-CO2. Then, the diffusivities of eucalyptol in SC-CO2 were determined in the ranges 202 – 252 bar and 313.15 – 333.15 K. The experimental data were analysed using D 12 predictive and correlation models.

Doutoramento em Engenharia Química Submitted by Bella Nolasco (bellanolasco@ua.pt) on 2014-05-05T09:08:49Z No. of bitstreams: 1 Diffusion coefficients in dense and supercritical fluids.pdf: 10906691 bytes, checksum: 8d7c330f092f8adae3de2158eb4a7ae2 (MD5) Made available in DSpace on 2014-05-05T09:08:49Z (GMT). No. of bitstreams: 1 Diffusion coefficients in dense and supercritical fluids.pdf: 10906691 bytes, checksum: 8d7c330f092f8adae3de2158eb4a7ae2 (MD5) Previous issue date: 2013-12-19

SummaryDuring development, neurons are constantly refining their connections in response to changes in activity. Experience-dependent plasticity is a key form of synaptic plasticity, involving changes in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) accumulation at synapses. Here, we report a critical role for the AMPAR auxiliary subunit stargazin in this plasticity. We show that stargazin is functional at the retinogeniculate synapse and that in the absence of stargazin, the refinement of the retinogeniculate synapse is specifically disrupted during the experience-dependent phase. Importantly, we found that stargazin expression and phosphorylation increased with visual deprivation and led to reduced AMPAR rectification at the retinogeniculate synapse. To test whether stargazin plays a role in homeostatic plasticity, we turned to cultured neurons and found that stargazin phosphorylation is essential for synaptic scaling. Overall, our data reveal an important role for stargazin in regulating AMPAR abundance and composition at glutamatergic synapses during homeostatic and experience-dependent plasticity.

Non-catalytic cellulosomal CBMs (carbohydrate-binding modules) are responsible for increasing the catalytic efficiency of cellulosic enzymes by selectively putting the substrate (a wide range of poly- and oligo-saccharides) and enzyme into close contact. In the present study we carried out an atomistic rationalization of the molecular determinants of ligand specificity for a family 11 CBM from thermophilic Clostridium thermocellum [CtCBM11 (C. thermocellum CBM11)], based on a NMR and molecular modelling approach. We have determined the NMR solution structure of CtCBM11 at 25°C and 50°C and derived information on the residues of the protein that are involved in ligand recognition and on the influence of the length of the saccharide chain on binding. We obtained models of the CtCBM11–cellohexaose and CtCBM11–cellotetraose complexes by docking in accordance with the NMR experimental data. Specific ligand–protein CH-π and Van der Waals interactions were found to be determinant for the stability of the complexes and for defining specificity. Using the order parameters derived from backbone dynamics analysis in the presence and absence of ligand and at 25°C and 50°C, we determined that the protein's backbone conformational entropy is slightly positive. This data in combination with the negative binding entropy calculated from ITC (isothermal titration calorimetry) studies supports a selection mechanism where a rigid protein selects a defined oligosaccharide conformation.

Dissertação de mestrado em Ciências – Formação Contínua de Professores (área de especialização em Física e Química), A temática da educação científica e tecnológica tem sido gradualmente introduzida nos diferentes níveis de ensino com o intuito de formar cidadãos capazes de compreender o mundo no qual estão inseridos. Neste âmbito procurou-se desenvolver o tema «nanociência e nanotecnologia», dado se tratar de um conceito atual e interdisciplinar, englobando desde as ciências exatas e experimentais às ciências humanas e sociais. No presente trabalho pretendeu-se demonstrar a importância deste tema, descrevendo e analisando uma proposta a ser aplicada ao nível de ensino pré-universitário destinada às aulas de ciências experimentais no 3º ciclo do ensino básico e no ensino secundário. As atividades desenvolvidas consistiram na síntese de nanopartículas de ouro e de óxido de ferro em solução aquosa e na produção de filmes finos de ouro sobre um substrato de vidro. A síntese das nanopartículas de ouro em solução aquosa foi efectuada segundo um método clássico (atribuído a Turkevich) possibilitando a obtenção de soluções coloidais com nanopartículas de ouro de diferentes dimensões. Foi estudado o respetivo comportamento ótico quando submetida à luz branca e à radiação monocromática polarizada (radiação laser) tal como a cor e a observação do efeito de Tyndall. A atividade desenvolvida, em laboratório, foi filmada para a elaboração de um vídeo de apoio didático para posterior utilização para fins demonstrativos e, posteriormente, realizada em contexto escolar por alunos do 10º ano do ensino regular. A deposição de filmes finos de ouro sobre substrato de vidro pelo método de evaporação térmica resistiva teve como objetivo a obtenção de amostras que permitissem comparar a cor exibida por estas nanopartículas com as dispersas em meio coloidal com as desse mesmo material a nível macroscópico. Os resultados obtidos neste trabalho foram divulgados na forma de um poster apresentado na Semana de la Nanociencia y la Nanotecnologia, realizado em Medellin, Colômbia . A síntese de nanopartículas de óxido de ferro por via química foi dirigida para a obtenção de um material ferrofluido estudando o seu comportamento na presença de um campo magnético gerado por um símples íman. Este comportamento foi comparado com o exibido por partículas macroscópicas deste mesmo material, sendo este comportamento posteriormente observado por alunos do 9º ano. Desenvolveu-se e implementou-se um inquérito de opinião aplicado a uma amostra de professores do grupo disciplinar de Física e Quimica, a nível nacional, dos quais foram obtidas 161 respostas. A análise estatística e descritiva dos dados recolhidos permitiu obter algumas conclusões quanto à opinião dos mesmos sobre implementação de temas como a nanociência e nanotecnologia em níveis de ensino pré-universitário., The theme of the scientific and technological education has been gradually introduced in the different teaching level aiming to form citizens able to understand the world in which they are inserted. Is in this context the subject «nanoscience and nanotecnology» is an actual theme involving concept that are interdisciplinar spreaded from experimental sciences till the human and social studies. The present work intends to demonstrate the importance of this subject and describes the pratical activities developed to be applied in laboratory class of experimental sciences of a pre-universitary teaching level (3rd cycle of the basic teaching and secondary levels). The activities developed were the synthesis of aqueous colloidal solutions of gold nanoparticles and iron oxide nanoparticles and to produce thin gold films deposited over a surface. Using the Turkevich method a colloidal solutions with gold nanoparticles of different sizes were synthesized. Their optical behavior when submited to white light and monochromatic polarized radiation (laser radiation) was studied and the color and Tyndall effect were observed. The laboratorial activity was filmed to make a video for teaching purposes which was made available on Youtube and was later performed at the lab by 10th grade students. Physical vapor deposition (PVD) technique was used to produce thin gold deposition over glass substrate. The purpose of this activity was to supply samples that allow the comparison of the color shown by the gold nanoparticles deposited in a solid substrate with those dispersed in colloidal aqueous solution as well with the observed gold macroscopic characteristics. The results obtained in was presented in a poster in Semana de la Nanociencia y la Nanotecnologia, occured at Medellin, Colômbia . The synthesis of an aqueous iron oxide nanoparticles by chemical method was directed to obtain a ferrofluid material which behaviour was studied in the presence of magnetic fields using a simple magnet. This behaviour was compared with the observed characteristics of similar materials with macro dimensions. The magnetic behaviour of the produced ferrofluid and of the magnetite was explored at the 9th grade student classes. An inquire was developed and sent to teachers of Physics and Chemistry across Portugal and 161 replies were collected. The statistical and descriptive treatment of the obtained answers allowed to conclued about the opinion on implementation of subjects like the nanoscience and nanotecnology at pre-universitary teaching levels.

PEst-C/EQB/LA0006/2011 This work was supported by Fundação para a Ciência e a Tecnologia Grants PEst-C/EQB/LA0006/2011, PTDC/BIA-PRO/69732/2006, PTDC/QUI-BIQ/100359/2008, and PTDC/BIA-PRO/103980/2008. Supported by Fundação para a Ciência e a Tecnologia individual fellowship SFRH/BD/38667/2007. Protein-protein interactions play a pivotal role in a large number of biological processes exemplified by the assembly of the cellulosome. Integration of cellulosomal components occurs through the binding of type I cohesin modules located in a non-catalytic molecular scaffold to type I dockerin modules located at the C terminus of cellulosomal enzymes. The majority of type I dockerins display internal symmetry reflected by the presence of two essentially identical cohesin-binding surfaces. Here we report the crystal structures of two novel Clostridium thermocellum type I cohesin-dockerin complexes (CohOlpC-Doc124A and CohOlpA-Doc918). The data revealed that the two dockerins, Doc918 and Doc124A, are unusual because they lack the structural symmetry required to support a dual binding mode. Thus, in both cases, cohesin recognition is dominated by residues located at positions 11, 12, and 19 of one of the dockerin binding surfaces. The alternative binding mode is not possible (Doc918) or highly limited (Doc124A) because residues that assume the critical interacting positions, when dockerins are reoriented by 180°, make steric clashes with the cohesin. In common with a third dockerin (Doc258) that also presents a single binding mode, Doc124A directs the appended cellulase, Cel124A, to the surface of C. thermocellum and not to cellulosomes because it binds preferentially to type I cohesins located at the cell envelope. Although there are a few exceptions, such as Doc918 described here, these data suggest that there is considerable selective pressure for the evolution of a dual binding mode in type I dockerins that direct enzymes into cellulosomes. publishersversion published

Trafficking of NMDA receptors to the surface of neurons and to synapses is critical for proper brain function and activity-dependent plasticity. Recent evidence suggests that surface trafficking of other ionotropic glutamate receptors requires ligand binding for exit from the endoplasmic reticulum. Here, we show that glutamate binding to GluN2 is required for trafficking of NMDA receptors to the cell surface. We expressed a panel of GluN2B ligand binding mutants in heterologous cells with GluN1 or in rat cultured neurons and found that surface expression correlates with glutamate efficacy. Such a correlation was found even in the presence of dominant negative dynamin to inhibit endocytosis and surface expression correlated with Golgi localization, indicating differences in forward trafficking. Co-expression of wild type GluN2B did not enhance surface expression of the mutants, suggesting that glutamate must bind to both GluN2 subunits in a tetramer and that surface expression is limited by the least avid of the two glutamate binding sites. Surface trafficking of a constitutively closed cleft GluN2B was indistinguishable from that of wild type, suggesting that glutamate concentrations are typically not limiting for forward trafficking. YFP-GluN2B expressed in hippocampal neurons from GluN2B(-/-) mice rescued synaptic accumulation at similar levels to wild type. Under these conditions, surface synaptic accumulation of YFP-GluN2B mutants also correlated with apparent glutamate affinity. Altogether, these results indicate that glutamate controls forward trafficking of NMDA receptors to the cell surface and to synapses and raise the intriguing idea that NMDA receptors may be functional at intracellular sites.

Glutamate receptors of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type mediate fast excitatory synaptic transmission in the CNS. Synaptic strength is modulated by AMPA receptor binding partners, which regulate receptor synaptic targeting and functional properties. We identify Contactin-associated protein 1 (Caspr1) as an AMPA receptor interactor. Caspr1 is present in synapses and interacts with AMPA receptors in brain synaptic fractions. Coexpression of Caspr1 with GluA1 increases the amplitude of glutamate-evoked currents. Caspr1 overexpression in hippocampal neurons increases the number and size of synaptic GluA1 clusters, whereas knockdown of Caspr1 decreases the intensity of synaptic GluA1 clusters. Hence, Caspr1 is a regulator of the trafficking of AMPA receptors to synapses.

Cellulosomes are highly efficient nanomachines that play a fundamental role during the anaerobic deconstruction of complex plant cell wall carbohydrates. The assembly of these complex nanomachines results from the very tight binding of repetitive cohesin modules, located in a noncatalytic molecular scaffold, and dockerin domains located at the C-terminus of the enzyme components of the cellulosome. The number of enzymes found in a cellulosome varies but may reach more than 100 catalytic subunits if cellulosomes are further organized in polycellulosomes, through a second type of cohesin-dockerin interaction. Structural studies have revealed how the cohesin-dockerin interaction mediates cellulosome assembly and cell-surface attachment, while retaining the flexibility required to potentiate catalytic synergy within the complex. Methods that might be applied for the production, purification, and structure determination of cohesin-dockerin complexes are described here.

Diabetic retinopathy and diabetic encephalopathy are two common complications of diabetes mellitus. The impairment of glutamatergic neurotransmission in the retina and hippocampus has been suggested to be involved in the pathogenesis of these diabetic complications. In this study, we investigated the effect of elevated glucose concentration and diabetes on the protein content and surface expression of AMPA receptor subunits in the rat retina and hippocampus. We have used two models, cultured retinal and hippocampal cells exposed to elevated glucose concentration and an animal model of streptozotocin-induced type 1 diabetes. The immunoreactivity of GluA1, GluA2 and GluA4 was evaluated by Western blot and immunocytochemistry. The levels of these subunits at the plasma membrane were evaluated by biotinylation and purification of plasma membrane-associated proteins. Elevated glucose concentration increased the total levels of GluA2 subunit of AMPA receptors in retinal neural cells, but not of the subunits GluA1 or GluA4. However, at the plasma membrane, elevated glucose concentration induced an increase of all AMPA receptor subunits. In cultured hippocampal neurons, elevated glucose concentration did not induce significant alterations in the levels of AMPA receptor subunits. In the retinas of diabetic rats there were no persistent changes in the levels of AMPA receptor subunits comparing to aged-matched control retinas. Also, no consistent changes were detected in the levels of GluA1, GluA2 or GluA4 in the hippocampus of diabetic rats. We demonstrate that elevated glucose concentration induces early changes in AMPA receptor subunits, mainly in GluA2 subunit, in retinal neural cells. Conversely, hippocampal neurons seem to remain unaffected by elevated glucose concentration, concerning the expression of AMPA receptors, suggesting that AMPA receptors are more susceptible to the stress caused by elevated glucose concentration in retinal cells than in hippocampal neurons.

The authors acknowledge financial support from Fundacao para a Ciencia e a Tecnologia, Portugal, through grants PTDC/BIA-PRO/69732/2006, PTDC/QUI-BIQ/100359/2008, and the individual fellowship SFRH/BD/38667/2007 (to J.L.A.B.); the U.K. Biological and Biotechnological Sciences Research Council (studentship to A. C.); the US Department of Energy (Grant 00562492); and the Israel Science Foundation (Grant 966/09). The authors also acknowledge the European Synchrotron Radiation Facility, Grenoble, France (beamline ID14-EH4) for access and technical support during data collection. Clostridium thermocellum is a well-characterized cellulose-degrading microorganism. The genome sequence of C. thermocellum encodes a number of proteins that contain type I dockerin domains, which implies that they are components of the cellulose-degrading apparatus, but display no significant sequence similarity to known plant cell wall-degrading enzymes. Here, we report the biochemical properties and crystal structure of one of these proteins, designated CtCel124. The protein was shown to be an endo-acting cellulase that displays a single displacement mechanism and acts in synergy with Cel48S, the major cellulosomal exo-cellulase. The crystal structure of CtCel124 in complex with two cellotriose molecules, determined to 1.5 Å, displays a superhelical fold in which a constellation of α-helices encircle a central helix that houses the catalytic apparatus. The catalytic acid, Glu96, is located at the C-terminus of the central helix, but there is no candidate catalytic base. The substrate-binding cleft can be divided into two discrete topographical domains in which the bound cellotriose molecules display twisted and linear conformations, respectively, suggesting that the enzyme may target the interface between crystalline and disordered regions of cellulose. publishersversion published

10 páginas, 4 figuras, 3 suplementos figuras -- PAGS nros. 571-580, The cellulosome is a highly elaborate cell-bound multienzyme complex that efficiently orchestrates the deconstruction of cellulose and hemicellulose, two of the nature's most abundant polymers. Understanding the intricacy of these nanomachines evolved by anaerobic microbes could sustain the development of an effective process for the conversion of lignocellulosic biomass to bio-ethanol. In Clostridium thermocellum, cellulosome assembly is mediated by high-affinity protein:protein interactions (> 109 M− 1) between dockerin modules found in the catalytic subunits and cohesin modules located in a non-catalytic protein scaffold termed CipA. Whereas the atomic structures of several cellulosomal components have been elucidated, the structural organization of the complete cellulosome remains elusive. Here, we reveal that a large fragment of the cellulosome presents a mostly compact conformation in solution, by solving the three-dimensional structure of a C. thermocellum mini-cellulosome comprising three consecutive cohesin modules, each bound to one Cel8A cellulase, at 35 Å resolution by cryo-electron microscopy. Interestingly, the three cellulosomal catalytic domains are found alternately projected outward from the CipA scaffold in opposite directions, in an arrangement that could expand the area of the substrate accessible to the catalytic domains. In addition, the cellulosome can transit from this compact conformation to a multitude of diverse and flexible structures, where the linkers between cohesin modules are extended and flexible. Thus, structural transitions controlled by changes in the degree of flexibility of linkers connecting consecutive cohesin modules could regulate the efficiency of substrate recognition and hydrolysis, This work was funded by the Spanish Ministry of Science and Innovation (SAF2008-00451 to O.L.), the Red Temática de Investigación Cooperativa en Cáncer from the Instituto de Salud Carlos III (RD06/0020/1001 to O.L.), the Autonomous Region of Madrid (CAM S-BIO-0214-2006 to O.L.), the Human Frontiers Science Program (RGP39/2008 to O.L.) and the Portuguese Science and Technology Foundation (project PTDC/QUI-BIQ/100359/2008). F.M.V.D. was supported by grant SFRH//BPD/26508/2006 from the Portuguese Science and Technology Foundation. B.G.A. has been supported by a JAE-DOC contract of the Consejo Superior de Investigaciones Científicas and a Juan de la Cierva contract from the Spanish Ministry of Science

Macromolecular X-ray crystallography is an important and powerful technique in drug discovery, used by pharmaceutical companies in the discovery process of new medicines. The detailed analysis of crystal structures of protein-ligand complexes allows the study of the specific interactions of a particular drug with its protein target at the atomic level. It is used to design and improve drugs. The starting point of these studies is the preparation of suitable crystals of complexes with potential ligands, which can be achieved by using different strategies described in this chapter. In addition, an introduction to X-ray crystallography is given, highlighting the fundamental steps necessary to determine the three-dimensional structure of protein-ligand complexes, as well as some of the tools and criteria to validate crystal structures available in databases.

Glutamate receptors of the AMPA-type mediate fast excitatory synaptic transmission in the central nervous system and play key roles in synaptic plasticity. The binding of these receptors to a variety of proteins is known to regulate their targeting to the synapse and consequently to modulate synaptic strength, as well as to modify receptor characteristics. In this study, a proteomic screening was conducted in order to identify new binding partners for GluR4 AMPA receptor subunit. Immunoprecipitation of GluR4 and associated proteins was performed using rat cerebellum lysates and an heterologous systems overexpressing GluR4 AMPA receptor subunit. Isolated immuno-complexes were resolved by 1-D SDS-PAGE, and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). This approach led to the identification of several interactors, most of which are novel AMPA receptor partners, namely, cytoskeleton proteins, motor proteins, RNA processing proteins which are part of neuronal RNA granules, and kinases, among others. This study unravels new constituents of the macromolecular complex of long-form calcium-permeable AMPA receptors.

Macromolecular X-ray crystallography is an important and powerful technique in drug discovery, used by pharmaceutical companies in the discovery process of new medicines. The detailed analysis of crystal structures of protein-ligand complexes allows the study of the specific interactions of a particular drug with its protein target at the atomic level. It is used to design and improve drugs. The starting point of these studies is the preparation of suitable crystals of complexes with potential ligands, which can be achieved by using different strategies described in this chapter. In addition, an introduction to X-ray crystallography is given, highlighting the fundamental steps necessary to determine the three-dimensional structure of protein-ligand complexes, as well as some of the tools and criteria to validate crystal structures available in databases.

The authors acknowledge financial support from Fundacao para a Ciencia e Tecnologia, Portugal through grant SFRH/BPD/30239/2006 and project PTDC/QUI/64203/2006. The authors also acknowledge the ESRF, Grenoble (beamlines ID14- EH2, ID14- EH4, ID23- EH1 and ID-29) for access and technical support during data collection. Human SOUL (hSOUL) is a 23 kDa haem-binding protein that was first identified as the PP23 protein isolated from human full-term placentas. Here, the overexpression, purification and crystallization of hSOUL are reported. The crystals belonged to space group P6422, with unit-cell parameters a = b = 145, c = 60 Å and one protein molecule in the asymmetric unit. X-ray diffraction data were collected to 3.5 Å resolution at the ESRF. A preliminary model of the three-dimensional structure of hSOUL was obtained by molecular replacement using the structures of murine p22HBP (PDB codes 2gov and 2hva), obtained by solution NMR, as search models. publishersversion published

Machado-Joseph disease (MJD) is a fatal, dominant neurodegenerative disorder. MJD results from polyglutamine repeat expansion in the MJD-1 gene, conferring a toxic gain of function to the ataxin-3 protein. In this study, we aimed at overexpressing ataxin-3 in the rat brain using lentiviral vectors (LV), to generate an in vivo MJD genetic model and, to study the disorder in defined brain regions: substantia nigra, an area affected in MJD, cortex and striatum, regions not previously reported to be affected in MJD. LV encoding mutant or wild-type human ataxin-3 was injected in the brain of adult rats and the animals were tested for behavioral deficits and neuropathological abnormalities. Striatal pathology was confirmed in transgenic mice and human tissue. In substantia nigra, unilateral overexpression of mutant ataxin-3 led to: apomorphine-induced turning behavior; formation of ubiquitinated ataxin-3 aggregates; alpha-synuclein immunoreactivity; and loss of dopaminergic markers (TH and VMAT2). No neuropathological changes were observed upon wild-type ataxin-3 overexpression. Mutant ataxin-3 expression in striatum and cortex, resulted in accumulation of misfolded ataxin-3, and within striatum, loss of neuronal markers. Striatal pathology was confirmed by observation in MJD transgenic mice of ataxin-3 aggregates and substantial reduction of DARPP-32 immunoreactivity and, in human striata, by ataxin-3 inclusions, immunoreactive for ubiquitin and alpha-synuclein. This study demonstrates the use of LV encoding mutant ataxin-3 to produce a model of MJD and brings evidence of striatal pathology, suggesting that this region may contribute to dystonia and chorea observed in some MJD patients and may represent a target for therapies.

Mestrado em Engenharia Química Esta dissertação de mestrado integrado teve como principal objectivo a extracção de óleo de grainha de uva com dióxido de carbono supercrítico, tendo-se medido o rendimento da separação e avaliado a qualidade óleo obtido em termos de composição e concentração em triacilglicerídeos. Para além disso, com o intuito de aumentar o rendimento da extracção, foram realizados ensaios com a semente previamente tratada a pressão elevada. A modelação dos dados experimentais foi também efectuada. Para dar cumprimento aos objectivos citados, delineou-se o seguinte programa de trabalhos: (i) obter e preparar a semente (i.e., separação do mosto, lavagem, secagem, moagem e classificação por tamanhos); (ii) submeter a grainha a tratamentos a pressão elevada (HPP) e medir o rendimento da extracção via Soxhlet com n-hexano; (iii) efectuar a extracção supercrítica (SFE) do óleo de grainha de uva, utilizando amostras tratadas e não-tratadas a pressão elevada; (iv) determinar o perfil de triacilglicerídeos do óleo extraído por cromatografia em fase gasosa; (v) modelar os resultados experimentais. Primeiramente estudou-se o efeito da granulometria da semente moída no rendimento da extracção, tendo-se confirmado que é uma variável fundamental do processo: h ( p d = 0.75 mm) =11.59% e h ( p d = 0.85 mm) =6.72%. Das experiências de HPP concluiu-se que o rendimento final da extracção não é sensivelmente alterado para tratamentos até 3000 bar; a 5000 bar registou-se uma redução de 6.7% no valor de final h . As extracções supercríticas realizadas a 40 ºC e 180 bar com amostras de grainha tratada e não tratada a pressão elevada deram origem a rendimentos equivalentes aos do Soxhlet com n-hexano. Este facto mostrou que a SFE é um processo eficaz. As curvas cumulativas de extracção medidas com as duas amostras apresentaram os dois períodos típicos de extracção: no primeiro deles, aproximadamente linear, extraiu-se 90-92% do óleo disponível; o segundo, menos importante, atinge rapidamente um patamar. Comparando os primeiros períodos de extracção das duas SFE realizadas, constatou-se que a experiência conduzida com grainha pré-tratada a 1000 bar exibiu uma taxa de extracção 39.7% superior. Este facto permitiu concluir que, embora o HPP não afecte o rendimento final da extracção, a velocidade do processo é significativamente melhorada, permitindo reduzir o tempo necessário para remover a mesma quantidade de óleo. No que concerne à qualidade do óleo extraído, as análises mostraram que ele contém mais de 92% de triacilglicerídeos e que é particularmente rico em ácidos gordos insaturados (~80%), sendo os ácidos oleico (C18:1) e linoleico (C18:2) os mais abundantes (17-24% e 60-66%, respectivamente). A modelação da extracção supercrítica foi feita com base nos trabalhos de Perrut et al. (1997) e Sovová (2005). O modelo ajustou muito bem os dados experimentais, obtendose os erros absolutos médios seguintes: AAD(c/HPP) =2.36% e AAD(s/HPP) =2.72%. Com o modelo determinaram-se os perfis de concentração de óleo no leito e as curvas de eluição, a partir dos quais se calcularam os parâmetros fundamentais do processo. A zona de transferência de massa do leito, definida pelos limites de 5% e 95% da concentração máxima, corresponde a 7.2% (s/HPP) e a ~4.9% (c/HPP) do seu comprimento total. As velocidades de propagação das ondas de concentração através do leito foram 96 e 35 vezes superiores ao tempo de passagem do CO2. The main objective of this dissertation was the supercritical fluid extraction of grape seed oil using carbon dioxide, where the separation yield has been measured and the oil quality has been evaluated in terms of the composition and concentration of triacylglycerides. Furthermore, in order to increase the extraction yield, assays with pre-treated seed at high pressure have been carried out. With the purpose of accomplishing the cited objectives, the following working programme was established: (i) to obtain and prepare the seed (separation from musts, washing, drying, milling and classification); (ii) to carry out high pressure treatments (HPP) and measured the extraction yield via Soxhlet with n-hexane; (iii) to perform the supercritical fluid extraction (SFE) of the grape seed oil, using treated and untreated samples; (iv) to determine the triacylglycerides profile of the extracted oil by gas chromatography; (v) to model the experimental results. Firstly, the effect of the milled seed granulometry upon extraction yield has been studied, being confirmed that it is a fundamental process variable: h ( p d = 0.75 mm) =11.59% and h ( p d = 0.85 mm) =6.72%. From HPP experiments one concluded the final yield is not affected by treatments up to 3000 bar; at 5000 bar, final h was reduced by 6.7%. The supercritical fluid extractions carried out at 40 ºC and 180 bar with treated and untreated seed gave rise to yields equivalent to those obtained by Soxhlet using n-hexane, which proves the SFE is an efficient process. The cumulative extraction curves for both samples presented the two typical extraction periods: in the first one, approximately linear, it has been removed 90-92% of the available oil; the second, less important, reached a plateau rapidly. Comparing the first extraction periods of both SFEs performed, one concluded that the extraction rate for treated seed was 39.7% higher. Such fact allowed us to conclude that, although HPP does not influence final yield, the rate of the process is significantly improved, reducing the time needed to remove a fixed quantity of oil. With respect to the oil quality, the analysis showed it contains more than 92% of triacylglycerides and that it is particularly rich in unsaturated fatty acids (~80%), where oleic (C18:1) and linoleic (C18:2) acids are the most abundant (17-24% and 60-66%, respectively). Modelling has been performed on the basis of the works published by Perrut et al. (1997) and Sovová (2005). The model fitted data very well, giving rise to the following absolute average deviations: AAD(HPP) =2.36% e AAD(no HPP) =2.72%. With this model the concentration profiles in the bed and the breakthrough curves were determined, and the chief process parameters calculated. The mass transfer zone, defined using the limits of 5% and 95% of the maximum concentration, corresponds to 7.2% (no HPP) and ~4.9% (HPP) of the bed length. The velocities of the concentration waves through the bed were 96 and 35 times higher than the space time of CO2.

12 páginas, 5 figuras., Glutamate receptors of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type play an important role in synaptic plasticity and contribute to cell death under excitotoxic conditions. AMPA receptors form heterotetramers of four homologous subunits (GluR1–4), which exist in two functionally different isoforms, flip and flop, generated by alternative splicing. We identified transcripts for alternatively spliced isoforms of AMPA receptor subunits which lack both the flip and the flop exons, in hippocampal and retinal cultures. These transcripts originate AMPA receptor subunits lacking the flip/flop cassette, the fourth transmembrane domain and the intracellular C-terminus. Truncated GluR1 associates with full-length GluR1 and exerts a dominant negative effect, giving rise to non-functional receptors. Moreover, truncated GluR1 reaches the cell surface, but is not efficiently targeted to the synapse. Hippocampal neuronal transfection with truncated GluR1 resulted in a significant reduction in apoptotic neuronal death triggered by toxic concentrations of glutamate. Furthermore, mRNA coding for the truncated subunits is consistently detected in some regions of the brain in epileptic rats and in hippocampal neurons submitted to toxic concentrations of glutamate. The existence of truncated AMPA receptor subunits may constitute an intrinsic neuroprotective mechanism., This work was supported by Fundação para a Ciência e a Tecnologia, Portugal (SFRH/BD/1286/2000, POCTI/BCI/39127/ 2001 and POCI/SAU-NEU/58955/2004) and the BFU2006-07138 grant to JL.

Neuronal activity controls the strength of excitatory synapses by mechanisms that include changes in the postsynaptic responses mediated by AMPA receptors. These receptors account for most fast responses at excitatory synapses of the CNS, and their activity is regulated by various signaling pathways which control the electrophysiological properties of AMPA receptors and their interaction with numerous intracellular regulatory proteins. AMPA receptor phosphorylation/dephosphorylation and interaction with other proteins control their recycling and localization to defined postsynaptic sites, thereby regulating the strength of the synapse. This review focuses on recent advances in the understanding of the molecular mechanisms of regulation of AMPA receptors, and the implications in synaptic plasticity.

African swine fever virus (ASFV), a large enveloped DNA-containing virus, infects domestic and wild pigs, and multiplies in soft ticks, causing an economically relevant hemorrhagic disease. Evaluation of the nuclear import ability of ASFV p10 protein was the major purpose of the present work. Two approaches were used to determine if p10 protein is imported into the nucleus by an active process: a yeast-based nuclear import assay and the determination of the subcellular localization of p10 protein in mammalian cells by fluorescence microscopy. The results obtained clearly demonstrate that p10 protein is actively imported into the nucleus, both in yeast and mammalian cells. Experiments aiming at identifying the critical residues responsible for the nuclear import of ASFV p10 protein indicate that the amino acids comprised between the positions 71 and 77 are important, although not sufficient, for the protein active nuclear import. In ASFV-infected cells, the p10 protein strongly accumulates in the nucleus at late times post-infection, indicating that p10 protein may accomplish an important function inside the nucleus during the late phase of the viral replication cycle., This work was supported by a grant from the Portuguese Foundation for Science and Technology (POCTI/CVT/44854/2002). I. Nunes-Correia and A. Eulálio were recipients of fellowships from the Portuguese Foundation for Science and Technology. The work in V. Citovsky laboratory was supported by grants from the National Institutes of Health, National Science Foundation, US Department of Agriculture, US–Israel Binational Science Foundation (BSF), and US–Israel Binational Research and Development Fund (BARD). The work in M.L. Salas laboratory was supported by a grant from the Spanish Ministerio de Educatión y Ciencia (BFU2004-00298) and by an institutional grant from the Fundación Ramón Areces. J.M. Rodríguez was supported by the “Ramón y Cajal” program of the Ministerio de Educación y Ciencia.

The assembly of proteins that display complementary activities into macromolecular complexes is critical to cellular function. One such enzyme complex, of environmental significance, is the plant cell wall degrading apparatus of anaerobic bacteria, termed the cellulosome. The complex assembles through the interaction of enzyme-derived “type I dockerin” modules with the multiple “cohesin” modules of the scaffolding protein. Clostridium thermocellum type I dockerin modules contain a duplicated 22-residue sequence that comprises helix-1 and helix-3, respectively. The crystal structure of a C. thermocellum type I cohesin-dockerin complex showed that cohesin recognition was predominantly through helix-3 of the dockerin. The sequence duplication is reflected in near-perfect 2-fold structural symmetry, suggesting that both repeats could interact with cohesins by a common mechanism in wild-type (WT) proteins. Here, a helix-3 disrupted mutant dockerin is used to visualize the reverse binding in which the dockerin mutant is indeed rotated 180° relative to the WT dockerin such that helix-1 now dominates recognition of its protein partner. The dual binding mode is predicted to impart significant plasticity into the orientation of the catalytic subunits within this supramolecular assembly, which reflects the challenges presented by the degradation of a heterogeneous, recalcitrant, insoluble substrate by a tethered macromolecular complex.

Brain-derived neurotrophic factor (BDNF) plays an important role in synaptic plasticity in the hippocampus, but the mechanisms involved are not fully understood. The neurotrophin couples synaptic activation to changes in gene expression underlying long term potentiation and short term plasticity. Here we show that BDNF acutely up-regulates GluR1, GluR2, and GluR3 -amino-3-hydroxy-5-methyl-4- isoxazole propionic acid (AMPA) receptor subunits in 7-day tropomyosin-related kinase in vitro cultured hippocampal neurons. The increase in GluR1 and GluR2 protein levels in developing cultures was impaired by K252a, a Trk inhibitor, and by translation (emetine and anisomycin) and transcription ( -amanitine and actinomycin D) inhibitors. Accordingly, BDNF increased the mRNA levels for GluR1 and GluR2 subunits. Biotinylation studies showed that stimulation with BDNF for 30 min selectively increased the amount of GluR1 associated with the plasma membrane, and this effect was abrogated by emetine. Under the same conditions, BDNF induced GluR1 phosphorylation on Ser-831 through activation of protein kinase C and Ca2 -calmodulin-dependent protein kinase II. Chelation of endogenous extracellular BDNF with TrkB-IgG selectively decreased GluR1 protein levels in 14-day in vitro cultures of hippocampal neurons. Moreover, BDNF promoted synaptic delivery of homomeric GluR1 AMPA receptors in cultured organotypic slices, by a mechanism independent of NMDA receptor activation. Taken together, the results indicate that BDNF up-regulates the protein levels of AMPA receptor subunits in hippocampal neurons and induces the delivery of AMPA receptors to the synapse., This work was supported by Fundac¸a˜o para a Cieˆncia e a Tecnologia and Fundo Europeu de Desenvolvimento Regional (Grants POCTI/BCI/46466/ 2002 and SFRH/BD/9692/2002). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The periplasmic nitrate reductase from Cupriavidus necator (also known as Ralstonia eutropha) is a heterodimer that is able to reduce nitrate to nitrite. It comprises a 91 kDa catalytic subunit (NapA) and a 17 kDa subunit (NapB) that is involved in electron transfer. The larger subunit contains a molybdenum active site with a bis-molybdopterin guanine dinucleotide cofactor as well as one [4Fe-4S] cluster, while the small subunit is a di-haem c-type cytochrome. Crystals of the oxidized form of this enzyme were obtained using polyethylene glycol 3350 as precipitant. A single crystal grown at the High Throughput Crystallization Laboratory of the EMBL in Grenoble diffracted to beyond 1.5 A at the ESRF (ID14-1), which is the highest resolution reported to date for a nitrate reductase. The unit-cell parameters are a = 142.2, b = 82.4, c = 96.8 A, beta = 100.7 degrees, space group C2, and one heterodimer is present per asymmetric unit.

Polyglutamine expansion diseases are inherited neurodegenerative disorders caused by the expansion of CAG repeat mutations in the coding region of genes encoding for specific proteins, mostly of unknown function. One example is Machado-Joseph disease (MJD) or spinocerebellar ataxia 3, which was described in people of Portuguese descendents and is caused by expanded ataxin-3, a polyubiquitin-binding protein. Like other neurodegenerative diseases, MJD exhibits gradual progression of symptoms that finally result in the death of the patients. Despite the identification of the genetic defects, the molecular mechanisms by which the mutant protein initiates the pathogenic process remain to be elucidated. This chapter resumes some of the most important features of polyglutamine expansion diseases with a special emphasis on MJD pathogenesis. Particular relevance is given to ataxin-3 structure and function, the formation of aggregates of mutant ataxin-3, the characteristics of current disease animal models and the most recent therapeutic strategies proposed for the treatment of MJD.

NPY is present in the retina of different species but its role is not elucidated yet. In this work, using different rat retina in vitro models (whole retina, retinal cells in culture, microglial cell cultures, rat Müller cell line and retina endothelial cell line), we demonstrated that NPY staining is present in the retina in different cell types: neurons, macroglial, microglial and endothelial cells. Retinal cells in culture express NPY Y1, Y2, Y4 and Y5 receptors. Retina endothelial cells express all NPY receptors except NPY Y5 receptor. Moreover, NPY is released from retinal cells in culture upon depolarization. In this study we showed for the first time that NPY is present in rat retina microglial cells and also in rat Müller cells. These in vitro models may open new perspectives to study the physiology and the potential pathophysiological role of NPY in the retina. http://www.sciencedirect.com/science/article/B6T0B-4N0X5HW-3/1/e20d17a164f9188cfb71c43dcde59534