Divergence of chemical function in the alkaline phosphatase superfamily: Structure and mechanism of the P-C bond cleaving enzyme phosphonoacetate hydrolase

Phosphonates constitute a class of natural products that mimic the properties of the more common organophosphate ester metabolite yet are not readily degraded owing to the direct linkage of the phosphorus atom to the carbon atom. Phosphonate hydrolases have evolved to allow bacteria to utilize environmental phosphonates as a source of carbon and phosphorus. The work reported in this paper examines one such enzyme, phosphonoacetate hydrolase. By using a bioinformatic approach, we circumscribed the biological range of phosphonoacetate hydrolase to a select group of bacterial species from different classes of Proteobacteria. In addition, using gene context, we identified a novel 2-aminoethylphosphonate degradation pathway in which phosphonoacetate hydrolase is a participant. The X-ray structure of phosphonoformate-bound phosphonoacetate hydrolase was determined to reveal that this enzyme is most closely related to nucleotide pyrophosphatase/diesterase, a promiscuous two-zinc ion metalloenzyme of the alkaline phosphatase enzyme superfamily. The X-ray structure and metal ion specificity tests showed that phosphonoacetate hydrolase is also a two-zinc ion metalloenzyme. By using site-directed mutagenesis and P-32-labeling strategies, the catalytic nucleophile was shown to be Thr64. A structure-guided, site-directed mutation-based inquiry of the catalytic contributions of active site residues identified Lys126 and Lys128 as the most likely candidates for stabilization of the aci-carboxylate dianion leaving group. A catalytic mechanism is proposed which combines Lys12/Lys128 leaving group stabilization with zinc ion activation of the Thr64 nucleophile and the substrate phosphoryl group.

Extended contact effects as a function of closeness of relationship with ingroup contacts

Using survey data from Catholics and Protestants in Northern Ireland (N = 428), the authors examined the effects of extended contact via different types of ingroup contacts (neighbors, work colleagues, friends, and family members) and tested whether closeness to ingroup contacts moderated the effects of extended contact on outgroup trust. Results demonstrated that extended contact effects varied as a function of the relationship to ingroup contacts, and that extended contact interacted with closeness ratings in predicting outgroup trust. Consistent with hypotheses, extended contacts via more intimate ingroup relationships (i.e., friends and family) were overall more strongly related to outgroup trust than extended contacts via less intimate ingroup relations (i.e., neighbors and work colleagues). Moreover, within each level of intimacy extended contact was related to outgroup trust only at high, and not at low, levels of rated closeness to ingroup contacts. The theoretical contributions, limitations and practical implications of these findings are discussed.

Low pressure carbon dioxide solubility in pure electrolyte solvents for lithium-ion batteries as a function of temperature. Measurement and prediction

Experimental values for the carbon dioxide solubility in eight pure electrolyte solvents for lithium ion batteries – such as ethylene carbonate (EC), propylene carbonate (PC), dimethyl carbonate (DMC), ethyl methyl carbonate (EMC), diethyl carbonate (DEC), ?-butyrolactone (?BL), ethyl acetate (EA) and methyl propionate (MP) – are reported as a function of temperature from (283 to 353) K and atmospheric pressure. Based on experimental solubility data, the Henry’s law constant of the carbon dioxide in these solvents was then deduced and compared with reported values from the literature, as well as with those predicted by using COSMO-RS methodology within COSMOthermX software and those calculated by the Peng–Robinson equation of state implemented into Aspen plus. From this work, it appears that the CO2 solubility is higher in linear carbonates (such as DMC, EMC, DEC) than in cyclic ones (EC, PC, ?BL). Furthermore, the highest CO2 solubility was obtained in MP and EA solvents, which are comparable to the solubility values reported in classical ionicliquids. The precision and accuracy of the experimental values, considered as the per cent of the relative average absolute deviations of the Henry’s law constants from appropriate smoothing equations and from literature values, are close to (1% and 15%), respectively. From the variation of the Henry’s law constants with temperature, the partial molar thermodynamic functions of dissolution such as the standard Gibbs free energy, the enthalpy, and the entropy are calculated, as well as the mixing enthalpy of the solvent with CO2 in its hypothetical liquid state.

Visual function and dysfunction in early and late age-related maculopathy

Late age-related maculopathy (ARM) is responsible for the majority of blind registrations in the Western world among persons over 50 years of age. It has devastating effects on quality of life and independence and is becoming a major public health concern. Current treatment options are limited and most aim to slow progression rather than restore vision; therefore, early detection to identify those patients most suitable for these interventions is essential. In this work, we review the literature encompassing the investigation of visual function in ARM in order to highlight those visual function parameters which are affected very early in the disease process. We pay particular attention to measures of acuity, contrast sensitivity (CS), cone function, electrophysiology, visual adaptation, central visual field sensitivity and metamorphopsia. We also consider the impact of bilateral late ARM on visual function as well as the relationship between measures of vision function and self-reported visual functioning. Much interest has centred on the identification of functional changes which may predict progression to neovascular disease; therefore, we outline the longitudinal studies, which to date have reported dark-adaptation time, short-wavelength cone sensitivity, colour-match area effect, dark-adapted foveal sensitivity, foveal flicker sensitivity, slow recovery from glare and slower foveal electroretinogram implicit time as functional risk factors for the development of neovascular disease. Despite progress in this area, we emphasise the need for longitudinal studies designed in light of developments in disease classification and retinal imaging, which would ensure the correct classification of cases and controls, and provide increased understanding of the natural course and progression of the disease and further elucidate the structure-function relationships in this devastating disorder.

Low pressure carbon dioxide solubility in lithium-ion batteries based electrolytes as a function of temperature. Measurement and prediction

We present in this study the effect of nature and concentration of lithium salt, such as the lithium hexafluorophosphate, LiPF6; lithium tris(pentafluoroethane)-trifluorurophosphate LiFAP; lithium bis(trifluoromethylsulfonyl)imide, LiTFSI, on the CO2 solubility in four electrolytes for lithium ion batteries based on pure solvent that include ethylene carbonate (EC), dimethyl carbonate (DMC), ethyl methyl carbonate (EMC), diethyl carbonate (DEC), as well as, in the EC:DMC, EC:EMC and EC:DEC (50:50) wt.% binary mixtures as a function of temperature from (283 to 353) K and atmospheric pressure. Based on experimental solubility values, the Henry’s law constant of the carbon dioxide in these solutions with the presence or absence of lithium salt was then deduced and compared with reported values from the literature, as well as with those predicted by using COSMO-RS methodology within COSMOThermX software. From this study, it appears that the addition of 1 mol · dm-3 LiPF6 salt in alkylcarbonate solvents decreases their CO2 capture capacity. By using the same experimental conditions, an opposite CO2 solubility trend was generally observed in the case of the addition of LiFAP or LiTFSI salts in these solutions. Additionally, in all solutions investigated during this work, the CO2 solubility is greater in electrolytes containing the LiFAP salt, followed by those based on the LiTFSI case. The precision and accuracy of the experimental data reported therein, which are close to (1 and 15)%, respectively. From the variation of the Henry’s law constant with temperature, the partial molar thermodynamic functions of dissolution such as the standard Gibbs energy, the enthalpy, and the entropy, as well as the mixing enthalpy of the solvent with CO2 in its hypothetical liquid state were calculated. Finally, a quantitative analysis of the CO2 solubility evolution was carried out in the EC:DMC (50:50) wt.% binary mixture as the function of the LiPF6 or LiTFSI concentration in solution to elucidate how ionic species modify the CO2 solubility in alkylcarbonates-based Li-ion electrolytes by investigating the salting effects at T = 298.15 K and atmospheric pressure.

The IQGAP-myosin light chain interaction: what is its function?

The first members of the IQGAP family of proteins were
characterised over 15 years ago. It is now known that these molecules act
at the interface between cellular signalling pathways and the actin
cytoskeleton. They bind to a diverse range of signalling molecules –
including those involved in calcium, GTPase, kinase and growth factor
signalling. One intriguing interaction is that between mammalian
IQGAP1 and the myosin essential light chain isoform, Mlc1sa. Although
this has been demonstrated in vitro, its in vivo role is not known. Indeed,
it would be tempting to dismiss it as an experimental artefact, except for
the existence of a parallel interaction in the budding yeast,
Saccharomyces cerevisae. In this organism, the IQGAP-like protein
(Iqg1p) interacts with a myosin essential light chain (Mlc1p). This interaction is critical for the correct execution of cytokinesis. IQGAP-like
proteins also play key roles in cytokinesis in other fungi. Recent work
implicating mammalian IQGAP1 in cytokinesis may help explain the role
of the interaction in higher eukarytotes.

Detecting the work statistics through Ramsey-like interferometry

Out-of-equilibrium statistical mechanics is attracting considerable interest due to the recent advances in the control and manipulations of systems at the quantum level. Recently, an interferometric scheme for the detection of the characteristic function of the work distribution following a time-dependent process has been proposed [L. Mazzola et al., Phys. Rev. Lett. 110 (2013) 230602]. There, it was demonstrated that the work statistics of a quantum system undergoing a process can be reconstructed by effectively mapping the characteristic function of work on the state of an ancillary qubit. Here, we expand that work in two important directions. We first apply the protocol to an interesting specific physical example consisting of a superconducting qubit dispersively coupled to the field of a microwave resonator, thus enlarging the class of situations for which our scheme would be key in the task highlighted above. We then account for the interaction of the system with an additional one (which might embody an environment), and generalize the protocol accordingly.

Low pressure methane solubility in lithium-ion batteries based solvents and electrolytes as a function of temperature. Measurement and prediction

The methane solubility in five pure electrolyte solvents and one binary solvent mixture for lithium ion batteries – such as ethylene carbonate (EC), propylene carbonate (PC), dimethyl carbonate (DMC), ethyl methyl carbonate (EMC), diethyl carbonate (DEC) and the (50:50 wt%) mixture of EC:DMC was studied experimentally at pressures close to atmospheric and as a function of temperature between (280 and 343) K by using an isochoric saturation technique. The effect of the selected anions of a lithium salt LiX (X = hexafluorophosphate,

INFLUENCE OF NOX4 NADPH OXIDASE ON GENERATION AND ANGIOGENIC FUNCTION OF INDUCED PLURIPOTENT STEM CELL-DERIVED ENDOTHELIAL CELLS

Human induced pluripotent stem (iPS) cell-derived endothelial cells (ECs) hold clear potential for therapeutic angiogenesis as a novel strategy for ischaemic disease. Recently, we have developed a novel method for direct reprogramming of partial iPS (PiPS) cells, which unlike iPS cells, are generated before pluripotency so do not form tumours, and may be differentiated into ECs with characteristic morphology and pro-angiogenic actions. Our previous work showed that PiPS-derived ECs are capable of forming vascular-like tubes both in vitro and in vivo and promoting re-endothelialisation of ischemic tissue, with greater effectiveness versus mature ECs.

Interestingly, our preliminary data demonstrate that Nox NADPH oxidases, which are reported to influence stem cell function, are progressively induced during PiPs/PiPS-EC differentiation and in response to hypoxia, with Nox4 demonstrating highest expression. As this isoform is an established regulator of angiogenesis, we hypothesize that Nox4 plays a key role in modulating PiPS-EC generation and angiogenic function.

The aim of this project is therefore to investigate: (1) the specific role of Nox4 in direct reprogramming of PiPS cells and differentiation to PiPS-ECs; (2) whether genetic manipulation of Nox4 influences in vitro function of PiPs-ECs and their ability to promote in vivo angiogenesis. This will be achieved by employing established in vitro functional assays and an experimental model of hindlimb ischaemia with assessment of relevant end-points. Identification of a key role for Nox4 in regulating PiPS-EC generation/function may inform selective targeting of this isoform to enhance the efficiency of PiPS-EC differentiation and their capacity to treat ischemic disease.

Theorizing temporary spatial clusters and institutional boundary-work in industrial markets

To better understand the nature of temporary spatial clusters (TSC’s) in industrial marketing settings, this conceptual paper first provides a theoretical synthesis of spatial understanding from the industrial marketing (IM) and economic geography (EG) fields, focusing particularly on Doreen Massey’s work on relational space. This leads to a conceptual schema for organizing the IM literature in terms of spatiality, and which also helps clarify the ontological nature of TSCs. We then move to introduce the notion of institutional boundary-work, drawing on the work of Thomas Gieryn, and Andrea Brighenti’s examination of territorology, to conceptualize the activities of market actors engaged in the ongoing social accomplishment of TSCs. Such activities, we suggest, involve these actors ‘marching’ boundaries to assume network influence and maintain market order in IM settings. In summary, therefore, our paper addresses two fundamental questions: i) How do we conceptualize the form of TSCs in IM settings? And, ii) what function(s) are TSCs performing (and how is this being undertaken) in IM? The paper closes by providing methodological guidance for how a research agenda on TSCs within IM activity might be developed, followed by a summary of the managerial implications that emerge from our theorizations.

Soil function and biodiversity: regional variations and climate changes

Embora o objetivo principal da proteção internacional dos solos seja proteger tanto as funções quanto a estrutura do solo, a atual abordagem trata principalmente da proteção ao nível estrutural. Há uma carência de estudos que contemplem a ligação das funções do solo com os níveis da comunidade. Além disso, é ainda desconhecido se as variáveis ambientais (ex: tipos de solo, condições climáticas) atuam nas funções do solo da mesma maneira que influenciam sua estrutura biológica. Ademais, as alterações climáticas poderão ter sozinhas ou combinadas com os poluentes, um grande efeito nos ecossistemas terrestres. O presente trabalho propõe estudar as funções e a estrutura biológica do solo quando impactados devido a estresse tóxico (poluição por Cu) e/ou alterações a fatores como a temperatura e abundância de organismos, de maneira a simular possíveis variações regionais ou climáticas. Para alcançar os objetivos principais 3 experiências utilizando diferentes densidades de E. crypticus e 2 gerações foram feitas (Capítulos II e III). Duas experiências com mesocosmos (SMS) decorreram durante 3 meses sob uma gama de diversas temperaturas (10 – 29°C), que representam temperaturas médias para Portugal e Dinamarca (Capítulos IV e V). Duas experiências de campo também foram realizadas com intuito de validar os SMSs (Capítulo VI). Resultados demonstraram que os efeitos do Cu na reprodução dos enquitraídeos dependem da densidade inicial de organismos, especialmente na 2ª geração. Entretanto, nos SMSs expostos a Cu, a densidade inicial é menos importante nos resultados finais. O aumento da temperatura alterou majoritariamente a fase inicial de crescimento populacional. Em períodos mais longos, a abundância estabilizou tornando-se menos influenciada pelas temperaturas. Períodos longos de exposição reforçaram os efeitos da temperatura, como por ex: diversas espécies foram similarmente afetadas a 29 ou 26°C quando expostas durante 28 ou 61 dias respectivamente. De forma geral, o Cu reduziu a abundância da maioria das espécies ao longo do tempo, com poucas exceções. Os resultados da decomposição da matéria orgânica (MO) e atividade alimentar associaram-se com a abundância de organismos em baixas temperaturas (10-23°C). Entretanto, com o aumento das temperaturas (19-29°C), este comportamento não foi claro e a abundância de espécies e atividade alimentar diminuíram enquanto a decomposição da MO aumentou. Além disso, os resultados observados nos SMSs foram confirmados no campo. Mais especificamente, alterações ocorreram na fase de crescimento (correspondente à Primavera) e a exposição ao Cu diminuiu os efeitos da temperatura. Metodologias mais complexas (ex: mais gerações e experiências com múltiplas espécies) apresentam muitos benefícios, mas também proporcionam respostas mais complexas, as quais exigem um maior “peso” de evidências para serem comprovadas.

‘It's been hell.’ Italian and British practice educators’ narratives of working with struggling or failing social work students in practice learning settings

Periods of assessed learning in practice settings are common requirements for social work students world wide. The ‘practice learning opportunity’ as it is known in the UK, and ‘tirocinio di servizio sociale’ as it is referred to in Italy, are important sites of gatekeeping in preventing unsuitable people from becoming social workers. The experience of assessing failing students in practice learning settings however, has been found to be particularly stressful and challenging for practice educators. This article documents findings from two qualitative studies that explored field educators’ experiences of working with struggling or failing social work students in Italy and England. The study finds both similarities and differences in the narratives of the assessors from the two countries Similarities include, unpleasant emotional experience of working with a failing student, internalisation of the students failing as the practice educators’ own failing, perceptions that the universities may hide negative information about students and lack of acknowledgement of the gatekeeping function inherent in the practice educator role. Differences include the level of emotionality experienced by educators, the way students are spoken about and the perceived role and responses of the university. Further comparative European research which focuses on practice education is indicated.

Brain-derived neurotrophic factor and adenosine signalling on amyloid-β peptide induced toxicity : impact on hippocampal function

Tese de doutoramento, Ciências Biomédicas (Neurociências), Universidade de Lisboa, Faculdade de Medicina, 2014

Biological membranes : biophysical properties and aquaporin function

Tese de doutoramento, Farmácia (Bioquímica), Universidade de Lisboa, Faculdade de Farmácia, 2014

Synaptic dysfunction in early encephalopathies : from genes to function

Tese de doutoramento, Medicina (Neurologia), Universidade de Lisboa, Faculdade de Medicina, 2015