Mining protein structure data

The principal topic of this work is the application of data mining techniques, in particular of machine learning, to the discovery of knowledge in a protein database. In the first chapter a general background is presented. Namely, in section 1.1 we overview the methodology of a Data Mining project and its main algorithms. In section 1.2 an introduction to the proteins and its supporting file formats is outlined. This chapter is concluded with section 1.3 which defines that main problem we pretend to address with this work: determine if an amino acid is exposed or buried in a protein, in a discrete way (i.e.: not continuous), for five exposition levels: 2%, 10%, 20%, 25% and 30%. In the second chapter, following closely the CRISP-DM methodology, whole the process of construction the database that supported this work is presented. Namely, it is described the process of loading data from the Protein Data Bank, DSSP and SCOP. Then an initial data exploration is performed and a simple prediction model (baseline) of the relative solvent accessibility of an amino acid is introduced. It is also introduced the Data Mining Table Creator, a program developed to produce the data mining tables required for this problem. In the third chapter the results obtained are analyzed with statistical significance tests. Initially the several used classifiers (Neural Networks, C5.0, CART and Chaid) are compared and it is concluded that C5.0 is the most suitable for the problem at stake. It is also compared the influence of parameters like the amino acid information level, the amino acid window size and the SCOP class type in the accuracy of the predictive models. The fourth chapter starts with a brief revision of the literature about amino acid relative solvent accessibility. Then, we overview the main results achieved and finally discuss about possible future work. The fifth and last chapter consists of appendices. Appendix A has the schema of the database that supported this thesis. Appendix B has a set of tables with additional information. Appendix C describes the software provided in the DVD accompanying this thesis that allows the reconstruction of the present work.

Superoxide reductase from the syphilis spirochete Treponema pallidum

Superoxide reductase is a 14 kDa metalloprotein containing a catalytic nonhaem iron centre [Fe(His)4Cys]. It is involved in defence mechanisms against oxygen toxicity, scavenging superoxide radicals from the cell. The oxidized form of Treponema pallidum superoxide reductase was crystallized in the presence of polyethylene glycol and magnesium chloride. Two crystal forms were obtained depending on the oxidizing agents used after purification: crystals grown in the presence of K3Fe(CN)6 belonged to space group P21 (unit-cell parameters a = 60.3, b = 59.9, c = 64.8 A ° , = 106.9 ) and diffracted beyond 1.60 A ° resolution, while crystals grown in the presence of Na2IrCl6 belonged to space group C2 (a = 119.4, b = 60.1, c = 65.6 A ° , = 104.9 ) and diffracted beyond 1.55 A ° . A highly redundant X-ray diffraction data set from the C2 crystal form collected on a copper rotating-anode generator ( = 1.542 A ° ) clearly defined the positions of the four Fe atoms present in the asymmetric unit by SAD methods. A MAD experiment at the iron absorption edge confirmed the positions of the previously determined iron sites and provided better phases for model building and refinement. Molecular replacement using the P21 data set was successful using a preliminary trace as a search model. A similar arrangement of the four protein molecules could be observed.

Bilirubin directly disrupts membrane lipid polarity and fluidity, protein order, and redox status in rat mitochondria

Background/Aims: Unconjugated bilirubin (UCB) impairs crucial aspects of cell function and induces apoptosis in primary cultured neurones. While mechanisms of cytotoxicity begin to unfold, mitochondria appear as potential primary targets. Methods: We used electron paramagnetic resonance spectroscopy analysis of isolated rat mitochondria to test the hypothesis that UCB physically interacts with mitochondria to induce structural membrane perturbation, leading to increased permeability, and subsequent release of apoptotic factors. Results: Our data demonstrate profound changes on mitochondrial membrane properties during incubation with UCB, including modified membrane lipid polarity and fluidity (P , 0:01), as well as disrupted protein mobility(P , 0:001). Consistent with increased permeability, cytochrome c was released from the intermembrane space(P , 0:01), perhaps uncoupling the respiratory chain and further increasing oxidative stress (P , 0:01). Both ursodeoxycholate, a mitochondrial-membrane stabilising agent, and cyclosporine A, an inhibitor of the permeability transition, almost completely abrogated UCB-induced perturbation. Conclusions: UCB directly interacts with mitochondria influencing membrane lipid and protein properties, redox status, and cytochrome c content. Thus, apoptosis induced by UCB may be mediated, at least in part, by physical perturbation of the mitochondrial membrane. These novel findings should ultimately prove useful to our evolving understanding of UCB cytotoxicity.

Viability of the use of thin-film A-SiC:H photodiodes for protein identification

In this paper, we present a multilayer device based on a-Si:H/a-SiC:H that operates as photodetector and optical filter. The use of such device in protein detection applications is relevant in Fluorescence Resonance Energy Transfer (FRET) measurements. This method demands the detection of fluorescent signals located at specific wavelengths bands in the visible part of the electromagnetic spectrum. The device operates in the visible range with a selective sensitivity dependent on electrical and optical bias. Several nanosensors were tested with a commercial spectrophotometer to assess the performance of FRET signals using glucose solutions of different concentrations. The proposed device was used to demonstrate the possibility of FRET signals detection, using visible signals of similar wavelength and intensity. The device sensitivity was tuned to enhance the wavelength band of interest using steady state optical bias at 400 nm. Results show the ability of the device to detect signals in this range. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molybdenum-containing proteins from Sulphate Reducing Bacteria: studying proteins with novel cofactors and revealing new features of old enzymes

Dissertação apresentada para a obtenção do Grau de Doutor em Bioquímica, especialidade de Bioquímica-Física pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Insights into the mechanims underlyng the antiproliferative potential of a Co(II) coordination compound bearing 1,10-Phenanthroline-5,6-Dione: DNA and protein interaction studies

The very high antiproliferative activity of [Co(Cl)(H2O)(phendione)(2)][BF4] (phendione is 1,10-phenanthroline-5,6-dione) against three human tumor cell lines (half-maximal inhibitory concentration below 1 mu M) and its slight selectivity for the colorectal tumor cell line compared with healthy human fibroblasts led us to explore the mechanisms of action underlying this promising antitumor potential. As previously shown by our group, this complex induces cell cycle arrest in S phase and subsequent cell death by apoptosis and it also reduces the expression of proteins typically upregulated in tumors. In the present work, we demonstrate that [Co(Cl)(phendione)(2)(H2O)][BF4] (1) does not reduce the viability of nontumorigenic breast epithelial cells by more than 85 % at 1 mu M, (2) promotes the upregulation of proapoptotic Bax and cell-cycle-related p21, and (3) induces release of lactate dehydrogenase, which is partially reversed by ursodeoxycholic acid. DNA interaction studies were performed to uncover the genotoxicity of the complex and demonstrate that even though it displays K (b) (+/- A standard error of the mean) of (3.48 +/- A 0.03) x 10(5) M-1 and is able to produce double-strand breaks in a concentration-dependent manner, it does not exert any clastogenic effect ex vivo, ruling out DNA as a major cellular target for the complex. Steady-state and time-resolved fluorescence spectroscopy studies are indicative of a strong and specific interaction of the complex with human serum albumin, involving one binding site, at a distance of approximately 1.5 nm for the Trp214 indole side chain with log K (b) similar to 4.7, thus suggesting that this complex can be efficiently transported by albumin in the blood plasma.

Localization studies of the FemXAB protein family in Staphylococcus aureus

Dissertação apresentada para a obtenção do Grau de Mestre em Genética Molecular e Biomedicina, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Protein-Polysaccharides of trametes versocolor: production and biological activities

Extracellular-(E-PPS) and intracellular-protein-polysaccharides (I-PPS) complexes were produced by Trametes versicolor in submerged cultures with different carbon sources. The highest extracellular-(EPS) and intracellular-polysaccharide (IPS) concentration in the complexes was obtained with tomato pomace culture. DPPH radical scavenging for E-PPS and I-PPS produced by liter of culture was equivallent to 2.115 +/- A 0.227 and 1.374 +/- A 0.364 g of ascorbic acid, respectively. These complexes showed a protector effect in the oxidation of erythrocyte membranes and had ability to inhibit the hemolysis and methemoglobin synthesis in stressed erythrocytes. These results suggest that extracellular- and intracellular- polysaccharides produced are important bioactive compounds with medicinal potential.

Liquid crystal beads constrained on thin cellulosic fibers: Electric field induced microrotors and N-I transition

We directly visualize the response of nematic liquid crystal drops of toroidal topology threaded in cellulosic fibers, suspended in air, to an AC electric field and at different temperatures over the N-I transition. This new liquid crystal system can exhibit non-trivial point defects, which can be energetically unstable against expanding into ring defects depending on the fiber constraining geometries. The director anchoring tangentially near the fiber surface and homeotropically at the air interface makes a hybrid shell distribution that in turn causes a ring disclination line around the main axis of the fiber at the center of the droplet. Upon application of an electric field, E, the disclination ring first expands and moves along the fiber main axis, followed by the appearance of a stable "spherical particle" object orbiting around the fiber at the center of the liquid crystal drop. The rotation speed of this particle was found to vary linearly with the applied voltage. This constrained liquid crystal geometry seems to meet the essential requirements in which soliton-like deformations can develop and exhibit stable orbiting in three dimensions upon application of an external electric field. On changing the temperature the system remains stable and allows the study of the defect evolution near the nematic-isotropic transition, showing qualitatively different behaviour on cooling and heating processes. The necklaces of such liquid crystal drops constitute excellent systems for the study of topological defects and their evolution and open new perspectives for application in microelectronics and photonics.

Production of recombinant protein hLIF in static and dynamic systems of animal cell culture

Thesis for the Degree of Master of Science in Biotechnology Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Integrating protein structural information

Dissertação apresentada para obtenção de Grau de Doutor em Bioquímica,Bioquímica Estrutural, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Protein folding, metal ions and conformational states: the case of a di-cluster ferredoxin

Dissertation presented to obtain the PhD degree in Biochemistry at the Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa

Phenylketonuria: Protein content and amino acids profile of dishes for phenylketonuric patients. The relevance of phenylalanine

Phenylketonuria is an inborn error of metabolism, involving, in most cases, a deficient activity of phenylalanine hydroxylase. Neonatal diagnosis and a prompt special diet (low phenylalanine and natural-protein restricted diets) are essential to the treatment. The lack of data concerning phenylalanine contents of processed foodstuffs is an additional limitation for an already very restrictive diet. Our goals were to quantify protein (Kjeldahl method) and amino acid (18) content (HPLC/fluorescence) in 16 dishes specifically conceived for phenylketonuric patients, and compare the most relevant results with those of several international food composition databases. As might be expected, all the meals contained low protein levels (0.67–3.15 g/100 g) with the highest ones occurring in boiled rice and potatoes. These foods also contained the highest amounts of phenylalanine (158.51 and 62.65 mg/100 g, respectively). In contrast to the other amino acids, it was possible to predict phenylalanine content based on protein alone. Slight deviations were observed when comparing results with the different food composition databases.

Nutritional composition of low protein and phenylalanine-restricted dishes prepared for phenylketonuric patients

Nutritional management is essential for Phenylketonuria (PKU) treatment, consisting in a semi-synthetic and low phenylalanine (Phe) diet, which includes strictly controlled amounts of low protein natural foods (essentially fruits and vegetables) supplemented with Phe-free protein substitutes and dietetic low-protein products. PKU diet has to be carefully planned, providing the best ingredient combinations, so that patients can achieve good metabolic control and an adequate nutritional status. Hereupon, it is mandatory to know the detailed composition of natural and/or cooked foodstuffs prepared specifically for these patients. We intended to evaluate sixteen dishes specifically prepared for PKU patients, regarding the nutritional composition, Phe and tyrosine (Tyr) contents, fatty acids profile, and vitamins E and B12 amounts. The nutritional composition of the cooked samples was 15.5–92.0 g/100 g, for moisture; 0.7–3.2 g/100 g, for protein; 0.1–25.0 g/100 g, for total fat; and 5.0–62.0 g/100 g, for total carbohydrates. Fatty acids profile and vitamin E amount reflected the type of fat used. All samples were poor in vitamin B12 (0.3–0.8 μg/100 g). Boiled rice presented the highest Phe content: 50.3 mg/g of protein. These data allow a more accurate calculation of the diet portions to be ingested by the patients according to their individual tolerance.