Artefacts induced on c-type haem proteins by electrode surfaces

J Biol Inorg Chem (2011) 16:209–215 DOI 10.1007/s00775-010-0717-z

Enzymatic activity mastered by altering metal coordination spheres

J Biol Inorg Chem (2008) 13:1185–1195 DOI 10.1007/s00775-008-0414-3

Doença de Chagas congênita: avaliação imunocitoquímica do parasitismo em tecidos de feto macerado

O método imunocitoquímico de peroxidase anti-peroxídase é aplicado, pela primeira vez, em tecidos de natimorto macerado de II-III graus para coloração específica de amastigotas do Trypanosoma cruzi, em caso de doença de Chagas congênita. Os resultados mostram que o encontro de ninhos de amastigotas é muito facilitado pelo método, mesmo em tecidos autolisados, onde a morfologia dos parasitas geralmente se mostra muito alterada; o método é recomendável, também nestes casos, para a avaliação mais precisa da intensidade do parasitismo.

Características das células parasitadas pelo Trypanosoma cruzi na parede da veia central das supra-renais de Chagásicos crônicos

Foram analisados os aspectos das células musculares parasitadas pelo Trypanosoma cruzi, na veia da supra-renal de chagásicos crônicos, através de exame ao microscópio óptico de lâminas coradas pela hematoxilina-eosina (HE), PAS, Feulgen e peroxidase-antiperoxidase (PAP) para antigenos do T. cruzi. Além das modificações nucleares descritas anteriormente, os leiomiócitos parasitados exibem alterações citoplasmáticas que podem ser vistas mesmo em células que albergam poucos parasitas. As formas amastigotas geralmente estão envoltas por halo claro e o citoplasma restante adquire aspecto granuloso ou reticular, basófilopelo HE, sendo sempre PAS e Feulgen negativos. Estes dados sugerem que o material basófilo no citoplasma deve ser RNA ribossômico. A periferia dos ninhos que mostram uma "membrana" com reação do PAP para antigenos do T. cruzi fortemente positiva, poderia ser devida a reação cruzada de material celular rechaçado para a periferia ou a difusão de antigenos do T. cruzi e sua adsorção à periferia celular. O material citoplasmático PAP positivo poderia resultar de artefato, de reação imunocitoquimica cruzada, de antigenos tripanossomóticos difundidos ou de antigenos tripanossoma-simile resultantes de interações entre o leiomiócito e o parasita.

Immunocytochemical identification of leishmania and Trypanosoma cruzi amastigotes in situ with homologous and heterologous polyclonal antibodies

The unlabelled antibody peroxidase-antiperoxidase method was used to study the immunocytochemical properties of Leishmania and Trypanosoma cruzi amastigotes in situ after tissues had been submitted to different fixation procedures. Antisera were obtained from rabbits chronically infected with different strains of T. cruzi or immunized with L. mexicana amazonensis and L. braziliensis guyanensis, and were applied on 5 µm thick sections. T. cruzi antigens were well stained by the three anti-T. cruzi sera and the two anti-heis.hmama.sera at optimum dilution between 1:1,000 and 1:2,000, regardless the parasite strain. Differently, the leishmanial antigens were revealed by Leishmania sera only at low dilutions (between 1:60 -1:160), whereas the anti-T. cruzi sera, at these low dilutions, gave rather weak stainings. Although there is no clear explanation for this immunocytochemical "reverse-monodirectional" cross-reactivity between Leishmania and T. cruzi, the present results show that polyclonal antibodies agains Leishmania species, when used for immunocytochemical detection of these parasites in situ, react more strongly with T. cruzi amastigotes than with the homologous amastigotes.

Imunobiossensores capacitivos interdigitais com camadas sensíveis micro e nano-estruturadas

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Prevalência de paracoccidioidomicose-infecção determinada através de teste sorológico em doadores de sangue na região Noroeste do Paraná, Brasil

No presente trabalho, empregamos uma metodologia de alta sensibilidade, o teste de ELISA clássico, com o objetivo de avaliar os níveis de anticorpos anti Paracoccidioides brasiliensis em indivíduos saudáveis, residentes na região Noroeste do Paraná. Foram analisados 680 soros de doadores de sangue aprovados pela triagem sorológica clássica de Bancos de Sangue. Os doadores eram residentes em quatro municípios. O exo-antígeno empregado foi o Ag7dias, soros diluídos 1/400, conjugado anti-IgG humana marcada com peroxidase e revelada com orto-fenilenodiamina. Foi possível detectar 181 (27%) soros com níveis de anticorpos acima do valor de corte. Essa porcentagem variou entre os municípios amostrados: Campo Mourão, Maringá, Cianorte e Umuarama. Nossos resultados, aliados às condições climáticas e geográficas favoráveis, além de relatos anteriores de casos de PCM autóctones da região, permitem sugerir que a região Noroeste do Paraná seja região endêmica para PCM.

Avaliação do uso de oligossacáridos na conservação pós-colheita de frutos

Os frutos são altamente perecíveis, recorrendo-se à conservação em atmosferas refrigeradas, prolongando o tempo de vida e permitindo o transporte e colocação faseada no mercado, reduzindo os desperdícios. O stress oxidativo leva à produção de metabolitos e enzimas antioxidativas pelas células vegetais como defesa. Neste estudo testaram-se os oligossacáridos de alginato de sódio e quitosano, produzidos por irradiação dos polissacáridos. Assumiu-se a hipótese que, estes compostos biologicamente ativos de estrutura similar a produtos de degradação de polissacáridos de paredes celulares de plantas e fungos, simulem condições de stress, funcionando como moléculas sinalizadoras acionando respostas para manter a homeostasia redox. Estes compostos foram testados como potenciais alternativas para aumentar o tempo de vida dos frutos quando aplicados em conservação pós-colheita. Este efeito foi avaliado em kiwis em dois estados distintos de maturação. No estado menos avançado, foram usados oligossacáridos provenientes de ambos os compostos; o tratamento com oligossacáridos derivados de alginato de sódio foi mais eficaz, retardando significativamente a perda de firmeza. No estado mais avançado foram também tratados com soluções de polissacáridos; os polissacáridos e oligossacáridos provenientes de quitosano foram os mais eficazes, causando menor amolecimento da polpa. A capacidade antioxidante foi determinada nas amostras que tinham sido sujeitas à temperatura de refrigeração. O teste DPPH mostrou uma redução da capacidade antioxidativa dependendo do estado de maturação, oligossacárido utilizado e tempo de tratamento. A atividade de enzimas envolvidas no stress antioxidativo foi determinada nas amostras/tratamentos que revelaram diferenças significativas na firmeza. A atividade da peroxidase de ascorbato e da catalase foi significativamente ativada em resposta aos tratamentos aplicados em kiwis de maturação mais avançado; a atividade da polifenoloxidase não foi significativamente ativada. A análise sensorial mostrou que os tratamentos reduziram a aceitação pelo “painel de provadores”.

Digital microfluidic devices: the role of the dielectric layer

Digital microfluidics (DMF) is a field which has emerged in the last decade as a re-liable and versatile tool for sensing applications based on liquid reactions. DMF allows the discrete displacement of droplets, over an array of electrodes, by the application of voltage, and also the dispensing from a reservoir, mixing, merging and splitting fluidic operations. The main drawback of these devices is due to the need of high driving volt-ages for droplet operations. In this work, alternative dielectric layers combinations were studied aiming the reduction of these driving voltages. DMF chips were designed, pro-duced and optimized according to the theory of electrowetting-on-dielectric, adopting different combinations of parylene-C and tantalum pentoxide (Ta2O5) as dielectric ma-terials, and Teflon as hydrophobic layer. With both devices’ configurations, i.e., Parylene as single dielectric, and multilayer chips combining Parylene and Ta2O5, it was possible to perform all the fluidic opera-tions in the microliter down to hundreds of nanoliters range. Multilayer chips presented significant reduction on driving voltages for droplet op-erations in silicone oil filler medium: from 70 V (parylene only) down to 30 V (parylene/Ta2O5) for dispensing; and from 50 V (parylene only) down to 15 V (parylene/Ta2O5) for movement. Peroxidase colorimetric reactions were successfully performed as proof-of-concept, using multilayer configuration devices.

Digital microfluidics on paper

This thesis is one of the first reports of digital microfluidics on paper and the first in which the chip’s circuit was screen printed unto the paper. The use of the screen printing technique, being a low cost and fast method for electrodes deposition, makes the all chip processing much more aligned with the low cost choice of paper as a substrate. Functioning chips were developed that were capable of working at as low as 50 V, performing all the digital microfluidics operations: movement, dispensing, merging and splitting of the droplets. Silver ink electrodes were screen printed unto paper substrates, covered by Parylene-C (through vapor deposition) as dielectric and Teflon AF 1600 (through spin coating) as hydrophobic layer. The morphology of different paper substrates, silver inks (with different annealing conditions) and Parylene deposition conditions were studied by optical microscopy, AFM, SEM and 3D profilometry. Resolution tests for the printing process and electrical characterization of the silver electrodes were also made. As a showcase of the applications potential of these chips as a biosensing device, a colorimetric peroxidase detection test was successfully done on chip, using 200 nL to 350 nL droplets dispensed from 1 μL drops.

Autoantibody profile in individuals with chronic hepatitis C

IntroductionAutoantibodies are often produced during infection with chronic hepatitis C virus (HCV), but it remains controversial whether they influence the biochemical profile and histological features of this disease. Therefore, this current study sought to describe these autoantibodies and evaluate their impact on the clinical and histological presentation of hepatitis C.MethodsThis cross-sectional analytical study assessed patients with HCV (RNA+) from October 2011 to July 2012.ResultsThis study included 66 patients, with a mean age of 53.2±10.5 years. Of these patients, 60.6% were male, and 54.3% presented with genotype 1. Non-organ-specific autoantibodies (NOSA) were detected in 24% of the patients; of these, 7.6% were anti-mitochondrial antibodies (AMA+), 26.7% were anti-smooth muscle antibodies (SMA+) and 6.8% were liver kidney microsomal type 1 antibodies (LKM1+). With respect to the thyroid autoantibodies, 7.4% were anti-peroxidase (ATPO+) antibodies, and none were anti-thyroglobulin (ATG+) antibodies. Regarding celiac disease autoantibodies, 5.8% were endomysial antibodies (EMA+), and no transglutaminase (TTG+) antibodies were detected. Cryoglobulins were found in 2.1% of patients. When NOSA+ individuals were compared to patients without the presence of NOSAs, they exhibited higher median alkaline phosphatase (0.7 vs. 0.6 xULN; p=0.041), lower median platelet counts (141,500.0 vs. 180,500.0/mm3; p=0.036), lower mean prothrombin activity (72.6±11.5% vs. 82.2±16.0%; p=0.012) and an increased prevalence of significant fibrosis (E≥2) (45.5% vs. 18.2%; p=0.012). There was also a tendency for a greater proportion of NOSA+ cases to have marked periportal activity (APP≥3) (44.5% vs. 15.6%; p=0.087).ConclusionsIn addition to the high prevalence of autoantibodies associated with HCV infection, it was observed that NOSA positivity was associated with a more severe histological and biochemical profile of hepatitis C infection.

Parasitic thyroid nodule in a patient with Hashimoto's chronic thyroiditis

A case of parasitic thyroid nodule is presented. The patient was a non symptomatic 53-year-old white woman, on irregular course of L-thyroxine to treat hypothyroidism due to Hashimoto's thyroiditis. Without a history of thyroid trauma or surgery, she presented a 1.6 x 0.7 x 0.5cm right pre-laryngeal lymph node-like mass which, on ultrasonography, appeared distinct from the gland. TSH, thyroid peroxidase antibody and thyroglobulin antibody serum levels were elevated and T4-free level was normal. Thyroid and total body 99mTc isonitrile scintiscan showed a topic thyroid without radionuclide uptake in the nodule. Fine-needle aspiration of the nodule showed epithelial cells with nuclear atypia and oncocytic changes plus intense lymphoid infiltration and germinative center formation, simulating lymph node metastasis of papillary thyroid carcinoma. Conventional biopsy revealed a parasitic thyroid nodule with Hashimoto's chronic thyroiditis. Parasitic thyroid nodule must always be remembered so that unnecessary surgical assessment and undesirable sequels may be avoided.

C-cells in colloid goiter

PURPOSE: The aim of this investigation was to quantitatively evaluate C-cells in colloid goiters, analyzing 36 thyroids that were obtained through thyroidectomy from 24 patients with goiter and 12 normal glands from adult patients without thyroid disease, which were used as the control group. MATERIAL AND METHODS: On average, 6 different thyroid areas were sampled and labeled by immunohistochemistry with a monoclonal anticalcitonin antibody, utilizing the avidin-biotin-peroxidase complex. C-cells were counted in fields measuring 1 square centimeter, and the mean number of cells per field was then calculated. Data were statistically analyzed using the Mann-Whitney test. RESULTS: In the colloid goiter group, the number of C-cells ranged from 0 to 23 per field, while in normal controls they ranged from 20 to 148 per field. CONCLUSIONS: These results demonstrate a significant decrease of C-cell number in the colloid goiter group compared with control group, indicating that the hyperplastic process is restricted to follicular cells, to the detriment of C-cells, which probably cease to receive trophic stimuli.

Enzymatic treatments to improve mechanical properties and surface hydrophobicity of jute fiber membranes

Fiber membranes prepared from jute fragments can be valuable, low cost, and renewable. They have broad application prospects in packing bags, geotextiles, filters, and composite reinforcements. Traditionally, chemical adhesives have been used to improve the properties of jute fiber membranes. A series of new laccase, laccase/mediator systems, and multi-enzyme synergisms were attempted. After the laccase treatment of jute fragments, the mechanical properties and surface hydrophobicity of the produced fiber membranes increased because of the cross-coupling of lignins with ether bonds mediated by laccase. The optimum conditions were a buffer pH of 4.5 and an incubation temperature of 60 °C with 0.92 U/mL laccase for 3 h. Laccase/guaiacol and laccase/alkali lignin treatments resulted in remarkable increases in the mechanical properties; in contrast, the laccase/2,2-azino-bis-(3-ethylthiazoline-6-sulfonate) (ABTS) and laccase/2,6-dimethoxyphenol treatments led to a decrease. The laccase/ guaiacol system was favorable to the surface hydrophobicity of jute fiber membranes. However, the laccase/alkali lignin system had the opposite effect. Xylanase/laccase and cellulase/laccase combined treatments were able to enhance both the mechanical properties and the surface hydrophobicity of jute fiber membranes. Among these, cellulase/laccase treatment performed better; compared to mechanical properties, the surface hydrophobicity of the jute fiber membranes showed only a slight increase after the enzymatic multi-step processes.

Determination of catalase activity and its inhibition by a simple manometric method

[Excerpt] Introduction: There has been a considerable amount of controversy about the use of manometric methods to measure catalase activity. As Maehly and Chance point out in their excellent review] the advantages of these methods is "... that they can be used for any kind of biological material, and purification of the enzyme is not required. The assay is independent of small amounts of peroxidase activity. It is fairly simple to perform, it is rapid and it can be adapted to continuous reading of the reaction". A variety of drawbacks are also listed by the same authors, viz, the inactivation of the enzyme under the experimental conditions and the time lag before a constant rate of oxygen evolution is reached. [...]