Produção de óleos e fillers proteicos a partir da raspa tripa da indústria de curtumes

A indústria de curtumes é fortemente geradora de resíduos sólidos e águas residuais pelo que a necessidade de criar alternativas para a sua valorização e minimização é premente e constante. Este trabalho teve, como principal objectivo, o aproveitamento da raspa tripa, resíduo da indústria de curtumes. Para tal, a raspa tripa foi sujeita a um processo de hidrólise térmica e/ou enzimática para obtenção de gordura e hidrolisado proteico. Na hidrólise da raspa tripa foi estudada a influência de vários factores como a quantidade de enzima e de água, a temperatura e o tempo. Constatou-se que foi para a temperatura de 60ºC, um tempo de quatro horas, uma quantidade de 2% de enzima e 100% de água, relativamente à massa de raspa utilizada, que se obteve o melhor resultado com um rendimento de extracção de 93%. A partir da gordura obtida foram produzidos óleos por sulfatação e sulfitação, designados por óleos sulfatados e sulfitados, respectivamente. O hidrolisado proteico obtido foi concentrado por evaporação para aumentar o seu teor de sólidos totais. Com este foram produzidos gluproteicos e fillers, por acção de glutaraldeído e do efeito sinérgico de outros produtos. Os óleos, o hidrolisado concentrado e seus derivados foram testados no processo de engorduramento e recurtume do couro. A avaliação dos resultados foi realizada por comparação dos resultados dos testes físico-mecânicos das amostras de pele obtidas com valores de referência e por comparação com um ensaio padrão em que se utilizaram produtos de referência do mercado. Os resultados foram muitos satisfatórios visto que foram superiores aos valores de referência e também superiores aos valores obtidos para o padrão salvo raras excepções. Reconheceu-se assim a aplicabilidade destes produtos no processo de recurtume e engorduramento que também corresponderam no que respeita a características importantes do couro como a firmeza de flor e o toque. No decorrer do trabalho constatou-se que as peles tratadas com hidrolisado proteico apresentavam uma cor mais intensa, pelo que se efectuou um estudo de colorimetria através do método CIELAB. Comprovou-se que o hidrolisado proteico e os gluproteicos quando utilizados no processo de recurtume, por substituição do Fortan A40, intensificam a cor da pele. A valorização da raspa tripa resulta numa dupla vantagem para a indústria dos curtumes. Se por um lado diminui o impacto ambiental que esta origina, por outro lado possibilita a produção de produtos que podem ser substitutos de outros produtos químicos que têm que ser adquiridos por esta indústria.

SMYD3 contributes to a more aggressive phenotype of prostate cancer and targets Cyclin D2 through H4K20me3

Prostate cancer (PCa) is one of the most incident cancers worldwide but clinical and pathological parameters have limited ability to discriminate between clinically significant and indolent PCa. Altered expression of histone methyltransferases and histone methylation patterns are involved in prostate carcinogenesis. SMYD3 transcript levels have prognostic value and discriminate among PCa with different clinical aggressiveness, so we decided to investigate its putative oncogenic role on PCa.We silenced SMYD3 and assess its impact through in vitro (cell viability, cell cycle, apoptosis, migration, invasion assays) and in vivo (tumor formation, angiogenesis). We evaluated SET domain's impact in PCa cells' phenotype. Histone marks deposition on SMYD3 putative target genes was assessed by ChIP analysis.Knockdown of SMYD3 attenuated malignant phenotype of LNCaP and PC3 cell lines. Deletions affecting the SET domain showed phenotypic impact similar to SMYD3 silencing, suggesting that tumorigenic effect is mediated through its histone methyltransferase activity. Moreover, CCND2 was identified as a putative target gene for SMYD3 transcriptional regulation, through trimethylation of H4K20.Our results support a proto-oncogenic role for SMYD3 in prostate carcinogenesis, mainly due to its methyltransferase enzymatic activity. Thus, SMYD3 overexpression is a potential biomarker for clinically aggressive disease and an attractive therapeutic target in PCa.

Molecular epidemiology of a nosocomial outbreak due to Enterobacter cloacae and Enterobacter agglomerans in Campinas, São Paulo, Brazil

A total of 73 isolates (57 Enterobacter cloacae and 16 Enterobacter agglomerans), recovered during an outbreak of bacteremia in the Campinas area, São Paulo, Brazil, were studied. Of these isolates, 61 were from parenteral nutrition solutions, 9 from blood cultures, 2 from a sealed bottle of parenteral nutrition solution, and one was of unknown origin. Of the 57 E. cloacae isolates, 54 were biotype 26, two were biotype 66 and one was non-typable. Of 39 E. cloacae isolates submitted to ribotyping, 87.2% showed the same banding pattern after cleavage with EcoRI and BamHI. No important differences were observed in the antimicrobial susceptibility patterns among E. cloacae isolates exhibiting the same biotype, serotype and ribotype. All E. agglomerans isolates, irrespective of their origin, showed same patterns when cleaved with EcoRI and BamHI. The results of this investigation suggest an intrinsic contamination of parenteral nutrition solutions and incriminate these products as a vehicle of infection in this outbreak.

Mitosis and protein Na-terminal acetylation

Dissertation presented to obtain the Ph.D degree in Developmental Biology

Imunossensores Eletroquímicos para o Diagnóstico Precoce e Descentralizado do Cancro da Mama

O cancro é uma das principais causas de morte em todo o mundo. Entre as mulheres, o cancro da mama é o mais frequente. A deteção precoce do cancro é de extrema importância na medida em que pode aumentar as possibilidades de cura dos pacientes e contribuir para a diminuição da taxa de mortalidade desta doença. Um método que tem contribuído para a deteção precoce do cancro é a análise de biomarcadores. Biomarcadores associados ao cancro da mama, como o Recetor 2 do Fator de Crescimento Epidérmico Humano (HER2) e o Antigénio Carbohidratado 15-3 (CA 15-3), podem ser detetados através de dispositivos como os biossensores. Neste trabalho foram desenvolvidos dois imunossensores eletroquímicos para a análise de HER2 e CA 15-3. Para ambos os sensores foram utilizados, como transdutores, elétrodos serigrafados de carbono. A superfície destes transdutores foi nanoestruturada com nanopartículas de ouro. Foram realizados imunoensaios não-competitivos (do tipo sandwich) em ambos os imunossensores, cuja estratégia consistiu na (i) imobilização do respetivo anticorpo de captura na superfície nanoestruturada dos elétrodos, (ii) bloqueio da superfície com caseína, (iii) incubação com uma mistura do analito (HER2 ou CA 15-3) e o respetivo anticorpo de deteção biotinilado, (iv) adição de estreptavidina conjugada com fosfatase alcalina (S-AP; a AP foi utilizada como marcador enzimático), (v) adição de uma mistura do substrato enzimático (3-indoxil fosfato) e nitrato de prata, e (vi) deteção do sinal analítico através da redissolução anódica, por voltametria de varrimento linear, da prata depositada enzimaticamente. Com as condições experimentais otimizadas, foi estabelecida a curva de calibração para a análise de HER2 em soro, entre 15 e 100 ng/mL, obtendo-se um limite de deteção de 4,4 ng/mL. Para o CA 15-3 a curva de calibração (em solução aquosa) foi estabelecida entre 15 e 250 U/mL, obtendo-se um limite de deteção de 37,5 U/mL. Tendo em conta o valor limite (cutoff value) estabelecido para o HER2 (15 ng/mL) pode-se comprovar a possível utilidade do imunossensor desenvolvido para o diagnóstico precoce e descentralizado do cancro da mama. No caso do CA 15-3 serão necessários estudos adicionais para se poder avaliar a utilidade do imunossensor para o diagnóstico do cancro da mama.

Magnetic nanoparticles for biocatalysis and bioseparation

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Advantages of the rapid HIV-1 test in occupational accidents with potentially contaminated material among health workers

In occupational accidents involving health professionals handling potentially contaminated material, the decision to start or to continue prophylactic medication against infection by Human Immunodeficiency Virus (HIV) has been based on the ELISA test applied to a blood sample from the source patient. In order to rationalize the prophylactic use of antiretroviral agents, a rapid serologic diagnostic test of HIV infection was tested by the enzymatic immunoabsorption method (SUDS HIV 1+2, MUREX®) and compared to conventional ELISA (Abbott HIV-1/ HIV-2 3rd Generation plus EIA®). A total of 592 cases of occupational accidents were recorded at the University Hospital of Ribeirão Preto from July 1998 to April 1999. Of these, 109 were simultaneously evaluated by the rapid test and by ELISA HIV. The rapid test was positive in three cases and was confirmed by ELISA and in one the result was inconclusive and later found to be negative by ELISA. In the 106 accidents in which the rapid test was negative no prophylactic medication was instituted, with an estimated reduction in costs of US$ 2,889.35. In addition to this advantage, the good correlation of the rapid test with ELISA, the shorter duration of stress and the absence of exposure of the health worker to the adverse effects of antiretroviral agents suggest the adoption of this test in Programs of Attention to Accidents with Potentially Contaminated Material.

P System antigenic determiners expression in Ascaris lumbricoides

The P System antigens have been detected in numerous parasites, bacterias and viruses, nevertheless the clinical significance is still unknown. The aim was to study the presence of P1 antigenic determiners in A. lumbricoides extracts by means of the use of 6 different monoclonal antibodies of well-known concentrations and Ig class. We worked with 14 A. lumbricoides extracts. Inhibition Agglutination Test was made in a bromelin enzymatic medium and 4 ºC temperature. Titre, Score and Sensitivity Parameter were determined for each monoclonal antibody against red cells suspension used as revealing system. Ten extracts inhibited the agglutination of all anti P1 monoclonal antibodies. The 4 remaining extracts only inhibited the agglutination of some of them. It is demonstrated that the extracts have P1 activity. This activity is independent of titre, Score, Sensitivity Parameter, concentration and Ig class and it depends on the epitope at which the monoclonal antibody is directed.

Myoglobin-biomimetic electroactive materials made by surface molecular imprinting on silica beads and their use as ionophores in polymeric membranes for potentiometric transduction

Myoglobin (Mb) is among the cardiac biomarkers playing a major role in urgent diagnosis of cardiovascular diseases. Its monitoring in point-of-care is therefore fundamental. Pursuing this goal, a novel biomimetic ionophore for the potentiometric transduction of Mb is presented. It was synthesized by surface molecular imprinting (SMI) with the purpose of developing highly efficient sensor layers for near-stereochemical recognition of Mb. The template (Mb) was imprinted on a silane surface that was covalently attached to silica beads by means of self-assembled monolayers. First the silica was modified with an external layer of aldehyde groups. Then, Mb was attached by reaction with its amine groups (on the external surface) and subsequent formation of imine bonds. The vacant places surrounding Mb were filled by polymerization of the silane monomers 3-aminopropyltrimethoxysilane (APTMS) and propyltrimethoxysilane (PTMS). Finally, the template was removed by imine cleavage after treatment with oxalic acid. The results materials were finely dispersed in plasticized PVC selective membranes and used as ionophores in potentiometric transduction. The best analytical features were found in HEPES buffer of pH 4. Under this condition, the limits of detection were of 1.3 × 10−6 mol/L for a linear response after 8.0 × 10−7 mol/L with an anionic slope of −65.9 mV/decade. The imprinting effect was tested by preparing non-imprinted (NI) particles and employing these materials as ionophores. The resulting membranes showed no ability to detect Mb. Good selectivity was observed towards creatinine, sacarose, fructose, galactose, sodium glutamate, and alanine. The analytical application was conducted successfully and showed accurate and precise results.

Co-infection between influenza virus and flagellated bacteria

Trypsin is required in the hemagglutinin (HA) cleavage to in vitro influenza viruses activation. This HA cleavage is necessary for virus cell entry by receptor-mediated endocytosis. Bacteria in the respiratory tract are potential sources of proteases that could contribute to the cleavage of influenza virus in vivo. From 47 samples collected from horses, pigs, and from humans, influenza presence was confirmed in 13 and these samples demonstrated co-infection of influenza with flagellated bacteria, Stenotrophomonas maltophilia from the beginning of the experiments. Despite treatment with antibiotics, the bacteria remained resistant in several of the co-infected samples (48.39%). These bacteria, considered opportunistic invaders from environmental sources, are associated with viral infections in upper respiratory tract of hosts. The protease (elastase), secreted by Stenotrophomonas maltophilia plays a role in the potentiation of influenza virus infection. Proteolytic activity was detected by casein agar test. Positive samples from animals and humans had either a potentiated influenza infectivity or cytopathic effect (CPE) in MDCK and NCI H292 cells, Stenotrophomonas maltophilia were always present. Virus and bacteria were observed ultrastructurally. These in vitro findings show that microbial proteases could contribute to respiratory complications by host protease activity increasing inflammation or destroying endogenous cell protease inhibitors.

Dissecção Coronária Espontânea Pós-Parto. A Propósito de um Caso Clínico

The authors report the clinical case of a 32 years old woman who suffered a spontaneous dissection of the common trunk, anterior descending, intermediary and circumflex coronary arteries, on the 6th post partum day. The diagnosis of acute myocardial infarction was made, through ECG and enzymatic studies and the patient was transferred to a Coronary Unit. Coronary angiography disclosed those lesions and the patient underwent a triple coronary bypass graft, under extracorporeal circulation. The post operative course was uneventful. Eight months after the operation she was found in good condition, free of symptoms and with a normal pattern of life.

BIiossensor Enzimático para avaliação da Capacidade Antioxidente

Os radicais livres formam-se naturalmente nos organismos vivos, pois a sua produção/geração está interligada com o processo de produção de energia (respiração), processos inflamatórios (fagocitose), regulação do crescimento celular, sinalização intercelular e síntese de substâncias biológicas relevantes. Estes também podem ser introduzidos por vias exógenas (poluição, radiação, tabaco, alimentação, etc). Os radicais livres têm capacidade de reagir com o material nucleico (ADN e ARN), proteínas e substâncias oxidáveis, causando danos oxidativos responsáveis pelo envelhecimento e originar doenças degenerativas, tais como, o cancro, arteriosclerose, artrite reumatoide, entre outras. De forma a combater os efeitos pejorativos provocados pelos radicais, os organismos vivos desenvolveram complexos sistemas de defesa antioxidante. Estes sistemas são constituídos por antioxidantes endógenos, produzidos pelos seres vivos, tais como enzimas ou por antioxidantes exógenos obtidos por via da alimentação (por exemplo o ácido ascórbico). Neste sentido, um antioxidante tem capacidade de eliminar ou reduzir a propagação da cadeia de geração de radicais livres. Neste trabalho foi desenvolvido um biossensor enzimático para a quantificação da capacidade antioxidante total de matrizes alimentares. A construção deste biossensor consistiu na eletroimobilização da adenina no elétrodo de pasta de carbono (EPC) ou na adsorção física da dA20 na superfície do EPC. O dano oxidativo foi induzido pelo radical hidroxilo gerado pela reação de Fenton. Nesta dissertação, foi estudada a capacidade de alguns antioxidantes em eliminar o efeito pejorativo dos radicais livres e combater a integridade das bases de adenina ou do dA20.Os antioxidantes estudados foram o ácido ascórbico e alguns ácidos fenólicos como o ácido hidroxibenzoico (ácido gálico) e ácidos hidroxicinâmicos (ácido cafeico e ácido cumárico). Estes antioxidantes têm a capacidade de neutralizar o radical hidroxilo e proteger a adenina/dA20 imobilizado na superfície do EPC. O comportamento da Lacase foi estudado na presença do ácido gálico e do ácido ascórbico. Os estudos eletroquímicos foram realizados através da voltametria de onda quadrada (VOQ), sendo que a interação entre a adenina/ou o dA20 imobilizada na superfície do EPC e os radicais livres na ausência e presença de antioxidantes foi avaliada por meio de mudanças no pico anódico produzido pela oxidação da adenina /dA20. Os resultados demonstraram que estes biossensores permitem a avaliação da capacidade antioxidante total em águas aromatizadas.

Secretion of five extracellular enzymes by strains of chromoblastomycosis agents

The gelatinase, urease, lipase, phospholipase and DNase activities of 11 chromoblastomycosis agents constituted by strains of Fonsecaea pedrosoi, F. compacta, Phialophora verrucosa, Cladosporium carrionii, Cladophialophora bantiana and Exophiala jeanselmei were analyzed and compared. All strains presented urease, gelatinase and lipase activity. Phospholipase activity was detected only on five of six strains of F. pedrosoi. DNase activity was not detected on the strains studied. Our results indicate that only phospholipase production, induced by egg yolk substrate, was useful for the differentiation of the taxonomically related species studied, based on their enzymatic profile.

Optimization of the Tartrate-Resistant Acid Phosphatase Detection by Histochemical Method

According to the new KDIGO (Kidney Disease Improving Global Outcomes) guidelines, the term of renal osteodystrophy, should be used exclusively in reference to the invasive diagnosis of bone abnormalities. Due to the low sensitivity and specificity of biochemical serum markers of bone remodelling,the performance of bone biopsies is highly stimulated in dialysis patients and after kidney transplantation. The tartrate-resistant acid phosphatase (TRACP) is an iso-enzyme of the group of acid phosphatases, which is highly expressed by activated osteoclasts and macrophages. TRACP in osteoclasts is in intracytoplasmic vesicles that transport the products of bone matrix degradation. Being present in activated osteoclasts, the identification of this enzyme by histochemistry in undecalcified bone biopsies is an excellent method to quantify the resorption of bone. Since it is an enzymatic histochemical method for a thermolabile enzyme, the temperature at which it is performed is particularly relevant. This study aimed to determine the optimal temperature for identification of TRACP in activated osteoclasts in undecalcified bone biopsies embedded in methylmethacrylate. We selected 10 cases of undecalcified bone biopsies from hemodialysis patients with the diagnosis of secondary hyperparathyroidism. Sections of 5 μm were stained to identify TRACP at different incubation temperatures (37º, 45º, 60º, 70º and 80ºC) for 30 minutes. Activated osteoclasts stained red and trabecular bone (mineralized bone) was contrasted with toluidine blue. This approach also increased the visibility of the trabecular bone resorption areas (Howship lacunae). Unlike what is suggested in the literature and in several international protocols, we found that the best results were obtained with temperatures between 60ºC and 70ºC. For technical reasons and according to the results of the present study, we recommended that, for an incubation time of 30 minutes, the reaction should be carried out at 60ºC. As active osteoclasts are usually scarce in a bone section, the standardization of the histochemistry method is of great relevance, to optimize the identification of these cells and increase the accuracy of the histomosphometric results. Our results, allowing an increase in osteoclasts contrast, also support the use of semi-automatic histomorphometric measurements.

Clinical and Pathological Findings in Women with Fabry Disease

Introduction. Fabry disease is a rare metabolic disorder caused by the genetic deficiency of the lysosomal hydrolase alpha-galactosidase A, located on chromosome X. Females with the defective gene are more than carriers and can develop a wide range of symptoms. Nevertheless, disease symptoms generally occur later and are less severe in women than in men. The enzyme deficiency manifests as a glycosphingolipidosis with progressive accumulation of glycosphingolipids and deposit of inclusion bodies in lysosomes giving a myelinlike appearance. Patients and Methods. Records of renal biopsies performed on adults from 1st January 2008 to 31st August 2011, were retrospectively examined at the Renal Pathology Laboratory. We retrieved biopsies diagnosed with Fabry disease and reviewed clinical and laboratory data and pathology findings. Results. Four female patients with a mean age of 49.3±4.5 (44-55) years were identified. The mean proteinuria was 0.75±0.3 g/24h (0.4-1.2) and estimated glomerular filtration rate (CKD EPI equation) was 71±15.7 ml/min/1.73m2 (48-83). Three patients experienced extra-renal organ involvement (cerebrovascular, cardiac, dermatologic, ophthalmologic and thyroid) with distinct severity degrees. Leukocyte α-GAL A activity was below normal range in the four cases but plasma and urinary enzymatic activity was normal. Light microscopy showed predominant vacuolisation of the podocyte cytoplasm and darkly staining granular inclusions on paraffin and plastic-embedded semi-thin sections. Electron microscopy showed in three patients the characteristic myelin-like inclusions in the podocyte cytoplasm and also focal podocyte foot process effacement. In one case the inclusions were also present in parietal glomerular cells, endothelial cells of peritubular capillary and arterioles. Conclusion. Clinical signs and symptoms are varied and can be severe among heterozygous females with Fabry disease. Intracellular accumulation of glycosphingolipids is a characteristic histologic finding of Fabry nephropathy. Since this disease is a potentially treatable condition, its early identification is imperative. We should consider it in the differential diagnosis of any patient presenting with proteinuria and/or chronic kidney disease, especially if there is a family history of kidney disease.