Universal tight binding model for chemical reactions in solution and at surfaces. I. Organic molecules

As is now well established, a first order expansion of the Hohenberg-Kohn total energy density functional about a trial input density, namely, the Harris-Foulkes functional, can be used to rationalize a non self consistent tight binding model. If the expansion is taken to second order then the energy and electron density matrix need to be calculated self consistently and from this functional one can derive a charge self consistent tight binding theory. In this paper we have used this to describe a polarizable ion tight binding model which has the benefit of treating charge transfer in point multipoles. This admits a ready description of ionic polarizability and crystal field splitting. It is necessary in constructing such a model to find a number of parameters that mimic their more exact counterparts in the density functional theory. We describe in detail how this is done using a combination of intuition, exact analytical fitting, and a genetic optimization algorithm. Having obtained model parameters we show that this constitutes a transferable scheme that can be applied rather universally to small and medium sized organic molecules. We have shown that the model gives a good account of static structural and dynamic vibrational properties of a library of molecules, and finally we demonstrate the model's capability by showing a real time simulation of an enolization reaction in aqueous solution. In two subsequent papers, we show that the model is a great deal more general in that it will describe solvents and solid substrates and that therefore we have created a self consistent quantum mechanical scheme that may be applied to simulations in heterogeneous catalysis.

Evaluation of the Factors Contributing to Levonorgestrel Binding in Addition Cure Silicone Elastomer Vaginal Rings

Background: Following progress of the dapivirine (DPV)-releasing silicone elastomer (SE) vaginal ring (VR) into Phase III clinical studies, there is now interest in developing next-generation rings that additionally provide contraception. Levonorgestrel (LNG) is a safe and effective progestin that is being widely considered for use as a hormonal contraceptive agent in future multipurpose prevention technology (MPT) products. Although LNG has previously been incorporated into various controlled release SE devices, minimal attention has focused on its propensity to irreversibly react with addition cure SE systems. Here, for the first time, we investigate this LNG binding phenomenon and outline strategies for overcoming it.
Methods: VRs containing various loadings of DPV and LNG were manufactured and in vitro release assessed. Different LNG-only SE samples were also prepared to assess the following parameters: (i) addition cure vs. condensation cure SEs; (ii) different types of addition cure SEs; (iii) mixing time, (iv) cure temperature, (v) cure time; and (vi) LNG particle size. After manufacture, the LNG-only samples were assayed for total drug content using a solvent extraction method. The SE curing reaction and the LNG binding reaction was probed using nuclear magnetic resonance (NMR) spectroscopy. Results:
Under certain drug/formulation/processing conditions, LNG was not recoverable from VRs. Further studies using non-ring samples showed that: (a) the phenomenon was only observed with addition cure SEs (and not condensation cure SEs); (b) the extent of binding was dependent upon the type of addition cure SE; (c) micronised LNG showed significantly greater binding than non-micronised LNG; (d) the extent of binding correlated with increased mixing time, cure time and cure temperature.
Conclusions: Careful control of the API characteristics, the SE composition, and the manufacturing conditions will be necessary to establish a practical VR formulation for controlled release of LNG.

Comparative biochemical analysis of three members of the Schistosoma mansoni TAL family: Differences in ion and drug binding properties

The tegumental allergen-like (TAL) proteins from Schistosoma mansoni are part of a family of calcium binding proteins found only in parasitic flatworms. These proteins have attracted interest as potential drug or vaccine targets, yet comparatively little is known about their biochemistry. Here, we compared the biochemical properties of three members of this family: SmTAL1 (Sm22.6), SmTAL2 (Sm21.7) and SmTAL3 (Sm20.8). Molecular modelling suggested that, despite similarities in domain organisation, there are differences in the three proteins’ structures. SmTAL1 was predicted to have two functional calcium binding sites and SmTAL2 was predicted to have one. Despite the presence of two EF-hand-like structures in SmTAL3, neither was predicted to be functional. These predictions were confirmed by native gel electrophoresis, intrinsic fluorescence and differential scanning fluorimetry: both SmTAL1 and SmTAL2 are able to bind calcium ions reversibly, but SmTAL3 is not. SmTAL1 is also able to interact with manganese, strontium, iron(II) and nickel ions. SmTAL2 has a different ion binding profile interacting with cadmium, manganese, magnesium, strontium and barium ions in addition to calcium. All three proteins form dimers and, in contrast to some Fasciola hepatica proteins from the same family; dimerization is not affected by calcium ions. SmTAL1 interacts with the anti-schistosomal drug praziquantel and the calmodulin antagonists trifluoperazine, chlorpromazine and W7. SmTAL2 interacts only with W7. SmTAL3 interacts with the aforementioned calmodulin antagonists and thiamylal, but not praziquantel. Overall, these data suggest that the proteins have different biochemical properties and thus, most likely, different in vivo functions.

Deregulation of genes related to iron and mitochondrial metabolism in refractory anemia with ring sideroblasts

The presence of SF3B1 gene mutations is a hallmark of refractory anemia with ring sideroblasts (RARS). However, the mechanisms responsible for iron accumulation that characterize the Myelodysplastic Syndrome with ring sideroblasts (MDS-RS) are not completely understood. In order to gain insight in the molecular basis of MDS-RS, an integrative study of the expression and mutational status of genes related to iron and mitochondrial metabolism was carried out. A total of 231 low-risk MDS patients and 81 controls were studied. Gene expression analysis revealed that iron metabolism and mitochondrial function had the highest number of genes deregulated in RARS patients compared to controls and the refractory cytopenias with unilineage dysplasia (RCUD). Thus mitochondrial transporters SLC25 (SLC25A37 and SLC25A38) and ALAD genes were over-expressed in RARS. Moreover, significant differences were observed between patients with SF3B1 mutations and patients without the mutations. The deregulation of genes involved in iron and mitochondrial metabolism provides new insights in our knowledge of MDS-RS. New variants that could be involved in the pathogenesis of these diseases have been identified.

An in situ ionic-liquid-assisted synthetic approach to iron fluoride/graphene hybrid nanostructures as superior cathode materials for lithium ion batteries

A tactful ionic-liquid (IL)-assisted approach to in situ synthesis of iron fluoride/graphene nanosheet (GNS) hybrid nanostructures is developed. To ensure uniform dispersion and tight anchoring of the iron fluoride on graphene, we employ an IL which serves not only as a green fluoride source for the crystallization of iron fluoride nanoparticles but also as a dispersant of GNSs. Owing to the electron transfer highways created between the nanoparticles and the GNSs, the iron fluoride/GNS hybrid cathodes exhibit a remarkable improvement in both capacity and rate performance (230 mAh g^-1 at 0.1 C and 74 mAh g^-1 at 40 C). The stable adhesion of iron fluoride nanoparticles on GNSs also introduces a significant improvement in long-term cyclic performance (115 mAh g^-1 after 250 cycles even at 10 C). The superior electrochemical performance of these iron fluoride/GNS hybrids as lithium ion battery cathodes is ascribed to the robust structure of the hybrid and the synergies between iron fluoride nanoparticles and graphene. © 2013 American Chemical Society.

Glycosylation of Vitamin D Binding Protein Reduced in Juvenile Idiopathic Arthritis Patients At Risk of Disease Extension.

Background/Purpose:Juvenile idiopathic arthritis (JIA) comprises a poorly understood group of chronic, childhood onset, autoimmune diseases with variable clinical outcomes. We investigated whether profiling of the synovial fluid (SF) proteome by a fluorescent dye based, two-dimensional gel (DIGE) approach could distinguish the subset of patients in whom inflammation extends to affect a large number of joints, early in the disease process. The post-translational modifications to candidate protein markers were verified by a novel deglycosylation strategy.Methods:SF samples from 57 patients were obtained around time of initial diagnosis of JIA. At 1 year from inclusion patients were categorized according to ILAR criteria as oligoarticular arthritis (n=26), extended oligoarticular (n=8) and polyarticular disease (n=18). SF samples were labeled with Cy dyes and separated by two-dimensional electrophoresis. Multivariate analyses were used to isolate a panel of proteins which distinguish patient subgroups. Proteins were identified using MALDI-TOF mass spectrometry with vitamin D binding protein (VDBP) expression and siaylation further verified by immunohistochemistry, ELISA test and immunoprecipitation. Candidate biomarkers were compared to conventional inflammation measure C-reactive protein (CRP). Sialic acid residues were enzymatically cleaved from immunopurified SF VDBP, enriched by hydrophilic interaction liquid chromatography (HILIC) and analysed by mass spectrometry.Results:Hierarchical clustering based on the expression levels of a set of 23 proteins segregated the extended-to-be oligoarticular from the oligoarticular patients. A cleaved isoform of VDBP, spot 873, is present at significantly reduced levels in the SF of oligoarticular patients at risk of disease extension, relative to other subgroups (p<0.05). Conversely total levels of vitamin D binding protein are elevated in plasma and ROC curves indicate an improved diagnostic sensitivity to detect patients at risk of disease extension, over both spot 873 and CRP levels. Sialysed forms of intact immunopurified VDBP were more prevalent in persistent oligoarticular patient synovial fluids.Conclusion:The data indicate that a subset of the synovial fluid proteome may be used to stratify patients to determine risk of disease extension. Reduced conversion of VDBP to a macrophage activation factor may represent a novel pathway contributing to increased risk of disease extension in JIA patients.

Ligand binding and signalling pathways of PTH receptors in sea bream (Sparus auratus) enterocytes

Whole animal studies have indicated that Ca2+ uptake by the gastrointestinal tract is regulated by the action of parathyroid hormone-related peptide (PTHrP) in teleost fish. We have characterised PTH receptors (PTHR) in piscine enterocytes and established, by using aminoterminal PTHrP peptides, the amino acid residues important for receptor activation and for stabilising the ligand/receptor complex. Ligand binding of 125I-(1–35tyr) PTHrP to the membrane fraction of isolated sea bream enterocytes revealed the existence of a single saturable high-affinity receptor (KD=2.59 nM; Bmax=71 fmol/mg protein). Reverse transcription/polymerase chain reaction with specific primers for sea bream PTH1R and PTH3R confirmed the mRNA expression of only the later receptor. Fugu (1–34) PTHrP increased cAMP levels in enterocytes but had no effect on total inositol phosphate accumulation. The aminoterminal peptides (2–34)PTHrP, (3–34)PTHrP and (7–34) PTHrP bound efficiently to the receptor but were severely defective in stimulating cAMP in enterocyte cells indicating that the first six residues of piscine (1–34)PTHrP, although not important for receptor binding, are essential for activation of the adenylate cyclase/phosphokinase A (AC-PKA)-receptor-coupled intracellular signalling pathway. Therefore, PTHrP in teleosts acts on the gastrointestinal tract through PTH3R and the AC-PKA intracellular signalling pathway and might regulate Ca2+ uptake at this site. Ligand-receptor binding and activity throughout the vertebrates appears to be allocated to the same amino acid residues of the amino-terminal domain of the PTHrP molecule.

The functional link between HFE and hepcidin- its contribution to iron metabolism regulation

Tese de doutoramento, Biologia (Biologia Molecular), Universidade de Lisboa, Faculdade de Ciências, 2013

The resilience of democratic institutions in Britain, Australia and the United States under conditions of total war

To what extent are democratic institutions resilient when nation states mobilise for war? Normative and empirical political theorists have long argued that wars strengthen the executive and threaten constitutional politics. In modern democracies, national assemblies are supposed to hold the executive to account by demanding explanations for events and policies; and by scrutinising, reviewing and, if necessary, revising legislative proposals intended to be binding on the host society or policies that have been implemented already. This article examines the extent to which the British and Australian parliaments and the United States Congress held their wartime executives to account during World War II. The research finds that under conditions approaching those of total war, these democratic institutions not only continued to exist, but also proved to be resilient in representing public concerns and holding their executives to account, however imperfectly and notwithstanding delegating huge powers. In consequence, executives—more so British and Australian ministers than President Roosevelt—were required to be placatory as institutional and political tensions within national assemblies and between assemblies and executives continued, and assemblies often asserted themselves. In short, even under the most onerous wartime conditions, democratic politics mattered and democratic institutions were resilient.

Determination of total petroleum hydrocarbons in soil from different locations using infrared spectrophotometry and gas chromatography

Total petroleum hydrocarbons (TPH) are important environmental contaminants which are toxic to human and environmental receptors. Several analytical methods have been used to quantify TPH levels in contaminated soils, specifically through infrared spectrometry (IR) and gas chromatography (GC). Despite being two of the most used techniques, some issues remain that have been inadequately studied: a) applicability of both techniques to soils contaminated with two distinct types of fuel (petrol and diesel), b) influence of the soil natural organic matter content on the results achieved by various analytical methods, and c) evaluation of the performance of both techniques in analyses of soils with different levels of contamination (presumably non-contaminated and potentially contaminated). The main objectives of this work were to answer these questions and to provide more complete information about the potentials and limitations of GC and IR techniques. The results led us to the following conclusions: a) IR analysis of soils contaminated with petrol is not suitable due to volatilisation losses, b) there is a significant influence of organic matter in IR analysis, and c) both techniques demonstrated the capacity to accurately quantify TPH in soils, irrespective of their contamination levels.

DNA-based biosensor for the electrocatalytic determination of antioxidant capacity in beverages

Reactive oxygen species (ROS) are produced as a consequence of normal aerobic metabolism and are able to induce DNA oxidative damage. At the cellular level, the evaluation of the protective effect of antioxidants can be achieved by examining the integrity of the DNA nucleobases using electrochemical techniques. Herein, the use of an adenine-rich oligonucleotide (dA21) adsorbed on carbon paste electrodes for the assessment of the antioxidant capacity is proposed. The method was based on the partial damage of a DNA layer adsorbed on the electrode surface by OH• radicals generated by Fenton reaction and the subsequent electrochemical oxidation of the intact adenine bases to generate an oxidation product that was able to catalyze the oxidation of NADH. The presence of antioxidant compounds scavenged hydroxyl radicals leaving more adenines unoxidized, and thus, increasing the electrocatalytic current of NADHmeasured by differential pulse voltammetry (DPV). Using ascorbic acid (AA) as a model antioxidant species, the detection of as low as 50nMof AA in aqueous solution was possible. The protection efficiency was evaluated for several antioxidant compounds. The biosensor was applied to the determination of the total antioxidant capacity (TAC) in beverages.

Control and comparison of the antioxidant capacity of beers

The purpose of the present work is to determine the antioxidant capacity (AC) of 27 commercial beers. The AC indicates the degree of protection of a certain organism against oxidative damage provoked by reactive oxygen and nitrogen species. Assays were carried out by the following methods: (i) total radical trapping antioxidant parameter (TRAP); (ii) trolox equivalent antioxidant capacity (TEAC); (iii) trolox equivalent antioxidant capacity (DPPH); (iv) ferric-ion reducing antioxidant parameter (FRAP); (v) cupric reducing antioxidant capacity (CUPRAC); (vi) oxygen radical absorbance capacity (ORAC). Ascorbic acid (AA), gallic acid (GA) and trolox (TR) were used as standards. All beers showed antioxidant power, but a wide range of ACs was observed. The effect of several factors upon these differences was studied. Statistical differences were found between ACs of beers of different colours. ORAC method provided always higher experimental ACs, of significant statistical differences to other assays.

Characterization of green zero-valent iron nanoparticles produced with tree leaf extracts

In the last decades nanotechnology has become increasingly important because it offers indisputable advantages to almost every area of expertise, including environmental remediation. In this area the synthesis of highly reactive nanomaterials (e.g. zero-valent iron nanoparticles, nZVI) is gaining the attention of the scientific community, service providers and other stakeholders. The synthesis of nZVI by the recently developed green bottom-up method is extremely promising. However, the lack of information about the characteristics of the synthetized particles hinders a wider and more extensive application. This work aims to evaluate the characteristics of nZVI synthesized through the green method using leaves from different trees. Considering the requirements of a product for environmental remediation the following characteristics were studied: size, shape, reactivity and agglomeration tendency. The mulberry and pomegranate leaf extracts produced the smallest nZVIs (5–10 nm), the peach, pear and vine leaf extracts produced the most reactive nZVIs while the ones produced with passion fruit, medlar and cherry extracts did not settle at high nZVI concentrations (931 and 266 ppm). Considering all tests, the nZVIs obtained from medlar and vine leaf extracts are the ones that could present better performances in the environmental remediation. The information gathered in this paper will be useful to choose the most appropriate leaf extracts and operational conditions for the application of the green nZVIs in environmental remediation.

The role of Di-iron proteins in pathogen resistance

Dissertation presented to obtain the Ph.D. degree in Biochemistry

Rapid [Gamma]-amino acid synthesis and the inhibition of the growth and development of oblique banded leaf-roller larvae

The hypothesis that rapid y-aminobutyric acid (GABA) accumulation is a plant defense against phytophagous insects was investigated. Simulation of mechanical damage resulting from phytophagous insect activity increased soybean (Glycine max L.) leaf GABA 10- to 25-fold within 1 to 4 min. Pulverizing leaf tissue resulted in a value of 2. 15 (±O. 11 SE) ~mol GABA per gram fresh weight. Increasing the GABA levels in a synthetic diet from 1.6 to 2.6 Jlffiol GABA per gram fresh weight reduced the growth rates, developmental rates, total biomass (50% reduction), and survival rates (30% reduction) of cultured Oblique banded leaf-roller (OBLR) (Choristonellra rosacealla Harris) larvae. In field experiments OBLR larvae were found predominantly on young terminal leaves which have a reduced capacity to produce GABA in response to mechanical damage. Glutamate decarboxylase (GAD) is a cytosolic enzyme which catalyses the decarboxylation of L-Glu to GABA. GAD is a calmodulin binding enzyme whose activity is stimulated dramatically by increased cytosolic H+ or Ca2 + ion concentrations. Phytophagous insect activity will disrupt the cellular compartmentation of H+ and Ca2 +, activate GAD and subsequent GABA accumulation. In animals GABA is a major inhibitory neurotransmitter. The possible mechanisms resulting in GABA inhibited growth and development of insects are discussed.