Electrochemical studies on small electron transfer proteins using membrane electrodes

Journal of Electroanalytical Chemistry 541 (2003) 153-162

Structural and mechanistic studies of iron containing proteins

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

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

Substituted P-Phenylene Ethynylene trimers as fluorescent sensors for nitroaromatic explosives

New sensory materials based on p-phenylene ethynylene trimers integrating calix[4]arene receptors (CALIX-PET) and tert-butylphenol (TBP-PET) moieties have been synthesized and their sensitivity and selectivity for the detection of nitroaromatic compounds (NACs) such as nitrobenzene (NB), 2,4-dinitrotoluene (2,4-DNT), 2,4,6-trinitrotoluene (TNT) and picric acid (PA) investigated in fluid phase and solid-state. It was found that both fluorophores displayed high sensitivities toward NACs detection in solution as evaluated by the Stern-Volmer formalism. For all the tested explosives, the ratio of fluorescence intensities (F-0/F) is a linear function of the quencher concentration only after appropriate correction of fluorescence quenching data for inner-filter effects. The quenching efficiencies for CALIX-PET and TBP-PET follow the order PA >> TNT > DNT > NB, which correlate well with the quenchers electron affinities as evaluated from their LUMOs energies thereby suggesting a photoinduced electron transfer as the dominant mechanism of fluorescence quenching. The selectivity of these sensors was checked against exemplar interferents possessing differentiated electronic properties (benzoic acid, 2,4-dichlorophenol and benzoquinone) and reduced quenching activity was detected. The quenching efficiencies and response times of the two fluorophores in the solid-state toward NB, 2,4-DNT and TNT vapors were evaluated through steady-state fluorescence quenching experiments with the materials dispersed in polymeric matrices or as neat films. The most significant fluorescence quenching responses were achieved for drop-casted films of TBP-PET upon exposure to nitroaromatics.

Peroxidase antibody test for mucocutaneous leishmaniasis serology performance indexes and comparison with a fluorescent antibody test: Comparação com o desempenho da reação de imunofluorescência

Performance indexes of the peroxidase antibody test were compared to that of the fluorescent antibody test. The peroxidase antibody test had a statistically higher sensitivity and negative predictive value and a higher efficiency than the fluorescent antibody test but its specificity and positive predictive value were within the 95% confidence limits for the values found for the fluorescent antibody test. Such differences did not change when Chagas' disease and visceral leishmaniasis sera were included in index calculations. Statistical analysis showed that the two tests have a substantial degree of agreement but the immunofluorescent test had a specificity index and a positive predictive value equal to 100.0% when Chagas' disease and visceral leishmaniasis sera were not included in the calculations of the performance index; in this instance, a positive test result equals a disclosure of the disease attribute due to the inexistence of false positive results. The enzyme/ protein ratio of the peroxidase conjugate, resulting in heavy or light-labeled conjugates may pose technical problems to its use in serology tests.

Elution of proteins from polyacrylamide eels: a simple and economic procedure

A simplified methodology for the quantitative electroelution of proteins from polyacrylamide gels is described. After staining with Coomassie Brilliant Blue R 250, the identified bands are excised from the gel and the proteins eluted using a procedure developed for use in conventional tube gel electrophoresis equipment.

Simplified fluorescent inhibition microtest for the titration of rabies neutralizing antibodies

A simplified fluorescence inhibition microtest (SFIMT) was standardized for the evaluation of antirabies serum neutralizing antibodies based on the rapid fluorescent focus inhibition test (RFFIT) and the fluorescence inhibition microtest (FIMT). The simplified test showed reproductibility similar to that of the FIMT with advantages as easier executation and quicker reading. A simple pre-treatment of Brazilian microplates produced for immune enzymatic assays (PROSIL) gave equivalent results and substantial coast reduction, in relation to imported plates (DIFCO). The simplified test can be easily implemented in less sophisticated laboratories, as alternative to the mouse serum neutralization test, still the most largely employed in Brazil, or even to others as RFFIT and FIMT.

Induction of iron regulated proteins during normal growth of Neisseria meningitidis in a chemically defined medium

The expression of iron regulated proteins (IRPs) in vitro has been obtained in the past by adding iron chelators to the culture after bacterial growth, in the presence of an organic iron source. We have investigated aspects concerning full expression of the meningococcal IRPs during normal growth, in defined conditions using Catlin medium, Mueller Hinton and Tryptic Soy Broth (TSB). The expression of IRPs varied between different strains with respect to Ethylenediamine Di-ortho-Hidroxy-phenyl-acetic acid (EDDA) concentrations, and according to culture medium, and also between different lots of TSB. For each strain, a specific set of IRPs were expressed and higher EDDA concentrations, or addition of glucose, or use of different culture media did not resulted in a differential expression of IRPs. We were not able to grow N. meningitidis under normal growth conditions using Desferal. We looked for a good yield of outer membrane vesicles (OMVs) expressing IRPs in iron-deficient Catlin medium containing EDDA and Hemin. Culture for 32 h at 30ºC after growing for 16 h at 37ºC supported good bacterial growth. Bacterial lysis was noted after additional 24 h at 30ºC. Approximately 4 times more OMVs was recoverable from a culture supernatant after 24 h at 30ºC than from the cells after 16 h at 37ºC. The IRP were as well expressed in OMVs from culture supernatant obtained after 24 h at 30ºC as from the cells after 16 h at 37ºC.

New fluorescent chemosensors based on bio-inspired ligands and macrocycles: from single molecules to nanoparticles

Dissertação apresentada para obtenção do grau de Doutor em Biotecnologia pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia. A presente dissertação foi preparada no âmbito do protocolo de acordo bilateral de educação avançada (ERASMUS) entre a Universidade de Vigo e a Universidade Nova de Lisboa

A proteomic approach toward the selection of proteins with enhanced intrinsic conformational stability

Journal of Proteome Research (2006)5: 2720-2726

Role of a novel disulfide bridge within the all-beta fold of soluble Rieske proteins

Journal of Biological Inorganic Chemistry (2010)15: 271-281

Natural and amyloid self-assembly of S100 proteins: structural basis of functional diversity

The S100 proteins are 10-12 kDa EF-hand proteins that act as central regulators in a multitude of cellular processes including cell survival, proliferation, differentiation and motility. Consequently, many S100 proteins are implicated and display marked changes in their expression levels in many types of cancer, neurodegenerative disorders, inflammatory and autoimmune diseases. The structure and function of S100 proteins are modulated by metal ions via Ca2+ binding through EF-hand motifs and binding of Zn2+ and Cu2+ at additional sites, usually at the homodimer interfaces. Ca2+ binding modulates S100 conformational opening and thus promotes and affects the interaction with p53, the receptor for advanced glycation endproducts and Toll-like receptor 4, among many others. Structural plasticity also occurs at the quaternary level, where several S100 proteins self-assemble into multiple oligomeric states, many being functionally relevant. Recently, we have found that the S100A8/A9 proteins are involved in amyloidogenic processes in corpora amylacea of prostate cancer patients, and undergo metal-mediated amyloid oligomerization and fibrillation in vitro. Here we review the unique chemical and structural properties of S100 proteins that underlie the conformational changes resulting in their oligomerization upon metal ion binding and ultimately in functional control. The possibility that S100 proteins have intrinsic amyloid-forming capacity is also addressed, as well as the hypothesis that amyloid self-assemblies may, under particular physiological conditions, affect the S100 functions within the cellular milieu.

Giardia duodenalis: INTER-STRAIN VARIABILITY OF PROTEINS, ANTIGENS, PROTEASES, ISOENZYMES AND NUCLEIC ACIDS

Giardia duodenalis isolates from asymptomatic or symptomatic patients and from animals present similarities and differences in the protein composition, antigenic profile, pattern of proteases and isoenzymes, as well as in nucleic acids analysis. In the present overview, these differences and similarities are reviewed with emphasis in the host-parasite interplay and possible mechanisms of virulence of the protozoon.

Protection of C57BL/10 mice by vaccination with association of purified proteins from Leishmania (Leishmania) amazonensis

In the past few years, induction of protective immunity to cutaneous leishmaniasis has been attempted by many researchers using a variety of antigenic preparations, such as living promastigotes or promastigote extracts, partially purified, or defined proteins. In this study, eleven proteins from Leishmania (Leishmania) amazonensis (LLa) with estimated molecular mass ranging from 97 to 13.5kDa were isolated by polyacrylamide gel electrophoresis and electro-elution. The proteins were associated as vaccine in different preparations with gp63 and BCG (Bacilli Calmette-Guérin). The antigenicity of these vaccines was measured by their ability to induce the production of IFN-g by lymphocyte from subjects vaccinated with Leishvacinâ . The immunogenicity was evaluated in vaccinated mice. C57BL/10 mice were vaccinated with three doses of each vaccine consisting of 30 mg of each protein at 15 days interval. One hundred mg of live BCG was only used in the first dose. Seven days after the last dose, they received a first challenge infection with 105 infective promastigotes and four months later, a second challenge was done. Two months after the second challenge, 42.86% of protection was obtained in the group of mice vaccinated with association of proteins of gp63+46+22kDa, gp63+13.5+25+42kDa, gp63+46+42kDa, gp63+66kDa, and gp63+97kDa; 57.14% of protection was demonstrated with gp63+46+97+13.5kDa, gp63+46+97kDa, gp63+46+33kDa, and 71.43% protection for gp63 plus all proteins. The vaccine of gp63+46+40kDa that did not protect the mice, despite the good specific stimulation of lymphocytes (LSI = 7.60) and 10.77UI/ml of IFN-g production. When crude extract of L. (L.) amazonensis was used with BCG a 57.14% of protection was found after the first challenge and 28.57% after the second, the same result was observed for gp63. The data obtained with the vaccines can suggest that the future vaccine probably have to contain, except the 40kDa, a cocktail of proteins that would protect mice against cutaneous leishmaniasis.

Metals in proteins from sulphate-reducing bacteria: adenylate kinase and ATP sulfurylase. Proteins containing cobalt, zinc, iron (II) ions

A Thesis submitted at the Faculty Science and Technology of the New University of Lisbon for a degree in Doctor of Philosophy in Biochemistry with specialization in Physical Biochemistry