Pseudomonas aeruginosa amidase: Aggregation in recombinant Escherichia coli

The effect of cultivation parameters such as temperature incubation, IPTG induction and ethanol shock on the production of Pseudomonasaeruginosa amidase (E.C.3.5.1.4) in a recombinant Escherichia coli strain in LB ampicillin culture medium was investigated. The highest yield of solubleamidase, relatively to other proteins, was obtained in the condition at 37 degrees C using 0.40 mM IPTG to induce growth, with ethanol. Our results demonstrate the formation of insoluble aggregates containing amidase, which was biologically active, in all tested growth conditions. Addition of ethanol at 25 degrees C in the culture medium improved amidase yield, which quantitatively aggregated in a biologically active form and exhibited in all conditions an increased specific activity relatively to the soluble form of the enzyme. Non-denaturing solubilization of the aggregated amidase was successfully achieved using L-arginine. The aggregates obtained from conditions at 37 degrees C by Furier transform infrared spectroscopy (FTIR) analysis demonstrated a lower content of intermolecular interactions, which facilitated the solubilization step applying non-denaturing conditions. The higher interactions exhibited in aggregates obtained at suboptimal conditions compromised the solubilization yield. This work provides an approach for the characterization and solubilization of novel reported biologically active aggregates of this amidase.

Protein-ligand docking study: diterpenes from Juniperus brevifolia as anticancer and antimicrobial agentes

REDCAT: Natural Products and related Redox Catalysts: Basic Research and Applications in Medicine and Agriculture, Aveiro, 25-27 Novembro de 2012.

Heat transfer coefficients from Newtonian and non-Newtonian fluids flowing in laminar regime in a helical coil

This study aimed to carry out experimental work to obtain, for Newtonian and non-Newtonian fluids, heat transfer coefficients, at constant wall temperature as boundary condition, in fully developed laminar flow inside a helical coil. The Newtonian fluids studied were aqueous solutions of glycerol, 25%, 36%, 43%, 59% and 78% (w/w) and the non-Newtonian fluids aqueous solutions of carboxymethylcellulose (CMC), a polymer, with concentrations 0.1%, 0.2%, 0.3%, 0.4% and 0.6% (w/w) and aqueous solutions of xanthan gum (XG), another polymer, with concentrations 0.1% and 0.2% (w/w). According to the rheological study performed, the polymer solutions had shear thinning behavior and different values of elasticity. The helical coil used has internal diameter, curvature ratio, length and pitch, respectively: 0.004575 m, 0.0263, 5.0 m and 11.34 mm. The Nusselt numbers for the CMC solutions are, on average, slightly higher than those for Newtonian fluids, for identical Prandtl and generalized Dean numbers. As outcome, the viscous component of the shear thinning polymer tends to potentiate the mixing effect of the Dean cells. The Nusselt numbers of the XG solutions are significant lower than those of the Newtonian solutions, for identical Prandtl and generalized Dean numbers. Therefore, the elastic component of the polymer tends to diminish the mixing effect of the Dean cells. A global correlation, for Nusselt number as a function of Péclet, generalized Dean and Weissenberg numbers for all Newtonian and non-Newtonian solutions studied, is presented.

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.

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.

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

Defective protein folding and function in metabolic disorders: studies on the mitochondrial flavoenzyme ETF

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

The role of ribonuclease R in bacterial adaptation to cold shock

RESUMO:Os microrganismos reagem à súbita descida de temperatura através de uma resposta adaptativa específica que assegura a sua sobrevivência em condições desfavoráveis. Esta adaptação inclui alterações na composição da membrana, na maquinaria de tradução e transcrição. A resposta ao choque térmico pelo frio induz uma repressão da transcrição. No entanto, a descida de temperatura induz a produção de um grupo de proteínas específicas que ajudam a ajustar/re-ajustar o metabolismo celular às novas condições ambientais. Em E. coli o processo de adaptação demora apenas quatro horas, no qual um grupo de proteínas específicas são induzidas. Depois desde período recomeça lentamente a produção de proteínas.A ribonuclease R, uma das proteínas induzidas durante o choque térmico pelo frio, é uma das principais ribonucleases em E. coli envolvidas na degradação do RNA. É uma exoribonuclease que degrada RNA de cadeia dupla, possui funções importantes na maturação e “turnover” do RNA, libertação de ribossomas e controlo de qualidade de proteínas e RNAs. O nível celular desta enzima aumenta até dez vezes após exposição ao frio e estabiliza em células na fase estacionária. A capacidade de degradar RNA de dupla cadeia é importante a baixas temperaturas quando as estruturas de RNA estão mais estáveis. No entanto, este mecanismo é desconhecido. Embora a resposta específica ao “cold shock” tenha sido descoberta há mais de duas décadas e o número de proteínas envolvidas sugerirem que esta adaptação é rápida e simples, continuamos longe de compreender este processo. No nosso trabalho pretendemos descobrir proteínas que interactuem com a RNase R em condições ambientais diferentes através do método “TAP-tag” e espectrometria de massa. A informação obtida pode ser utilizada para deduzir algumas das novas funções da RNase R durante a adaptação bacteriana ao frio e durante a fase estacionária. Mais importante ainda, RNase R poderá ser recrutada para um complexo de proteínas de elevado peso molecular durante o “cold-shock”.------------ABSTRACT:Microorganisms react to the rapid temperature downshift with a specific adaptative response that ensures their survival in unfavorable conditions. Adaptation includes changes in membrane composition, in translation and transcription machinery. Cold shock response leads to overall repression of translation. However, temperature downshift induces production of a set of specific proteins that help to tune cell metabolism and readjust it to the new environmental conditions. For Escherichia coli the adaptation process takes only about four hours with a relatively small set of specifically induced proteins involved. After this time, protein production resumes, although at a slower rate. One of the cold inducible proteins is RNase R, one of the main E. coli ribonucleases involved in RNA degradation. RNase R is an exoribonuclease that digest double stranded RNA, serves important functions in RNA maturation and turnover, release of stalled ribosomes by trans-translation, and RNA and protein quality control. The level of this enzyme increases about ten-fold after cold induction, and it is also stabilised in cells growing in stationary phase. The RNase R ability to digest structured RNA is important at low temperatures where RNA structures are stabilized but the exact role of this mechanism remains unclear. Although specific bacterial cold shock response was discovered over two decades ago and the number of proteins involved suggests that this adaptation is fast and simple, we are still far from understanding this process. In our work we aimed to discover the proteins interacting with RNase R in different environmental conditions using TAP tag method and mass spectrometry analysis. The information obtained can be used to deduce some of the new functions of RNase R during adaptation of bacteria to cold and in stationary growth phase. Most importantly RNase R can be recruited into a high molecular mass complex of protein in cold shock.

Electrophoretic protein patterns and numerical analysis of Candida albicans from the oral cavities of healthy children

The aim of this research was to evaluate the protein polymorphism degree among seventy-five C. albicans strains from healthy children oral cavities of five socioeconomic categories from eight schools (private and public) in Piracicaba city, São Paulo State, in order to identify C. albicans subspecies and their similarities in infantile population groups and to establish their possible dissemination route. Cell cultures were grown in YEPD medium, collected by centrifugation, and washed with cold saline solution. The whole-cell proteins were extracted by cell disruption, using glass beads and submitted to SDS-PAGE technique. After electrophoresis, the protein bands were stained with Coomassie-blue and analyzed by statistics package NTSYS-pc version 1.70 software. Similarity matrix and dendrogram were generated by using the Dice similarity coefficient and UPGMA algorithm, respectively, which made it possible to evaluate the similarity or intra-specific polymorphism degrees, based on whole-cell protein fingerprinting of C. albicans oral isolates. A total of 13 major phenons (clusters) were analyzed, according to their homogeneous (socioeconomic category and/or same school) and heterogeneous (distinct socioeconomic categories and/or schools) characteristics. Regarding to the social epidemiological aspect, the cluster composition showed higher similarities (0.788 < S D < 1.0) among C. albicans strains isolated from healthy children independent of their socioeconomic bases (high, medium, or low). Isolates of high similarity were not found in oral cavities from healthy children of social stratum A and D, B and D, or C and E. This may be explained by an absence of a dissemination route among these children. Geographically, some healthy children among identical and different schools (private and public) also are carriers of similar strains but such similarity was not found among other isolates from children from certain schools. These data may reflect a restricted dissemination route of these microorganisms in some groups of healthy scholars, which may be dependent of either socioeconomic categories or geographic site of each child. In contrast to the higher similarity, the lower similarity or higher polymorphism degree (0.499 < S D < 0.788) of protein profiles was shown in 23 (30.6%) C. albicans oral isolates. Considering the social epidemiological aspect, 42.1%, 41.7%, 26.6%, 23.5%, and 16.7% were isolates from children concerning to socioeconomic categories A, D, C, B, and E, respectively, and geographically, 63.6%, 50%, 33.3%, 33.3%, 30%, 25%, and 14.3% were isolates from children from schools LAE (Liceu Colégio Albert Einstein), MA (E.E.P.S.G. "Prof. Elias de Melo Ayres"), CS (E.E.P.G. "Prof. Carlos Sodero"), AV (Alphaville), HF (E.E.P.S.G. "Honorato Faustino), FMC (E.E.P.G. "Prof. Francisco Mariano da Costa"), and MEP (E.E.P.S.G. "Prof. Manasses Ephraim Pereira), respectively. Such results suggest a higher protein polymorphism degree among some strains isolated from healthy children independent of their socioeconomic strata or geographic sites. Complementary studies, involving healthy students and their families, teachers, servants, hygiene and nutritional habits must be done in order to establish the sources of such colonization patterns in population groups of healthy children. The whole-cell protein profile obtained by SDS-PAGE associated with computer-assisted numerical analysis may provide additional criteria for the taxonomic and epidemiological studies of C. albicans.

Crystallization and crystallographic analysis of the apo form of the orange protein (ORP) from desulfovibrio gigas

Acta Crystallographica Section F Structural Biology and Crystallization Communications Volume 65, Part 8

A copper protein and a cytochrome bind at the same site on bacterial cytochrome c peroxidase

Biochemistry, 2004, 43 (46), pp 14566–14576 DOI: 10.1021/bi0485833

Evaluation of CSF Neurotransmitters and Folate in 25 Patients with Rett Disorder and Effects of Treatment

Background: Rett disorder (RD) is a progressive neurodevelopmental entity caused by mutations in the MECP2 gene. It has been postulated that there are alterations in the levels of certain neurotransmitters and folate in the pathogenesis of this disease. Here we re-evaluated this hypothesis. Patients and Methods: We evaluated CSF folate, biogenic amines and pterines in 25 RD patients. Treatment with oral folinic acid was started in those cases with low folate. Patients were clinically evaluated and videotaped up to 6 months after therapy. Results: CSF folate was below the reference values in 32% of the patients. Six months after treatment no clinical improvement was observed. Three of the four patients with the R294X mutation had increased levels of a dopamine metabolite associated to a particular phenotype. Three patients had low levels of a serotonin metabolite. Two of them were treated with fluoxetine and one showed clinical improvement. No association was observed between CSF folate and these metabolites, after adjusting for the patients age and neopterin levels. Conclusion: Our results support that folinic acid supplementation has no significant effects on the course of the disease. We report discrete and novel neurotransmitter abnormalities that may contribute to the pathogenesis of RD highlighting the need for further studies on CSF neurotransmitters in clinically and genetically well characterized patients.

Effects of severe protein restriction in levels of parasitemia and in mortality of mice accutely infected with Trypanosoma cruzi

Adult mice were submitted to different degrees of protein restriction for five weeks (4.75, 9.5,14.25 and 19% of protein in isocaloric diets with normal content of mineral and vitamins), being subsequently infected with two strains of Trypanosoma cruzi: 10(5) trypomastigotes of Y strain or 14(5) trypomastigotes of CL strain. The same diet was maintained for all animals and the infection wasfollowed up by evaluation of blood parasites, mortality and intensity of lesions in the heart and skeleton muscle. Only severe protein restriction (4.75%) induced decrease in resistance to the infection with both the Y and CL strains of T. cruzi, which resulted in higher parasitemia and mortality. The inflammatory lesions in heart and skeleton muscle were less extensive in groups with severe protein restriction despite the increased number of parasite in muscle cells. Depression of immune mechanisms could be responsiblefor the reduced resistance and reduced inflammatory reaction after T. cruzi infection in severely protein restricted animals.

Thermal conditions affecting heat transfer in FDM/FFE: a contribution towards the numerical modelling of the process

The performance of parts produced by Free Form Extrusion (FFE), an increasingly popular additive manufacturing technique, depends mainly on their dimensional accuracy, surface quality and mechanical performance. These attributes are strongly influenced by the evolution of the filament temperature and deformation during deposition and solidification. Consequently, the availability of adequate process modelling software would offer a powerful tool to support efficient process set-up and optimisation. This work examines the contribution to the overall heat transfer of various thermal phenomena developing during the manufacturing sequence, including convection and radiation with the environment, conduction with support and between adjacent filaments, radiation between adjacent filaments and convection with entrapped air. The magnitude of the mechanical deformation is also studied. Once this exercise is completed, it is possible to select the material properties, process variables and thermal phenomena that should be taken in for effective numerical modelling of FFE.

Optimum dietary protein requirement for Amazonian Tambaqui, Colossoma macropomum Cuvier, 1818, fed fish meal free diets

Fish meal free diets were formulated to contain graded protein levels as 25% (diet 1), 30% (diet 2), 35% (diet 3) and 40% (diet 4). The diets were fed to tambaqui juveniles (Colossoma macropomum) (46.4 ± 6.3g) in randomly designed recirculating systems for 60 days, to determine the optimum protein requirement for the fish. The final weight of the fish, weight gain (28.1, 28.5, 32.2, 28.0g) and specific growth rate increased (P>0.05) consistently with increasing dietary protein up to treatment with 35% protein diet and then showed a declining trend. Feed intake followed the same trend resulting in best feed efficiency (62.5%) in fish fed diet with 35% protein. Similarly, the protein intake increased significantly with increasing dietary protein levels and reduced after the fish fed with 35% protein; while protein efficiency ratio (2.28, 1.99, 1.87, 1.74) decreased with increasing dietary protein levels. Carcass ash and protein had linear relationship with dietary protein levels while the lipid showed a decreasing trend. Ammonia content (0.68, 0.73, 0.81, 1.21 mg L-1) of the experimental waters also increased (P<0.05) with increasing protein levels while pH, dissolved oxygen and temperature remained fairly constant without any clear pattern of inclination. Broken-line estimation of the weight gain indicated 30% protein as the optimum requirement for the fish.