Blocking effects of polyunsaturated fatty acids on Na+ channels of neonatal rat ventricular myocytes.

Recent evidence indicates that polyunsaturated long-chain fatty acids (PUFAs) prevent lethal ischemia-induced cardiac arrhythmias in animals and probably in humans. To increase understanding of the mechanism(s) of this phenomenon, the effects of PUFAs on Na+ currents were assessed by the whole-cell patch-clamp technique in cultured neonatal rat ventricular myocytes. Extracellular application of the free 5,8,11,14,17-eicosapentaenoic acid (EPA) produced a concentration-dependent suppression of ventricular, voltage-activated Na+ currents (INa). After cardiac myocytes were treated with 5 or 10 microM EPA, the peak INa (elicited by a single-step voltage change with pulses from -80 to -30 mV) was decreased by 51% +/- 8% (P < 0.01; n = 10) and 64% +/- 5% (P < 0.001; n = 21), respectively, within 2 min. Likewise, the same concentrations of 4,7,10,16,19-docosahexaenoic acid produced the same inhibition of INa. By contrast, 5 and 10 microM arachidonic acid (AA) caused less inhibition of INa, but both n - 6 and n - 3 PUFAs inhibited INa significantly. A monounsaturated fatty acid and a saturated fatty acid did not. After washing out EPA, INa returned to the control level. Raising the concentration of EPA to 40 microM completely blocked INa. The IC50 of EPA was 4.8 microM. The inhibition of this Na+ channel was found to be dose and time, but not use dependent. Also, the EPA-induced inhibition of INa was voltage dependent, since 10 microM EPA produced 83% +/- 7% and 29% +/- 5% inhibition of INa elicited by pulses from -80 to -30 mV and from -150 to -30 mV, respectively, in single-step voltage changes. A concentration of 10 microM EPA shifted the steady-state inactivation curve of INa by -19 +/- 3 mV (n = 7; P < 0.01). These effects of PUFAs on INa may be important for their antiarrhythmic effect in vivo.

Thrombospondin 1 expression in transformed endothelial cells restores a normal phenotype and suppresses their tumorigenesis.

Murine endothelial cells are readily transformed in a single step by the polyomavirus oncogene encoding middle-sized tumor antigen. These cells (bEND.3) form tumors (hemangiomas) in mice which are lethal in newborn animals. The bEND.3 cells rapidly proliferate in culture and express little or no thrombospondin 1 (TS1). To determine the role of TS1 in regulation of endothelial cell phenotype, we stably transfected bEND.3 cells with a human TS1 expression vector. The cells expressing human TS1 were readily identified by their altered morphology and exhibited a slower growth rate and lower saturation density than the parental bEND.3 cells. The TS1-expressing cells also formed aligned cords of cells instead of clumps or cysts in Matrigel. Moreover, while the bEND.3 cells formed large tumors in nude mice within 48 hr, the TS1-expressing cells failed to form tumors even after 1 month. The TS1-transfected cells expressed transforming growth factor beta mRNA and bioactivity at levels similar to those of the parental or vector-transfected bEND.3 cells, indicating that the effects of TS1 expression are not due to the activation of transforming growth factor beta by TS1. TS1 expression resulted in a > 100-fold decrease in net fibrinolytic (urokinase-type plasminogen activator, uPA) activity due to more plasminogen-activator inhibitor 1 and less uPA secretion. TS1 thus appears to be an important regulator of endothelial cell phenotype required for maintaining the quiescent, differentiated state.

Mechanisms underlying the sensitivity of songbird forebrain neurons to temporal order.

Neurons in the songbird forebrain area HVc (hyperstriatum ventrale pars caudale or high vocal center) are sensitive to the temporal structure of the bird's own song and are capable of integrating auditory information over a period of several hundred milliseconds. Extracellular studies have shown that the responses of some HVc neurons depend on the combination and temporal order of syllables from the bird's own song, but little is known about the mechanisms underlying these response properties. To investigate these mechanisms, we recorded intracellular responses to a set of auditory stimuli designed to assess the degree of dependence of the responses on temporal context. This report provides evidence that HVc neurons encode information about temporal structure by using a variety of mechanisms including syllable-specific inhibition, excitatory postsynaptic potentials with a range of different time courses, and burst-firing nonlinearity. The data suggest that the sensitivity of HVc neurons to temporal combinations of syllables results from the interactions of several cells and does not arise in a single step from afferent inputs alone.

Os muitos nomes de Silvana: contribuições clínico-políticas da psicanálise sobre mulheres negras

Esta pesquisa aponta alguns dos efeitos subjetivos e estratégias singulares de resistência frente à desigualdade racial no nosso país, abordando as vicissitudes de inscrição no laço social de mulheres negras e pobres. É fruto de uma intervenção clínico-política com um grupo de adolescentes em uma Escola Municipal de Ensino Fundamental de São Paulo na qual foi se evidenciando, para nós, a necessidade de cada um desses adolescentes de defender intransigentemente a honra e o valor de suas mães frente aos outros membros do grupo. Tanto pelo seu excesso como pela sua repetição, essa situação nos sugeria um mal-estar e um não dito referido às configurações familiares e à posição destas mulheres nesta comunidade escolar, que nos levou a escutá-las. Tomando a indicação freudiana de que a psicologia individual seria também psicologia social e a formulação lacaniana de que podemos considerar o Inconsciente como sendo a Política, acreditamos ser indispensável escutar o sujeito levando em consideração o Outro, entendido tanto do ponto de vista sócio-histórico, como libidinal. Isso significa que não poderíamos escutar estas mulheres sem considerar o campo de desigualdades sociais e raciais no qual estavam inscritas discursivamente, o que nos exigiu uma interlocução fundamental tanto com pesquisas da antropologia social e da sociologia, como da história. A fala destas mulheres foi nos revelando que, além de outras identificações contingentes, o fato de serem reconhecidas e se reconhecerem como mulheres negras era um elemento fundamental nas suas vivências cotidianas. Uma vez que nosso passado escravista não teria sido suficientemente lembrado e admitido, alguns traços se fariam presentes através de uma transmissão simbólica, pelos subterrâneos da cultura, de uma posição de servidão a elas atribuída. Permaneceria de uma forma atualizada e insidiosa uma divisão racializada da nossa sociedade, ancorada na herança de uma cisão entre a mulher mundana cujo corpo seria visto como um corpo de gozo, mas sem valor social, a mucama, e a que seria valorizada socialmente à custa de um corpo assexuado, casta e educada, esposa do senhor de escravos. Apesar de tantos avanços, as conquistas femininas das últimas décadas não seriam totalmente estendidas a essas mulheres, negras e pobres, que seguiriam, frequentemente, apresentando no imaginário social um corpo ao qual se atribuiria a capacidade de satisfazer os desejos mais inconfessáveis de um homem à custa de ser visto como propriedade e domínio deste. A atitude racista se faria presente em relação a elas, entendida como o ato de segregação do gozo inadmitido de um sujeito no corpo de um outro, ou ainda, como Lacan apontou, impondo a um outro, seu modo de gozo. Mais do que uma identidade das mulheres negras, consideramos fundamental conceber a particularidade de um laço que se estabeleceria na relação com elas, na medida em que seu corpo seria capaz de despertar e revelar a relação do sujeito com o mais íntimo e insuportável de si mesmo: ela seria a estrangeira frente a um homem, por ser mulher; e seria estrangeira frente a uma mulher ou homem branco, por ser negra. A sua condição de estrangeira a deixaria assim como figura paradigmática de um Outro sexo, um sexo Outro, um gozo Outro, recaindo sobre ela as reações mais violentas de extirpação desse gozo. As estratégias de como manter o que seria próprio do gozo feminino não balizado pelo gozo fálico, posto que seria suplementar a ele frente a essa injunção de segregação e depreciação, seriam sempre singulares. Apresentamos um caso clínico, Silvana, apontando suas estratégias de resistência frente a um discurso social que a desqualificaria tentando lhe impor um estreitamento de sua vida erótica e sua redução a um modo único de gozo

Remote Control Electrodeposition: Patterning on Substrates without Direct Electrical Connections

Thesis (Ph.D.)--University of Washington, 2016-06

Growing semiconductor nanocrystals directly in a conducting polymer

We describe a single step method to synthesise lead sulphide (PbS) nanocrystals directly in the conjugated polymer poly (2-methoxy-5-(2'-ethyl-hexyloxy)-p-phenylene vinylene) (MEH-PPV). This method allows size control of the nanocrystal via co-solvent ratios. We find good agreement between nanocrystal sizes determined by transmission electron microscopy and sizes theoretically determined from the absorption edge of the nanocrystals. Finally we show that this synthesis technique is not restricted to MEH-PPV and demonstrate that nanocrystals can be grown in Poly(3-hexylthiophene-2,5-diyl) (P3HT). (C) 2005 Elsevier B.V. All rights reserved.

Mechanisms of acetohydroxyacid synthases

Acetohydroxyacid synthases are thiamin diphosphate- (ThDP-) dependent biosynthetic enzymes found in all autotrophic organisms. Over the past 4-5 years, their mechanisms have been clarified and illuminated by protein crystallography, engineered mutagenesis and detailed single-step kinetic analysis. Pairs of catalytic subunits form an intimate dimer containing two active sites, each of which lies across a dimer interface and involves both monomers. The ThDP adducts of pyruvate, acetaldehyde and the product acetohydroxyacids can be detected quantitatively after rapid quenching. Determination of the distribution of intermediates by NMR then makes it possible to calculate individual forward unimolecular rate constants. The enzyme is the target of several herbicides and structures of inhibitor-enzyme complexes explain the herbicide-enzyme interaction.

The production of male of-only offspring in beef cattle - a proof of principle project

The Australian beef industry places the greatest value in bulls, in comparison to cows, for prime beef production. Male carcasses can be sold for a larger profit due to their increased muscle mass. This project aims to demonstrate the feasibility of producing male animals that can sire male only offspring, through a transgenic approach in mice that could later be translated into livestock production systems. The mouse Sry (Sex determining region on the Y) gene has been shown to provide the initiating molecular signal leading to male sex determination in mammals. Sry has also been shown to cause sex reversal in XX mice transgenic for the gene. In this project Sry will be targeted to a locus not subject to X-inactivation on the X chromosome of XY mice. These mice will be bred to determine how the transgene is passed on, to determine expression of the transgene, and to assess its activity in causing XX sex reversal. The male mice transgenic for the Sry gene on their X chromosome will be produced using tetraploid aggregation, which in a single step produces 100% ES cell derived embryos. The same target locus can later be used to introduce the bovine SRY gene onto the X chromosome of bovidae species and using germ cell transplantation produce sex reversed animals. This would bypass the need for expensive chimera crosses and provide farmers with a stud bull capable of producing only sons.

On the relative complexity of labelled modal tableaux

We investigate the relative complexity of two free-variable labelled modal tableaux(KEM and Single Step Tableaux, SST). We discuss the reasons why p-simulation is not a proper measure of the relative complexity of tableaux-like proof systems, and we propose an improved comparison scale (p-search-simulation). Finally we show that KEM p-search-simulates SST while SST cannot p-search-simulate KEM.

Natural gradient matrix momentum

Natural gradient learning is an efficient and principled method for improving on-line learning. In practical applications there will be an increased cost required in estimating and inverting the Fisher information matrix. We propose to use the matrix momentum algorithm in order to carry out efficient inversion and study the efficacy of a single step estimation of the Fisher information matrix. We analyse the proposed algorithm in a two-layer network, using a statistical mechanics framework which allows us to describe analytically the learning dynamics, and compare performance with true natural gradient learning and standard gradient descent.

Switching the mode of metabolism in the yeast Saccharomyces cerevisiae

The biochemistry of most metabolic pathways is conserved from bacteria to humans, although the control mechanisms are adapted to the needs of each cell type. Oxygen depletion commonly controls the switch from respiration to fermentation. However, Saccharomyces cerevisiae also controls that switch in response to the external glucose level. We have generated an S. cerevisiae strain in which glucose uptake is dependent on a chimeric hexose transporter mediating reduced sugar uptake. This strain shows a fully respiratory metabolism also at high glucose levels as seen for aerobic organisms, and switches to fermentation only when oxygen is lacking. These observations illustrate that manipulating a single step can alter the mode of metabolism. The novel yeast strain is an excellent tool to study the mechanisms underlying glucose-induced signal transduction. © 2004 European Molecular Biology Organization.

Generation of primary hepatocyte microarrays by piezoelectric printing

We demonstrate a single-step method for the generation of collagen and poly-l-Lysine (PLL) micropatterns on a poly(ethylene glycol) (PEG) functionalized glass surface for cell based assays. The method involves establishing a reliable silanization method to create an effective non-adhesive PEG layer on glass that inhibits cell attachment, followed by the spotting of collagen or PLL solutions using non-contact piezoelectric printing. We show for the first time that the spotted protein micropatterns remain stable on the PEG surface even after extensive washing, thus significantly simplifying protein pattern formation. We found that adherence and spreading of NIH-3T3 fibroblasts was confined to PLL and collagen areas of the micropatterns. In contrast, primary rat hepatocytes adhered and spread only on collagen micropatterns, where they formed uniform, well defined functionally active cell arrays. The differing affinity of hepatocytes and NIH-3T3 fibroblasts for collagen and PLL patterns was used to develop a simple technique for creating a co-culture of the two cell types. This has the potential to form structured arrays that mimic the in vivo hepatic environment and is easily integrated within a miniaturized analytical platform for developing high throughput toxicity analysis in vitro.

Combined bioreaction and separation in centrifugal fields

The aim of this work has been to investigate the principle of combined centrifugal bioreaction-separation. The production of dextran and fructose by the action of the enzyme dextransucrase on sucrose was employed to elucidate some of the principles of this type of process. Dextran is a valuable pharmaceutical product used mainly as a blood volume expander and blood flow improver whilst fructose is an important dietary product. The development of a single step process capable of the simultaneous biosynthesis of dextran and the separation of the fructose by-product should improve dextran yields whilst reducing capital and processing costs. This thesis shows for the first time that it is possible to conduct successful bioreaction-separations using a rate-zonal centrifugation technique. By layering thin zones of dextrasucrase enzyme onto sucrose gradients and centrifuging, very high molecular weight (MW) dextran-enzyme complexes were formed that rapidly sedimented through the sucrose substrate gradients under the influence of the applied centrifugal field. The low MW fructose by-product sedimented at reduced rates and was thus separated from the enzyme and dextran during the reaction. The MW distribution of dextran recovered from the centrifugal bioreactor was compared with that from a conventional batch bioreactor. The results indicated that the centrifugal bioreactor produced up to 100% more clinical dextran with MWs of between 12 000 and 98 000 at 20% w/w sucrose concentrations than conventional bioreactors. This was due to the removal of acceptor fructose molecules from the sedimenting reaction zone by the action of the centrifugal field. Higher proportions of unwanted lower MW dextran were found in the conventional bioreactor than in the centrifugal bioreactor-separator. The process was studied on a number of alternative centrifugal systems. A zonal rotor fitted with a reorienting gradient core proved most successful for the evaluation of bioreactor performance. Results indicated that viscosity build-up in the reactor must be minimised in order to increase the yields of dextran per unit time and improve product separation. A preliminary attempt at modelling the process has also been made.

Novel bioadhesive formulations for mucosal and parenteral delivery of vaccines

In this work we have established the efficient mucosal delivery of vaccines using absorption enhancers and chitosan. In addition, the use of chitosan was shown to enhance the action of other known adjuvants, such as CTB or Quil-A. Collectively, the results presented herein indicate that chitosan has excellent potential as a mucosal adjuvant. We have evaluated a number of absorption enhancers for their adjuvant activity in vivo. Polyornithine was shown to engender high scrum immune reasons to nasally delivered antigens, with higher molecular weight polyornithine facilitating the best results. We have demonstrated for the first time that vitamin E TPGS can act as mucosal adjuvant. Deoxycholic acid, cyclodextrins and acylcarnitines were also identified as effective mucosal adjuvants and showed enhanced immune responses to nasally delivered TT, DT and Yersinia pestis V and F1 antigens. Previously, none of these agents, common in their action as absorption enhancing agents, have been shown to have immunopotentiating activity for mucosal immunisation. We have successfully developed novel surface modified microspheres using chitosan as an emulsion stabiliser during the preparation of PLA microspheres. It was found that immune responses could be substantially increased, effectively exploiting the immunopenetrating characteristics of both chitosan and PLA microspheres in the same delivery vehicle. In the same study, comparison of intranasal and intramuscular routes of administration showed that with these formulations, the nasal route could be as effective as intramuscular delivery, highlighting the potential of mucosal administration for these particulate delivery systems. Chitosan was co-administered with polymer microspheres. It was demonstrated that this strategy facilitates markedly enhanced immune responses in both magnitude and duration following intramuscular administration. We conclude that this combination shows potential for single dose administration of vaccines. In another study, we have shown that the addition of chitosan to alum adsorbed TT was able to enhance immune responses. PLA micro/nanospheres were prepared and characterised with discreet particle size ranges. A smaller particle size was shown to facilitate higher scrum IgG responses following nasal administration. A lower antigen loading was additionally identified as being preferential for the induction of immune responses in combination with the smaller particle size. This may be due to the fact that the number of particles will be increased when antigen loading is low, which may in turn facilitate a more widespread uptake of particles. PLA lamellar particles were prepared and characterised. Adsorbed TT was evaluated for the potential to engender immune responses in vivo. These formulations were shown to generate effective immune responses following intramuscular administration. Positively charged polyethylcyanoacrylate and PLA nanoparticies were designed and characterised and their potential as delivery vehicles for DNA vaccines was investigated. Successful preparation of particles with narrow size distribution and positive surface charge (imparted by the inclusion of chitosan) was achieved. In the evaluation of antibody responses to DNA encoded antigen in the presence of alum administered intranasally, discrimination between the groups was only seen following intramuscular boosting with the corresponding protein. Our study showed that DNA vaccines in the presence of either alum or Quil-A may advantageously influence priming of the immune system by a mucosal route. The potential for the combination of adjuvants, Quil-A and chitosan, to enhance antibody responses to plasmid encoded antigen co-administered with the corresponding protein antigen was shown and this is worthy of further investigation. The findings here have identified novel adjuvants and approaches to vaccine delivery. In particular, chitosan or vitamin E TPGS are shown here to have considerable promise as non-toxic, safe mucosal adjuvants. In addition, biodegradable mucoadhesive delivery systems, surface modified with chitosan in a single step process, may have application for other uses such as drug and gene delivery.

Femtosecond laser inscribed superstructure fibre gratings

We demonstrate the development of femtosecond laser inscribed superstructure fiber gratings (fsSFG) in silica optical fibre. We utilise a single step process, to inscribe low loss and polarisation independent, sampled gratings in optical fibres using the point by point femtosecond laser inscription method. Our approach results in a controlled modulated index change with complete suppression of any overlapping LPG structure leading to highly symmetric superstructure spectra, with the grating reflection well within the Fourier design limit. We also solve Maxwell's equations and calculate the back reflection spectrum using the bidirectional beam propagation method (BiBPM). Experimental results validate our numerical analysis and the estimation of inscription parameters such as ac index modulation, wavelength and the relative peak strength. We also explore how changes in the grating's period influence the reflection spectrum.