Structural characterization and comparative analysis of human and piscine cartilage acidic protein (CRTAC1/CRTAC2)

Dissertação de mestrado, Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014

Quercetin as an inhibitor of snake venom secretory phospholipase A2

As polyphenolic compounds isolated from plants extracts, flavonoids have been applied to various pharmaceutical uses in recent decades due to their anti-inflammatory, cancer preventive, and cardiovascular protective activities. In this study, we evaluated the effects of the flavonoid quercetin on Crotalus durissus terrificus secretory phospholipase A2 (sPLA2), an important protein involved in the release of arachidonic acid from phospholipid membranes. The protein was chemically modified by treatment with quercetin, which resulted in modifications in the secondary structure as evidenced through circular dichroism. In addition, quercetin was able to inhibit the enzymatic activity and some pharmacological activities of sPLA2, including its antibacterial activity, its ability to induce platelet aggregation, and its myotoxicity by approximately 40%, but was not able to reduce the inflammatory and neurotoxic activities of sPLA2. These results suggest the existence of two pharmacological sites in the protein, one that is correlated with the enzymatic site and another that is distinct from it. We also performed molecular docking to better understand the possible interactions between quercetin and sPLA2. Our docking data showed the existence of hydrogen-bonded, polar interactions and hydrophobic interactions, suggesting that other flavonoids with similar structures could bind to sPLA2. Further research is warranted to investigate the potential use of flavonoids as sPLA2 inhibitors. (C) 2010 Elsevier B.V. All rights reserved.

The periplasmic domain of the barrel assembly machinery protein A (BamA) from Escherichia coli assists folding of outer membrane protein A

The assembly of outer membranes of the cell wall of Gram-negative bacteria and of various organelles of eukaryotic cells requires the evolutionarily conserved β-barrel-assembly machinery (BAM) complex. This thesis describes the biochemical and biophysical properties of the periplasmic domain of the β-barrel assembly machinery protein A (PD-BamA) of the E. coli BAM complex, its effect on insertion and folding of the Outer membrane protein A (OmpA) into lipid bilayers and the identification of regions of PD-BamA that may be involved in protein-protein interactions. The secondary structure of PD-BamA in mixed lipid bilayers, analyzed by Circular dichroism (CD) spectroscopy, contained less β-sheet at an increased content of phosphatidylglycerol (PG) in the lipid membrane. This result showed membrane binding, albeit only in the presence of negatively charged lipids. Fluorescence spectroscopy demonstrated that PD-BamA only binds to lipid bilayers containing the negatively charged DOPG, confirming the results of CD spectroscopy. PD-BamA did not bind to zwitterionic but overall neutral lipid bilayers. PD-BamA bound to OmpA at a stoichiometry of 1:1. PD-BamA strongly facilitated insertion and folding of OmpA into lipid membranes. Kinetics of PD-BamA mediated folding of OmpA was well described by two parallel folding processes, a fast folding process and a slow folding process, differing by 2-3 orders of magnitude in their rate constants. The folding yields of OmpA depended on the concentration of lipid membranes and also on the lipid head groups. The presence of PD-BamA resulted in increased folding yields of OmpA in negatively charged DOPG, but PD-BamA did not affect the folding kinetics of OmpA into bilayers of zwitterionic but overall neutral lipids. The efficiency of folding and insertion of OmpA into lipid bilayers strongly depended on the ratio PD-BamA/OmpA and was optimal at equimolar concentrations of PD-BamA and OmpA. To examine complexes of unfolded OmpA with PD-BamA in more detail, site-directed spectroscopy was used to explore contact regions in both, PD-BamA and OmpA. Similarly, contact regions were also investigated for another protein complex formed by PD-BamA and the lipoprotein BamD. The obtained data suggest, that the site of interaction on PD-BamA for OmpA might be oriented towards the exterior environment away from the preceding POTRA domains, but that PD-BamA is oriented with its short α-helix α1 of POTRA domain 5 towards the C-terminal end of BamD.

Estudio de las modificaciones conformacionales y su influencia en la actividad inmunológica de péptidos derivados de las familias de las proteínas spect-1 y -2, siap-1 y -2 de plasmodium falciparum

SPECT-1 y -2 y SIAP-1 y -2 son proteínas pertenecientes al esporozoíto de Plasmodium falciparum causante de la malaria más agresiva en los humanos. Estas proteínas están involucradas en el paso del parásito a través de las células del hospedero humano y en la invasión del hepatocito, haciéndolas blancos atractivos para ser estudiadas. Péptidos conservados de alta capacidad de unión (cHABPs) a células HeLa y HepG2 derivados de estas moléculas son no inmunogénicos porque son incapaces de generar una respuesta inmunitaria, pero son claves para el parásito porque cumplen una función importante durante la infección del hospedero humano. En este trabajo se encontró que algunos cHABPs pertenecientes a las proteínas SPECT-1 y -2, están posiblemente involucrados con la unión y formación de poros sobre la membrana de las células hospederas, ayudando al esporozoíto a abrirse paso través de las células del hospedero. Por otro lado, con el fin de cambiar el comportamiento inmunológico de cHABPs derivados de SPECT-1 y -2 y SIAP-1 y -2, se obtuvieron nuevos péptidos mediante el reemplazo de aminoácidos críticos por otros residuos cuya masa molecular sea similar, pero diferente en su polaridad. En este trabajo se reporta que dichas modificaciones promovieron cambios en la estructura secundaria (determinada por DC o 1H-RMN) de los péptidos modificados (mHABPs) cuando se comparó con la estructura de los cHABPs nativos; adicionalmente, estos mHABPs invirtieron su comportamiento inmunológico convirtiéndose en péptidos inmunogénicos inductores de anticuerpos. Lo que permite establecer la existencia de una relación entre la estructura que adoptan estos mHABPs con su actividad inmunológica. Además, algunos de los mHABPs estudiados aquí, pueden ser candidatos a ser incluidos en la vacuna contra la malaria químicamente sintetizada multi-epitope y multi-estadio que se está desarrollando en la Fundación Instituto de Inmunología de Colombia (FIDIC).

Genes involved in germline regulative pathways in metazoa: an integrative approach to investigate a key feature of animal biology

In Metazoa, the germline represents the cell lineage devoted to transmission of genetic heredity across generations. Its functions intuitively evoke the crucial roles that it plays in the development of a new organism and in the evolution of the species. Germline establishment is tightly tied to animal multicellularity itself, in which the complex differentiation of cell lineages is favoured by the confinement of totipotency in specific cell populations. In the present thesis, I addressed the subject of germline characterization in animals through different approaches, in an attempt to cover different sides and scales. First, I investigated the extent and nature of shared differentially transcribed molecular factors in 10 different species germline-related lineages. I observed that newly evolved genes are less likely to be involved in germline-related mechanisms and that the mostly shared transcriptional signal across the species considered was the upregulation of genes associated to proper DNA replication, instead of the expected transcriptional and post-transcriptional regulation, that apparently have a higher level of lineage-specificity. I then focused on the evolutionary history of Tudor domain containing proteins, a gene family that underwent germline-associated expansions in animals. Using data from 24 holozoan phyla, I could confirm the previously proposed evolution of the Tudor domain secondary structure. Also, I associated lineage-specific family reductions and expansions to peculiar genomic dynamics and to the evolution of germline-associated piRNA pathway of retrotransposon silencing. Lastly, I characterized and investigated the expression of the Tudor protein TDRD7 in the clam Ruditapes philippinarum. Through immunolocalization, I could compare its expression profiles in gametogenic specimens to the previously characterized germline marker vasa. Combining results with literature, I proposed that, in this species, TDRD7 is involved in the assembly of germ granules, i.e. cytoplasmic structures associated to germline differentiation in virtually all animals, but whose assemblers can be taxon specific.

Protein-based nanomaterials as protein heterogeneous nucleants and structural studies on enzymes from two photosynthetic organisms

Proteins, the most essential biological macromolecules, are involved in nearly every aspect of life. The elucidation of their three-dimensional structures through X-ray analysis has significantly contributed to our understanding of fundamental mechanisms in life processes. However, the obstacle of obtaining high-resolution protein crystals remains significant. Thus, searching for materials that can effectively induce nucleation of crystals is a promising and active field. This thesis work characterizes and prepares albumin nanoparticles as heterogeneous nucleants for protein crystallization. These stable Bovine Serum Albumin nanoparticles were synthesized via the desolvation method, purified efficiently, and characterized in terms of dimension, morphology, and secondary structure. The ability of BSA-NPs to induce macromolecule nucleation was tested on three model proteins, exhibiting significant results, with larger NPs inducing more nucleation. The second part of this work focuses on the structural study, mainly through X-ray crystallography, of five chloroplast and cytosolic enzymes involved in the fundamental cellular processes of two photosynthetic organisms, Chlamydomonas reinhardtii and Arabidopsis thaliana. The structures of three enzymes involved in the Calvin-Benson-Bassham Cycle, phosphoribulokinase, troseposphatisomerase, and ribulosiophosphate epimerase from Chlamydomonas reinhardtii, were solved to investigate their catalytic and regulatory mechanisms. Additionally, the structure of nitrosylated-CrTPI made it possible to identify Cys14 as a target for nitrosylation, and the crystallographic structure of CrRPE was solved for the first time, providing insights into its catalytic and regulatory properties. Finally, the structure of S-nitrosoglutathione reductase, AtGSNOR, was compared with that of AtADH1, revealing differences in their catalytic sites. Overall, seven crystallographic structures, including partially oxidized CrPRK, CrPRK/ATP, CrPRK/ADP/Ru5P, CrTPI-nitrosylated, apo-CrRPE, apo-AtGSNOR, and AtADH1-NADH, were solved and are yet to be deposited in the PDB.

Mu-Chlorido-Bridged Dimanganese(II) complexes of the schiff base derived from [2+2] condensation of 2,6-diformyl-4-methylphenol and 1,3-bis(3-aminopropyl)tetramethyldisloxane: structure, magnetismo, electrochemical behaviour, and catalytic oxidation of secondary alcohols

The reaction of 2,6-diformyl-4-methylphenol with 1,3-bis(3-aminopropyl)tetramethyldisiloxane in the presence of MnCl2 in a 1:1:2 molar ratio in methanol afforded a dinuclear -chlorido-bridged manganese(II) complex of the macrocyclic [2+2] condensation product (H2L), namely, [Mn2Cl2(H2L)(HL)]Cl center dot 3H(2)O (1). The latter afforded a new compound, namely, [Mn2Cl2(H2L)(2)][MnCl4]center dot 4CH(3)CN center dot 0.5CHCl(3 center dot)0.4H(2)O (2), after recrystallisation from 1:1 CHCl3/CH3CN. The co-existence of the free and complexed azomethine groups, phenolato donors, mu-chlorido bridges, and the disiloxane unit were well evidenced by ESI mass spectrometry and FTIR spectroscopy and confirmed by X-ray crystallography. The magnetic measurements revealed an antiferromagnetic interaction between the two high-spin (S = 5/2, g = 2) manganese(II) ions through the mu-chlorido bridging ligands. The electrochemical behaviour of 1 and 2 has been studied, and details of their redox properties are reported. Both compounds act as catalysts or catalyst precursors in the solvent-free low-power microwave-assisted oxidation of selected secondary alcohols, for example, 1-phenylethanol, cyclohexanol, 2- and 3-octanol, to the corresponding ketones in the absence of solvent. The highest yield of 72% was achieved for 1-phenylethanol by using a maximum of 1% molar ratio of catalyst relative to substrate.

µ-Chlorido-bridged dimanganese(II) complexes of the schiff base derived from [2+2] condensation of 2,6-diformyl-4-methylphenol and 1,3-bis(3-aminopropyl)tetramethyldisiloxane: structure, magnetismo, electrochemical behaviour, and catalytic oxidation of secondary alcohols

The reaction of 2,6-diformyl-4-methylphenol with 1,3-bis(3-aminopropyl)tetramethyldisiloxane in the presence of MnCl2 in a 1:1:2 molar ratio in methanol afforded a dinuclear -chlorido-bridged manganese(II) complex of the macrocyclic [2+2] condensation product (H2L), namely, [Mn2Cl2(H2L)(HL)]Cl center dot 3H(2)O (1). The latter afforded a new compound, namely, [Mn2Cl2(H2L)(2)][MnCl4]center dot 4CH(3)CN center dot 0.5CHCl(3 center dot)0.4H(2)O (2), after recrystallisation from 1:1 CHCl3/CH3CN. The co-existence of the free and complexed azomethine groups, phenolato donors, mu-chlorido bridges, and the disiloxane unit were well evidenced by ESI mass spectrometry and FTIR spectroscopy and confirmed by X-ray crystallography. The magnetic measurements revealed an antiferromagnetic interaction between the two high-spin (S = 5/2, g = 2) manganese(II) ions through the mu-chlorido bridging ligands. The electrochemical behaviour of 1 and 2 has been studied, and details of their redox properties are reported. Both compounds act as catalysts or catalyst precursors in the solvent-free low-power microwave-assisted oxidation of selected secondary alcohols, for example, 1-phenylethanol, cyclohexanol, 2- and 3-octanol, to the corresponding ketones in the absence of solvent. The highest yield of 72% was achieved for 1-phenylethanol by using a maximum of 1% molar ratio of catalyst relative to substrate.

Floristic and structure of an Amazonian primary forest and a chronosequence of secondary succession

ABSTRACT The analysis of changes in species composition and vegetation structure in chronosequences improves knowledge on the regeneration patterns following land abandonment in the Amazon. Here, the objective was to perform floristic-structural analysis in mature forests (with/without timber exploitation) and secondary successions (initial, intermediate and advanced vegetation regrowth) in the Tapajós region. The regrowth age and plot locations were determined using Landsat-5/Thematic Mapper images (1984-2012). For floristic analysis, we determined the sample sufficiency and the Shannon-Weaver (H'), Pielou evenness (J), Value of Importance (VI) and Fisher's alpha (α) indices. We applied the Non-metric Multidimensional Scaling (NMDS) for similarity ordination. For structural analysis, the diameter at the breast height (DBH), total tree height (Ht), basal area (BA) and the aboveground biomass (AGB) were obtained. We inspected the differences in floristic-structural attributes using Tukey and Kolmogorov-Smirnov tests. The results showed an increase in the H', J and α indices from initial regrowth to mature forests of the order of 47%, 33% and 91%, respectively. The advanced regrowth had more species in common with the intermediate stage than with the mature forest. Statistically significant differences between initial and intermediate stages (p<0.05) were observed for DBH, BA and Ht. The recovery of carbon stocks showed an AGB variation from 14.97 t ha-1 (initial regrowth) to 321.47 t ha-1 (mature forests). In addition to AGB, Ht was also important to discriminate the typologies.

Isoelectric focusing of alpha-1 acid glycoprotein (orosomucoid) in immobilized pH-gradients with 8M urea: detection of its desialylated variants using an alkaline phosphatase-linked secondary antibody system.

A method allowing a clear separation of the different variants of desialylated alpha 1-acid glycoprotein (orosomucoid) has been developed using isoelectric focusing in immobilized pH gradients, supplemented with 8 M urea and 2% v/v 2-mercaptoethanol. Immunoblotting with two antibody-steps afforded high sensitivity and permitted the detection of about 700 pg of alpha 1-acid glycoprotein in a 20 microL plasma sample diluted 1:28 672. A one year old bloodstrain, kept at room temperature, could easily be phenotyped.

Diversity and structure of the tree community of a fragment of tropical secondary forest of the brazilian Atlantic Forest domain 15 and 40 years after logging

Two adjacent tracts of tropical secondary forest, situated in Itambé do Mato Dentro, south-eastern Brazil, which had been regenerating for 15 and 40 years after clearing, were compared with the purpose of detecting differences in species diversity and composition, species guild composition (regeneration, stratification and dispersion), and stand structure. Four and three 1,125 m² plots laid on the 15- and 40-year-old stands, respectively, sampled 2,430 trees with diameter at the base of the stem > 5 cm. The number of species (S = 199) was high for this forest type and significantly higher for the older stand. Tree density was significantly higher in the younger stand, particularly for smaller trees, whereas the two stands did not differ in both basal area and volume per hectare. Trees of shade-tolerant and understory species were significantly more abundant in the older stand. Though sharing a large proportion of species (49%), the two stands differed significantly in the abundance of many species. Live stumps probably contributed to the relatively quick restoration of some forest characteristics, particularly species diversity, basal area and volume.

Structure effects of self-assembled Prussian blue confined in highly organized mesoporous TiO(2) on the electrocatalytic properties towards H(2)O(2) detection

Here we report the derivatization of mesoporous TiO(2) thin films for the preparation of H(2)O(2) amperometric sensors. The coordination of the bifunctional ligand 1,10 phenantroline, 5,6 dione on the surface Ti(IV) ions provides open coordination sites for Fe(II) cations which are the starting point for the growth of a layer of Prussian blue polymer. The porous structure of the mesoporous TiO(2) allows the growth, ion by ion of the coordination polymer. Up to four layer of Prussian blue can be deposit without losing the porous structure of the film, which results in an enhanced response of these materials as H(2)O(2) sensors. These porous confined PB modified electrodes are robust sensors that exhibit good reproducibility, environmental stability and high sensitivity towards H(2)O(2) detection. (C) 2010 Elsevier B.V. All rights reserved.

The influence of hydrogen plasma pre-treatment on the structure of BDND electrode surface applied for phenol detection

The surface properties of boron-doped nanocrystalline diamond films treated with H(2) plasma was investigated in regard to their electrochemical response for phenol oxidation. The surface of these films is relatively flat formed by crystallites with sizes of about 40 nm. X-ray photoelectron spectroscopy analyses showed that electrode surface has a high amount of C-H bonds. This behavior is in agreement with Mott-Schottky plot measurements concerning the flat band potential that presented a value as expected for hydrogenated diamond surface. This electrode presented the phenol detection limit of 0.08 mg L(-1) for low phenol concentrations from 40 to 250 mu mol L(-1).

HPLC-EICD: An Useful Tool for the Pursuit of Novel Analytical Strategies for the Detection of Antioxidant Secondary Metabolites

Living cells are continuously exposed to a variety of challenges that exert oxidative stress and are directly related with senescence and the onset of various pathological conditions such as coronary heart disease, rheumatoid arthritis and cancer. Nevertheless, living organisms have developed a complex antioxidant network to counteract reactive species that are detrimental to life. With the aim of bio-prospecting plant species from the Brazilian Cerrado and Atlantic Forest, we have established a methodology to detect secondary antioxidant metabolites in crude extracts and fractions obtained from plant species. Combining HPLC with an electrochemical detector allowed us to detect micromolecules that showed antioxidant activities in Chimarrhis turbinata (DC) leaf extracts. Comparison with purified flavonoid standards led us to identify the compounds in their natural matrices giving valuable information on their antioxidant capacity.

Damage detection in a benchmark structure using AR-ARX models and statistical pattern recognition

Structural health monitoring (SHM) is related to the ability of monitoring the state and deciding the level of damage or deterioration within aerospace, civil and mechanical systems. In this sense, this paper deals with the application of a two-step auto-regressive and auto-regressive with exogenous inputs (AR-ARX) model for linear prediction of damage diagnosis in structural systems. This damage detection algorithm is based on the. monitoring of residual error as damage-sensitive indexes, obtained through vibration response measurements. In complex structures there are. many positions under observation and a large amount of data to be handed, making difficult the visualization of the signals. This paper also investigates data compression by using principal component analysis. In order to establish a threshold value, a fuzzy c-means clustering is taken to quantify the damage-sensitive index in an unsupervised learning mode. Tests are made in a benchmark problem, as proposed by IASC-ASCE with different damage patterns. The diagnosis that was obtained showed high correlation with the actual integrity state of the structure. Copyright © 2007 by ABCM.