Isoelectric Point Determination for Glossoscolex paulistus Extracellular Hemoglobin: Oligomeric Stability in Acidic pH and Relevance to Protein-Surfactant Interactions

The extracellular hemoglobin from Glossoscolex paulistus (HbGp) has a molecular mass of 3.6 M Da, It has a high oligomeric stability at pH 7.0 and low autoxidation rates, as compared to vertebrate hemoglobins. In this work, fluorescence and light scattering experiments were performed with the three oxidation forms of HbGp exposed to acidic pH. Our focus is on the HbGp stability at acidic pH and also on the determination of the isoelectric point (pI) of the protein. Our results show that the protein in the cyanomet form is more stable than in the other two forms, in the whole range. Our zeta-potential data are consistent with light scattering results. Average values apt obtained by different techniques were 5.6 +/- 0.5, 5.4 +/- 0.2 and 5.2 +/- 0.5 for the oxy, met, and cyanomet forms. Dynamic light scattering (DLS) experiments have shown that, at pH 6.0, the aggregation (oligomeric) state of oxy-, met- and cyanomet-HbGp remains the same as that at 7.0. The interaction between the oxy-HbGp and ionic surfactants at pH 5.0 and 6.0 was also monitored in the present study. At pH 5,0, below the protein pI, the effects of sodium dodecyl sulfate (SDS) and cetyltrimethylammonium chloride (CTAC) are inverted when compared to pH 7.0. For CTAC, in acid pH 5.0, no precipitation is observed, while for SDS an intense light scattering appears due to a precipitation process. HbGp interacts strongly with the cationic surfactant at pH 7.0 and with the anionic one at pH 5.0. This effect is due to the predominance, in the protein surface, of residues presenting opposite charges to the surfactant headgroups. This information can be relevant for the development of extracellular hemoglobin-based artificial blood substitutes.

Empresas de controle familiar e informed trading: evidências de short selling no mercado brasileiro?

O objetivo desse trabalho é testar se no mercado brasileiro, empresas familiares são mais suscetíveis a insider trading.Testes feitos no mercado americano evidenciaram efeito do controle familiar no conteúdo informacional embutido em montagem de posições vendidas de companhias abertas. Lá, foram encontrados níveis acima do normal de posições short em companhias de controle familiar principalmente em momentos que antecipavam resultados negativos que iriam ser publicados. Não encontramos evidências claras de que o fato da companhia ter controle familiar poderia levá-la a apresentar ou não insider trading, já que por limitação do modelo não é possível comparar o nível de anormal short para empresas de controle familiar e outras pois essa variável é excluída do modelo. Entretanto, observamos nos modelos em painel fixo com interações que existe diferença do efeito de algumas variáveis de controle para empresas de controle familiar ou não sobre outras variáveis de controle o que poderia mostrar que alguma influência o controle familiar poderia ter sobre o insider trading. Testamos também se empresas de controle estatal apresentavam maior volume médio diário anormal de posições vendidas em momentos que antecediam surpresas de resultado, e também não encontramos evidências claras e diretas que isso acontecia.

Functionalization of dendrimers for improved gene delivery to mesenchymal stem cell

Disease, injury, and age problems compromise human quality of life and continuously motivate the search for new and more efficacious therapeutic approaches. The field of Tissue Regeneration and Engineering has greatly evolved over the last years, mainly due to the combination of the important advances verified in Biomaterials Science and Engineering with those of Cell and Molecular Biology. In particular, a new and promising area arose – Nanomedicine – that takes advantage of the extremely small size and especial chemical and physical properties of Nanomaterials, offering powerful tools for health improvement. Research on Stem Cells, the self-renewing progenitors of body tissues, is also challenging to the medical and scientific communities, being expectable the appearance of new and exciting stem cell-based therapies in the next years. The control of cell behavior (namely, of cell proliferation and differentiation) is of key importance in devising strategies for Tissue Regeneration and Engineering. Cytokines, growth factors, transcription factors and other signaling molecules, most of them proteins, have been identified and found to regulate and support tissue development and regeneration. However, the application of these molecules in long-term regenerative processes requires their continuous presence at high concentrations as they usually present short half-lives at physiological conditions and may be rapidly cleared from the body. Alternatively, genes encoding such proteins can be introduced inside cells and be expressed using cell’s machinery, allowing an extended and more sustained production of the protein of interest (gene therapy). Genetic engineering of stem cells is particularly attractive because of their self-renewal capability and differentiation potential. For Tissue Regeneration and Engineering purposes, the patient’s own stem cells can be genetically engineered in vitro and, after, introduced in the body (with or without a scaffold) where they will not only modulate the behavior of native cells (stem cell-mediated gene therapy), but also directly participate in tissue repair. Cells can be genetically engineered using viral and non-viral systems. Viruses, as a result of millions of years of evolution, are very effective for the delivery of genes in several types of cells, including cells from primary sources. However, the risks associated with their use (like infection and immunogenic reactions) are driving the search for non-viral systems that will efficiently deliver genetic material into cells. Among them, chemical methods that are promising and being investigated use cationic molecules as carriers for DNA. In this case, gene delivery and gene expression level remain relatively low when primary cells are used. The main goal of this thesis was to develop and assess the in vitro potential of polyamidoamine (PAMAM) dendrimers based carriers to deliver genes to mesenchymal stem cells (MSCs). PAMAM dendrimers are monodispersive, hyperbranched and nanospherical molecules presenting unique characteristics that make them very attractive vehicles for both drug and gene delivery. Although they have been explored for gene delivery in a wide range of cell lines, the interaction and the usefulness of these molecules in the delivery of genes to MSCs remains a field to be explored. Adult MSCs were chosen for the studies due to their potential biomedical applications (they are considered multipotent cells) and because they present several advantages over embryonic stem cells, such as easy accessibility and the inexistence of ethical restrictions to their use. This thesis is divided in 5 interconnected chapters. Chapter I provides an overview of the current literature concerning the various non-viral systems investigated for gene delivery in MSCs. Attention is devoted to physical methods, as well as to chemical methods that make use of polymers (natural and synthetic), liposomes, and inorganic nanoparticles as gene delivery vectors. Also, it summarizes the current applications of genetically engineered mesenchymal stem cells using non-viral systems in regenerative medicine, with special focus on bone tissue regeneration. In Chapter II, the potential of native PAMAM dendrimers with amine termini to transfect MSCs is evaluated. The level of transfection achieved with the dendrimers is, in a first step, studied using a plasmid DNA (pDNA) encoding for the β-galactosidase reporter gene. The effect of dendrimer’s generation, cell passage number, and N:P ratio (where N= number of primary amines in the dendrimer; P= number of phosphate groups in the pDNA backbone) on the level of transfection is evaluated, being the values always very low. In a second step, a pDNA encoding for bone morphogenetic protein-2, a protein that is known for its role in MSCs proliferation and differentiation, is used. The BMP-2 content produced by transfected cells is evaluated by an ELISA assay and its effect on the osteogenic markers is analyzed through several classical assays including alkaline phosphatase activity (an early marker of osteogenesis), osteocalcin production, calcium deposition and mineralized nodules formation (late osteogenesis markers). Results show that a low transfection level is enough to induce in vitro osteogenic differentiation in MSCs. Next, from Chapter III to Chapter V, studies are shown where several strategies are adopted to change the interaction of PAMAM dendrimers with MSCs cell membrane and, as a consequence, to enhance the levels of gene delivery. In Chapter III, generations 5 and 6 of PAMAM dendrimers are surface functionalized with arginine-glycine-aspartic acid (RGD) containing peptides – experiments with dendrimers conjugated to 4, 8 and 16 RGD units were performed. The underlying concept is that by including the RGD integrin-binding motif in the design of the vectors and by forming RGD clusters, the level of transfection will increase as MSCs highly express integrins at their surface. Results show that cellular uptake of functionalized dendrimers and gene expression is enhanced in comparison with the native dendrimers. Furthermore, gene expression is dependent on both the electrostatic interaction established between the dendrimer moiety and the cell surface and the nanocluster RGD density. In Chapter IV, a new family of gene delivery vectors is synthesized consisting of a PAMAM dendrimer (generation 5) core randomly linked at the periphery to alkyl hydrophobic chains that vary in length and number. Herein, the idea is to take advantage of both the cationic nature of the dendrimer and the capacity of lipids to interact with biological membranes. These new vectors show a remarkable capacity for internalizing pDNA, being this effect positively correlated with the –CH2– content present in the hydrophobic corona. Gene expression is also greatly enhanced using the new vectors but, in this case, the higher efficiency is shown by the vectors containing the smallest hydrophobic chains. Finally, chapter V reports the synthesis, characterization and evaluation of novel gene delivery vectors based on PAMAM dendrimers (generation 5) conjugated to peptides with high affinity for MSCs membrane binding - for comparison, experiments are also done with a peptide with low affinity binding properties. These systems present low cytotoxicity and transfection efficiencies superior to those of native dendrimers and partially degraded dendrimers (Superfect®, a commercial product). Furthermore, with this biomimetic approach, the process of gene delivery is shown to be cell surface receptor-mediated. Overall, results show the potential of PAMAM dendrimers to be used, as such or modified, in Tissue Regeneration and Engineering. To our knowledge, this is the first time that PAMAM dendrimers are studied as gene delivery vehicles in this context and using, as target, a cell type with clinical relevancy. It is shown that the cationic nature of PAMAM dendrimers with amine termini can be synergistically combined with surface engineering approaches, which will ultimately result in suitable interactions with the cytoplasmic membrane and enhanced pDNA cellular entry and gene expression. Nevertheless, the quantity of pDNA detected inside cell nucleus is always very small when compared with the bigger amount reaching cytoplasm (accumulation of pDNA is evident in the perinuclear region), suggesting that the main barrier to transfection is the nuclear membrane. Future work can then be envisaged based on the versatility of these systems as biomedical molecular materials, such as the conjugation of PAMAM dendrimers to molecules able to bind nuclear membrane receptors and to promote nuclear translocation.

Population structure, habitat use and conservation of short-finned pilot whales Globicephala macrorhynchus in the Archipelago of Madeira

This thesis provides information on the grouping structure, survival, abundance, dive characteristics and habitat preferences of short-finned pilot whales occurring in the oceanic archipelago of Madeira (Portugal, NE Atlantic), based on data collected between 2001-2011, and contributes for its conservation. Photo-identification methods and genetic analyses demonstrated that there is a large degree of variability in site fidelity, including resident, regular visitor and transient whales, and that they may not be genetically isolated. It is proposed that the pilot whales encountered in Madeira belong to a single population encompassing several clans, possibly three clans of island-associated (i.e. resident and regular visitor) whales and others of transients, each containing two to three matrilineal pods. Mark-recapture methods estimated that the island-associated community is composed of less than 150 individuals and that their survival rate is within the range of other long-lived cetacean species, and that around 300 whales of different residency patterns uses the southern area of the island of Madeira from mid-summer to mid-autumn. No significant trend was observed between years. Time-depth recorders deployed in adult whales during daytime revealed that they spend over ¾ of their time at the surface, that they have a low diving rate, and that transient whales also forage during their passage. The analyses of visual data collected from nautical and aerial line-transect surveys indicate a core/preferred habitat area in the south-east of the island of Madeira. That area is used for resting, socializing, foraging, breeding, calving and birthing. Thus, that area should be considered as an important habitat for this species, at least seasonally (during autumn) when the species is more abundant, and included in conservation plans. No direct threat needing urgent measures was identified, although the impact of some activities like whale-watching or marine traffic should be assessed.

Crossover between macroscopic and mesoscopic regimes of vortex interactions in type-II superconductors

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A new short and simple health-related quality of life measurement for paediatric rheumatic diseases: initial validation in juvenile idiopathic arthritis

Objective. To develop and validate a new short and simple measure of health-related quality of life (HRQL) in children with juvenile idiopathic arthritis (JIA).Methods. The Paediatric Rheumatology Quality of Life Scale (PRQL) is a 10-item questionnaire that explores HRQL in two domains: physical health (PhH) and psychosocial health (PsH). Validation of the parent proxy report and child self-report versions of the instrument was accomplished by evaluating 472 JIA patients and similar to 800 healthy children. Validation analyses included assessment of feasibility, face and content validity; construct and discriminative ability; internal structure and consistency; test-retest reliability; responsiveness to clinical change; and minimal clinically important difference.Results. The PRQL was found to be feasible and to possess both face and content validity. The PRQL score correlated in the predicted range with most of the other JIA outcome measures, thereby demonstrating good construct validity, and discriminated well between different levels of disease severity. Assessment of internal structure (factor analysis) revealed that the PhH and PsH subscales identify two unambiguously separated domains. The internal consistency (Cronbach's alpha) was 0.86. The intraclass correlation coefficient for test-retest reliability was 0.91. The PRQL revealed fair responsiveness, with a standardized response mean of 0.67 in improved patients. Overall, the PRQL appeared to be more able to capture physical HRQL than psychosocial HRQL.Conclusion. The PRQL was found to possess good measurement properties and is, therefore, a valid instrument for the assessment of HRQL in children with JIA. This tool is primarily proposed for use in standard clinical care.

Performance, carcass yield, and meat quality of free-range broilers fed wet grain corn silage

This study aimed at evaluating the effect of total replacement of dry corn by wet grain corn silage (WGCS) in the feed of label broilers older than 28 days of age on performance, mortality, carcass, parts, breast meat and thighs meat yields, and meat quality. A mixed-sex flock of 448 ISA S 757-N (naked-neck ISA JA Label) day-old chicks was randomly distributed in to randomized block experimental design with four treatments (T1 - with no WGCS; T2 - WGCS between 28 and 83 days; T3 - WGCS between 42 and 83 days; and T4 - WGCS between 63 and 83 days) and four replicates of 28 birds each. Birds were raised under the same management and feeding conditions until 28 days of age, when they started to have free access to paddock with pasture (at least 3m²/bird) and to be fed the experimental diets. Feed and water were offered ad libitum throughout the rearing period, which was divided in three stages: starter (1 to 28 days), grower (29 to 63 days), and finisher (64 to 83 days) according to the feeding schedule. During the short periods of WGCS use (group T2 during grower stage and T4 during the finisher stage), performance and mortality results were similar as to those of the control group (T1). At the end of the experiment, it was observed that the extended use of WGCS (T2 and T3) determined a negative effect on feed conversion ratio. However, the best results of breast meat yield were observed with birds fed WGCS since 28 days (T2). It was concluded that WGCS can replace dry corn grain for short periods during the grower and finisher stages with no impairment of meat quality and yield in slow growth broilers.

Monitoring the positioning of short polycationic peptides in model lipid bilayers by combining hydrogen/deuterium exchange and electrospray ionization mass spectrometry

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Intermolecular interactions and characterization of the novel factor Xa exosite involved in macromolecular recognition and inhibition: Crystal structure of human Gla-domainless factor Xa complexed with the anticoagulant protein NAPc2 from the hematophagous nematode Ancylostoma caninum

NAPc2, an anticoagulant protein from the hematophagous nematode Ancylostoma caninum evaluated in phase-II/IIa clinical trials, inhibits the extrinsic blood coagulation pathway by a two step mechanism, initially interacting with the hitherto uncharacterized factor Xa exosite involved in macromolecular recognition and subsequently inhibiting factor VIIa (K-i = 8.4 pM) of the factor VIIa/tissue factor complex. NAPc2 is highly flexible, becoming partially ordered and undergoing significant structural changes in the C terminus upon binding to the factor Xa exosite. In the crystal structure of the ternary factor Xa/NAPc2/selectide complex, the binding interface consists of an intermolecular antiparallel beta-sheet formed by the segment of the polypeptide chain consisting of residues 74-80 of NAPc2 with the residues 86-93 of factor Xa that is additional maintained by contacts between the short helical segment (residues 67-73) and a turn (residues 26-29) of NAPc2 with the short C-terminal helix of factor Xa (residues 233-243). This exosite is physiologically highly relevant for the recognition and inhibition of factor X/Xa by macromolecular substrates and provides a structural motif for the development of a new class of inhibitors for the treatment of deep vein thrombosis and angioplasty. (c) 2006 Elsevier Ltd. All rights reserved.

The Origin of Nonmonotonic Complex Behavior and the Effects of Nonnative Interactions on the Diffusive Properties of Protein Folding

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Atomic Bose-Einstein condensation with three-body interactions and collective excitations

The stability of a Bose-Einstein condensed state of trapped ultra-cold atoms is investigated under the assumption of an attractive two-body and a repulsive three-body interaction. The Ginzburg-Pitaevskii-Gross (GPG) nonlinear Schrodinger equation is extended to include an effective potential dependent on the square of the density and solved numerically for the s-wave. The lowest collective mode excitations are determined and their dependences on the number of atoms and on the strength of the three-body force are studied. The addition of three-body dynamics can allow the number of condensed atoms to increase considerably, even when the strength of the three-body force is very small compared with the strength of the two-body force. We study in detail the first-order liquid-gas phase transition for the condensed state, which can happen in a critical range of the effective three-body force parameter.

Double parton interactions in gamma+3 jet events in p(p)over-bar collisions at root s=1.96 TeV

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Developmental lead exposure: behavioral alterations in the short and long term

Wistar dams were exposed to 500 ppm of Pb, as Ph acetate, or 660 ppm Na acetate in drinking water during pregnancy and lactation. Male pups at 23 (weaned) or 70 days (adult) of age were submitted to behavioral evaluation and Pb determination. The behaviors evaluated were: locomotor activity (open-field test), motor coordination (rotarod test), exploratory behavior (holeboard test), anxiety (elevated plus maze and social interaction tests), and learning and memory (shuttle box). Ph levels were measured in the blood and cerebral regions (hippocampus and striatum) of dams and pups. The results of the present report demonstrated that exposure to Ph during pregnancy and lactation induces in weaned pups hyperactivity, decreased exploratory behavior, and impairment of learning and memory. These alterations were observed at blood Ph levels in the range that may be attained in children chronically exposed to low levels of Pb (21 +/- 3 mug/dl). Regarding adults, the results demonstrated that the regimen of exposure adopted induces anxiety in these animals at nondetectable blood Ph levels. (C) 2001 Elsevier B.V. All rights reserved.

Modelling short-term temporal changes of bare soil CO2 emissions in a tropical agrosystem by using meteorological data

Determining the variability of carbon dioxide emission from soils is an important task as soils are among the largest sources of carbon in biosphere. In this work the temporal variability of bare soil CO2 emissions was measured over a 3-week period. Temporal changes in soil CO2 emission were modelled in terms of the changes that occurred in solar radiation (SR), air temperature (T-air), air humidity (AR), evaporation (EVAP) and atmospheric pressure (ATM) registered during the time period that the experiment was conducted. The multiple regression analysis (backward elimination procedure) includes almost all the meteorological variables and their interactions into the final model (R-2 = 0.98), but solar radiation showed to be the one of the most relevant variables. The present study indicates that meteorological data could be taken into account as the main forces driving the temporal variability of carbon dioxide emission from bare soils, where microbial activity is the sole source of carbon dioxide emitted. (C) 2003 Elsevier B.V. All rights reserved.

Influence of ionic interactions on the photoinduced Birefringence of poly[1-[4-(3-carboxy-4 hydroxyphenylazo) benzene sulfonamido]-1 2-ethanediyl, sodium salt] films

Electrostatic interactions govern most properties of polyelectrolyte films, as in the photoinduced bire-fringence of azo-containing polymers. In this paper we report a systematic investigation of optical storage characteristics of cast and layer-by-layer (LbL) films of poly[1 -[4-(3-carboxy-4 hydroxypheny-lazo) benzene sulfonamido]-1,2-ethanediyl, sodium salt] (PAZO). Birefringence was photoinduced faster in PAZO cast films prepared at high pHs, with the characteristic writing times decreasing almost linearly with the pH in the range between 4 and 9. This was attributed to an increased free volume for the azochromophores with the enhanced electrostatic repulsion in PAZO charged to a greater extent. In contrast, in LbL films of PAZO alternated with poly(allylamine hydrochloride) (PAH), the electrostatic interactions between the oppositely charged polymers hampered photoisomerization and molecular rearrangement, thus leading to a slower writing kinetics for highly charged PAH or PAZO.