Estudo das interações entre proteína NS1 do hRSV e flavonóides utilizando métodos biofísicos

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

Expressão, purificação e caracterização estrutural de proteínas codificadas por dois fitovírus

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudos funcionais da rgs-CaM, uma calmodulina envolvida na supressão de silenciamento por RNA

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

Chemical and thermal influence of the [4Fe-4S](2+) cluster of A/G-specific adenine glycosylase from Corynebacterium pseudotuberculosis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Expression, purification and characterization of cold shock protein A of Corynebacterium pseudotuberculosis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The putative Leishmania telomerase RNA (leishTER) undergoes trans-splicing and contains a conserved template sequence

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

Caracterização biofísica da interação entre a proteína M2-1 do Vírus Sincicial Respiratório Humano (HRSV) com quercetina

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

An evaluation of 3-rhamnosylquertetin, a glycolylated form of quercitin, against the myotoxic and edematogenic effects of sPLA2s from Crotalus durissus terrificus

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Development of an Electrochemical Insulin Sensor Based on a High Affinity DNA Sequence Found in the Insulin-linked Polymorphic Region

The detection of pertinent biomarkers has the potential provide an early indication of disease progression before considerable damage has been incurred. A decrease in an individual’s sensitivity to insulin, which may be quantified as the ratio of insulin to glucose in the blood after a glucose pulse, has recently been reported as an early predictor of insulin-dependent diabetes mellitus. Routine measurement of insulin levels is therefore desirable in the care of diabetes-prone individuals. A rapid, simple, and reagentless method for insulin detection would allow for wide-spread screenings that provide earlier signs of diabetes onset. The aim of this thesis is to develop a folding-base electrochemical sensor for the detection of insulin. The sensor described herein consists of a DNA probe immobilized on a gold disc electrode via an alkanethiol linker and embedded in an alkanethiol self-assembled monolayer. The probe is labeled with a redox reporter, which readily transfers electrons to the gold electrode in the absence of insulin. In the presence of insulin, electron transfer is inhibited, presumably due to a binding-induced conformational or dynamic change in the DNA probe that significantly alters the electron-tunneling pathway. A 28-base segment of the insulin-linked polymorphic region that has been reported to bind insulin with high affinity serves as the capture element of the DNA probe. Three probe constructs that vary in their secondary structure and position of the redox label are evaluated for their utility as insulin-sensing elements on the electrochemical platform. The effects of probe modification on secondary structure are also evaluated using circular dichroism spectroscopy.

Patterns and processes of diversification in a widespread and ecologically diverse avian group, the buteonine hawks (Aves, Accipitridae)

Buteonine hawks represent one of the most diverse groups in the Accipitridae, with 58 species distributed in a variety of habitats on almost all continents. Variations in migratory behavior, remarkable dispersal capability, and unusual diversity in Central and South America make buteonine hawks an excellent model for studies in avian evolution. To evaluate the history of their global radiation, we used an integrative approach that coupled estimation of the phylogeny using a large sequence database (based on 6411 bp of mitochondrial markers and one nuclear intron from 54 species), divergence time estimates, and ancestral state reconstructions. Our findings suggest that Neotropical buteonines resulted from a long evolutionary process that began in the Miocene and extended to the Pleistocene. Colonization of the Nearctic, and eventually the Old World, occurred from South America, promoted by the evolution of seasonal movements and development of land bridges. Migratory behavior evolved several times and may have contributed not only to colonization of the Holarctic, but also derivation of insular species. In the Neotropics, diversification of the buteonines included four disjunction events across the Andes. Adaptation of monophyletic taxa to wet environments occurred more than once, and some relationships indicate an evolutionary connection among mangroves, coastal and varzea environments. On the other hand, groups occupying the same biome, forest, or open vegetation habitats are not monophyletic. Refuges or sea-level changes or a combination of both was responsible for recent speciation in Amazonian taxa. In view of the lack of concordance between phylogeny and classification, we propose numerous taxonomic changes. (C) 2009 Elsevier Inc. All rights reserved.

OmpL1 Is an Extracellular Matrix- and Plasminogen-Interacting Protein of Leptospira spp.

Leptospirosis is a zoonosis with multisystem involvement caused by pathogenic strains of the genus Leptospira. OmpL1 is an outer membrane protein of Leptospira spp. that is expressed during infection. In this work, we investigated novel features of this protein. We describe that OmpL1 is a novel leptospiral extracellular matrix (ECM)-binding protein and a plasminogen (PLG) receptor. The recombinant protein was expressed in Escherichia coli BL21(DE3) Star/pLysS as inclusion bodies, refolded, and purified by metal-chelating chromatography. The protein presented a typical beta-strand secondary structure, as evaluated by circular dichroism spectroscopy. The recombinant protein reacted with antibodies in serum samples from convalescent leptospirosis patients with a high specificity compared to serum samples from individuals with unrelated diseases. These data strengthen the usefulness of OmpL1 as a diagnostic marker of leptospirosis. The characterization of the immunogenicity of recombinant OmpL1 in inoculated BALB/c mice showed that the protein has the capacity to elicit humoral and cellular immune responses, as denoted by high antibody titers and the proliferation of lymphocytes. We demonstrate that OmpL1 has the ability to mediate attachment to laminin and plasma fibronectin, with KD (equilibrium dissociation constant) values of 2,099.93 +/- 871.03 nM and 1,239.23 +/- 506.85 nM, respectively. OmpL1 is also a PLG receptor, with a KD of 368.63 +/- 121.23 nM, capable of generating enzymatically active plasmin. This is the first report that shows and characterizes OmpL1 as an ECM-interacting and a PLG-binding protein of Leptospira spp. that may play a role in bacterial pathogenesis when expressed during infection.

Septin C-Terminal Domain Interactions: Implications for Filament Stability and Assembly

Septins form a conserved family of filament forming GTP binding proteins found in a wide range of eukaryotic cells. They share a common structural architecture consisting of an N-terminal domain, a central GTP binding domain and a C-terminal domain, which is often predicted to adopt a coiled-coil conformation, at least in part. The crystal structure of the human SEPT2/SEPT6/SEPT7 heterocomplex has revealed the importance of the GTP binding domain in filament formation, but surprisingly no electron density was observed for the C-terminal domains and their function remains obscure. The dearth of structural information concerning the C-terminal region has motivated the present study in which the putative C-terminal domains of human SEPT2, SEPT6 and SEPT7 were expressed in E. coli and purified to homogeneity. The thermal stability and secondary structure content of the domains were studied by circular dichroism spectroscopy, and homo- and hetero-interactions were investigated by size exclusion chromatography, chemical cross-linking, analytical ultracentrifugation and surface plasmon resonance. Our results show that SEPT6-C and SEPT7-C are able to form both homo- and heterodimers with a high alpha-helical content in solution. The heterodimer is elongated and considerably more stable than the homodimers, with a K (D) of 15.8 nM. On the other hand, the homodimer SEPT2-C has a much lower affinity, with a K (D) of 4 mu M, and a moderate alpha-helical content. Our findings present the first direct experimental evidence toward better understanding the biophysical properties and coiled-coil pairings of such domains and their potential role in filament assembly and stability.

Rananos expression pattern during oogenesis and early embryonic development in Rhynchosciara americana

The Dipteran a native Brazilian insect that has become a valuable model system for developmental biology research because it provides an interesting opportunity to study a different type of insect oogenesis. Sequences from a cDNA library that was constructed with poly A + RNA from the ovaries of larvae at different ages were analyzed. Molecular characterization confirmed interesting findings, such as the presence of . The gene encodes a conserved RNA-binding protein that is required during early development for the maintenance and division of the primordial germ cells of Diptera. plays an important role in specifying the posterior regions of insect embryos and is important for abdomen formation. In the present work, we showed the spatial and temporal expression profiles of this important gene, which is involved in oogenesis and early development. Data mining techniques were used to obtain the complete sequence of . Bioinformatic tools were used to determine the following: (1) the secondary structure of the 3'-untranslated region of the mRNA, (2) the encoded protein of the isolated gene, (3) the conserved zinc-finger domains of the Nanos protein, and (4) phylogenetic analyses. Furthermore, RNA in situ hybridization and immunolocalization were used to determine mRNA and protein expression in the tissues that were studied and to define as a germ cell molecular marker.

Fumarate hydratase isoforms of Leishmania major: Subcellular localization, structural and kinetic properties

Fumarate hydratases (FHs; EC 4.2.1.2) are enzymes that catalyze the reversible hydration of fumarate to S-malate. Parasitic protists that belong to the genus Leishmania and are responsible for a complex of vector-borne diseases named leishmaniases possess two genes that encode distinct putative FH enzymes. Genome sequence analysis of Leishmania major Friedlin reveals the existence of genes LmjF24.0320 and LmjF29.1960 encoding the putative enzymes LmFH-1 and LmFH-2, respectively. In the present work, the FH activity of both L. major enzymes has been confirmed. Circular dichroism studies suggest important differences in terms of secondary structure content when comparing LmFH isoforms and even larger differences when comparing them to the homologous human enzyme. CD melting experiments revealed that both LmFH isoforms are thermolabile enzymes. The catalytic efficiency under aerobic and anaerobic environments suggests that they are both highly sensitive to oxidation and damaged by oxygen. Intracellular localization studies located LmFH-1 in the mitochondrion, whereas LmFH-2 was found predominantly in the cytosol with possibly also some in glycosomes. The high degree of sequence conservation in different Leishmania species, together with the relevance of FH activity for the energy metabolism in these parasites suggest that FHs might be exploited as targets for broad-spectrum antileishmanial drugs. (c) 2012 Elsevier B.V. All rights reserved.

Conformational stability of recombinant manganese superoxide dismutase from the filamentous fungus Trichoderma reesei

Superoxide dismutases (SODS; EC 1.15.1.1) are part of the antioxidant system of aerobic organisms and are used as a defense against oxidative injury caused by reactive oxygen species (ROS). The cloning and sequencing of the 788-bp genomic DNA from Trichoderma reesei strain QM9414 (anamorph of Hypocrea jecorina) revealed an open reading frame encoding a protein of 212 amino acid residues, with 65-90% similarity to manganese superoxide dismutase from other filamentous fungi. The TrMnSOD was purified and shown to be stable from 20 to 90 degrees C for 1 h at pH from 8 to 11.5, while maintaining its biological activity. (C) 2011 Elsevier B.V. All rights reserved.