Tab. 1+2+3: Selected principal soil properties in the oil exploitation region of KomiArcticOil (Usinsk) in the Russian tundra

Oil polluted and not oil polluted soils (crude oil hydrocarbons contents: 20-92500 mg/kg dry soil mass) under natural grass and forest vegetation and in a bog in the Russian tundra were compared in their principal soil ecological parameters, the oil content and the microbial indicators. CFE biomass-C, dehydrogenase and arylsulfatase activity were enhanced with the occurrence of crude oil. Using these parameters for purposes of controlling remediation and recultivation success it is not possible to distinguish bctween promotion of microbial activity by oil carbon or soil organic carbon (SOC). For this reason we think that these parameters are not appropriate to indicate a soil damage by an oil impact. In contrast the metabolie quotient (qC02), calculated as the ratio between soil basal respiration and the SIR biomass-C was adequate to indicate a high crude oil contamination in soil. Also, the ß-glucosidase activity (parameter ß-GL/SOC) was correlated negatively with oil in soil. The indication of a soil damage by using the stress parameter qCO, or the specific enzyme activities (activity/SOC) minimizes the promotion effect of the recent SOC content on microbial parameters. Both biomass methods (SIR, CFE) have technical problems in application for crude oil-contaminated and subarctic soils. CFE does not reflect the low C_mic level of the cold tundra soils. We recommend to test every method for its suitability before any data collection in series as well as application for cold soils and the application of ecophysiological ratios as R_mic/C_mic, C_mic/SOC or enzymatic activity/SOC instead of absolute data.

Metabolic and hematologic consequences of colectomy associated to hepatectomy in rats

To investigate the influence of partial colectomy associated with hepatectomy on the biodistribution of the 99mTc-phytate, on metabolic parameters, as well as labeling and morphology of red blood cells. METHODS: Wistar rats were distributed into three groups (each with six), nominated as colectomy, colectomy+hepatectomy and sham. In the 30th postoperative day all rats were injected with 99mTc-phytate 0.1mL i.v. (radioactivity 0.66 MBq). After 15 minutes, liver sample was harvested and weighed. Percentage radioactivity per gram of tissue (%ATI/g) was determined using an automatic gammacounter. Serum AST, ALT, alkaline phosphatase and red blood cells labeling were determined. RESULTS: The liver %ATI/g and red blood cells labeling were lower in colectomy and colectomy+hepatectomy rats than in sham rats (p <0.05), and no difference was detected comparing the colectomy and colectomy+hepatectomy groups. Red blood cells morphology did not differ among groups. Serum levels of AST, ALT and alkaline fosfatase were significantly higher in colectomy+hepatectomy than in colectomy rats (p<0.001). CONCLUSION: Hepatectomy associated with colectomy lowered the uptake of radiopharmaceutical in liver and in red blood cells in rats, coinciding with changes in liver enzymatic activity.

Les impacts de mitochondries étrangères sur le phénotype : une étude holistique chez le ventre rouge du nord Chrosomus eos

Les organismes cybrides souffrent généralement d'une altération de la spécificité des interactions mito-nucléaires, résultant en une détérioration du phénotype. Toutefois, diverses études démontrent que le transfert de mitochondries peut occasionnellement être positif. À l'heure actuelle, de nombreuses questions demeurent quant au degré d'influence de ces transferts sur les différents niveaux d'organisation du phénotype. Afin de répondre à ces questions, les formes sauvages et cybrides du poisson Chrosomus eos sont étudiées. Ainsi, le premier volet de ce projet de recherche démontre un impact des mitochondries Chrosomus neogaeus à différents niveaux d'organisation du phénotype des poissons C. eos, lorsque les formes sauvages et cybrides sont retrouvées en allopatrie. Le deuxième volet de cette thèse révèle, quant à lui, que ces modifications phénotypiques ne sont pas suffisantes pour induire un évènement de spéciation entre les deux biotypes, lorsqu'en sympatrie. De plus, cette étude suggère que la coévolution mito-nucléaire peut ne pas être une condition sine qua non à la perpétuation des individus en milieu naturel. Finalement, l'approche holistique considérée dans le troisième volet de cette recherche atteste de l'influence des mitochondries C. neogaeus à différents niveaux d'organisation du phénotype de C. eos, lorsque les formes sauvages et cybrides sont sympatriques. Cette influence est moins prononcée que celle observée à partir de biotypes allopatriques. Combinés, ces chapitres contribuent à une meilleure compréhension des liens existant entre les mitochondries et le phénotype d'un individu.

Tubular Urinary Enzymes in Acute Post-infectious Glomerulonephritis

Tubular function of 17 pediatric patients with a mild form of acute post-infectious glomerulonephritis was prospectively evaluated by assessment of the urinary activity of proximal and distal tubule enzymes. Neutral-like endopeptidase (NEP-like) and angiotensin-converting enzyme (ACE) were the proximal tubule enzymes assessed, while prolyl-endopeptidase (PE) and serine-endopeptidase H1 and H2 were the distal tubule enzymes analyzed. Urine was collected at diagnosis (T0) and after 2 (T2) and 6 (T6) months of follow-up. NEP-like enzyme activity (nmol/mg creatinine; median±quartile range) was increased at diagnosis, and this remained stable during the first 6 months (T0 18.30±83.26, T2 17.32±49.56, T6 23.38±107.18). Urinary activity of the other enzymes was as follows: ACE (mU/ml per mg creatinine) T0 0.08±0.16, T2 0.06±0.10, T6 0.18±0.29; PE (nmol/mg creatinine) T0 6.70±84.87, T2 9.55±69.00, T6 13.67±28.70; serine-endopeptidase H1 (nmol/mg creatinine) T0 7.86±26.95, T2 17.17±59.37, T6 18.19± 79.14; and serine-thiol-endopeptidase H2 (nmol/mg creatinine) T0 3.06±21.97, T2 12.06±32.42, T6 16.22± 44.06. Thirty other healthy children matched for age and gender were considered as a control group. This group was assessed once and the results were: NEP-like activity 6.05±10.54, ACE 0.11±0.22, PE 7.10±13.36, H1 5.00±17.30, and H2 6.00±20.16. In conclusion, we observed that NEP-like and H1 enzymes exhibited significant increased urinary activity 6 months after the diagnosis. This increase occurred in spite of the disappearance of clinical symptoms, which occurred 2 months after the diagnosis. We believe that the increase in urinary enzymatic activity could be a manifestation of a silent tubular dysfunction following an episode of acute post-infectious glomerulonephritis.

Potencial do óleo de Aloysia citriodora Palau no controle de fitopatógenos e na indução de resistência ao tombamento de plântulas de feijão, pepino e beterraba

The crops are affected by pests and diseases that decrease productivity. Among them are the damping off of seedlings that can occur in pre and post-emergence. In bean crops, cucumber and beet these diseases occur, being caused by various pathogens, especialy fitopathogenic fungi. Several measures are used for the controle of such diseases, among them, is the chemical seed treatment fungicides. However, society has become increasingly concerned about the quality and food and environmental contamination, generation a growting search for sensitive products to humans and the environment. The use of essential oils to control plant pathogens is an example of alternative tested by science in the search for less aggressive technologies. This study aimed to evaluate the efficiency of the use of essential oil Aloysia citriodora, in control of pathogens causing damping off in beans, cucumber and beet. This thesis was divided in four chapters, the introductory first, and the other addressing the control of Pythium sp. in beans, Sclerotinia sclerotiorum on cucumber, and Fusarium sp. on beet. The methodology consisted of four experiments in each pathosystem, with all the work done at the Federal Technological University of Parana, Campus Dois Vizinhos. In the first experiment evaluated the fungistatic and fungicidal effect of the essential oil of A. citriodora on PDA in vitro in mycelial growth of pathogens studied. In the second experiment evaluated the in vitro effect of essential oil concentrations of A. citriodora in BD medium on microscope slides, on the germination of sporangia Pythium sp. and conidia Fusarium sp., and in Petri dishes with PDA medium, the sclerotia germination speed index of S. sclerotiorum. In the third experiment, we evaluated in germination test in paper roll (PR), the phytotoxic effect or not the use of essential oil concentrations of A. citriodora in dry bean seed, cucumber and beet. The variables used to assess this experiment were the germination percentage, mediun green mass per plant and average length of seedlings. In the fourth experiment we assessed the effect of treating bean seeds, cucumber and beet with essential oil contents of A. citriodora, seeds in their subsequent substrates contamined with pathogens studied, Pythium sp., S. sclerotiorum and Fusarium sp. In this experiment we used the following variables: percentage of emergence, percentage of post-emergence damping off, green average mass per plant, average length per plant and biochemical analyzes. The biochemistry of plant tissues evaluated were as follows: protein content, enzymatic activities of peroxidases, phenylalanine ammonia-liase (PAL), chitinases and β-1,3-glucanases. The in vitro results show that the essential oil has fungistatic and fungicidal effect on mycelial growth, on sporangia germination, conidia and sclerotia of the pathogens studied in this work, wich may be related to its major components, citral and limonene. The oil also exhibits low phytotoxicity to seeds of the species studied, only in beans decreases germination in most studied dosage (0,25%), cucumber also in the higher dosage (0,25%) reduce the length of seedlings, and beet there were no negative effects to the seedlings. In the test in substrate contaminated with the pathogens, the use of essential oil: increased germination and decreased post emergence damping off of beans seedlings; at a concentration of 0,0625% decreases post emergence damping off in cucumber. In biochemical analyzes found an increase in the enzymatic activity of peroxidases and β-1,3-glucanases on beans, and glucanases on cucumber, and increased enzyme activity of peroxidases on beet, showing action in resistance induction at damping off.

Aplicação foliar de molibdênio em adubos verdes e o desempenho do feijoeiro em sucessão

The knowledge of molybdenum application in legumes on the availability of N, by BNF, increased enzymatic activity and the residual effect caused on crops growth and yield can contribute to the greater scientific understanding involved in green manure processes. The aim of this study was to evaluate the Mo application and the N from Crotalaria juncea and Canavalia ensiformis green manures on common bean performance. Were conducted field experiments for the crops succession system (green manures - common bean) and laboratory essays for the enzymatic activities. Green manure production was installed in a factorial arrangement 2 x 4, with two green manure legumes species, sunnhemp (Crotalaria juncea) and jack beans (Canavalia ensiformis), and four Mo doses (0, 40, 80, 120 g ha-1) in the form of sodium molybdate (Na2MoO4), foliar applied, in a randomized block design with four replicates. For succession crop (common bean) additional treatment was added, beans grown without any fertilization, following the same experimental design from the previous crop. The dry matter decomposition and the N mineralization of green manure were monitored through collection of residues over time, by using the litter bags method. In laboratory were carried out tests of nitrate reductase activity in green manures and common beans at 90 and 66 days after sowing, respectively. The sunnhemp responded linearly positively to the application of Mo as the dry matter and N accumulation. While the jack beans presented a negative quadratic response for dry matter and there was no adjustment of regression models to N. The jack beans showed a higher decomposition rate and N mineralization compared to sunnhemp. The half lives for decomposing 50% of dry matter on the soil was 123 and 104 days to sunnhemp and jack beans, respectively, and 50% of N present in the residues was mineralized at 93 and 85 days. In common bean, differed from the control for number of pods the dose of 40 g ha-1 of Mo in both species of green manures and the dose 80 g ha-1 of Mo in jack beans. For number of grains only in sunnhemp on the dose of 40 g ha-1 of Mo differ from the control. The nitrate reductase activity was influenced by developmental stage of green manure species. In common bean, the activity of nitrate reductase was up to three times higher than the dose 0 g ha-1 of Mo compared to treatment with application of Mo in both species. There was no effect of Mo doses or species of green manure on common bean yield.

Produção enzimática de biodiesel a partir de resíduos agro-industriais usando a lipase imobilizada de origem microbiana

Trabalho Final de Mestrado para obtenção do grau de Mestre em Engenharia Química e Biológica

Metabolic and hematologic consequences of colectomy associated to hepatectomy in rats

To investigate the influence of partial colectomy associated with hepatectomy on the biodistribution of the 99mTc-phytate, on metabolic parameters, as well as labeling and morphology of red blood cells. METHODS: Wistar rats were distributed into three groups (each with six), nominated as colectomy, colectomy+hepatectomy and sham. In the 30th postoperative day all rats were injected with 99mTc-phytate 0.1mL i.v. (radioactivity 0.66 MBq). After 15 minutes, liver sample was harvested and weighed. Percentage radioactivity per gram of tissue (%ATI/g) was determined using an automatic gammacounter. Serum AST, ALT, alkaline phosphatase and red blood cells labeling were determined. RESULTS: The liver %ATI/g and red blood cells labeling were lower in colectomy and colectomy+hepatectomy rats than in sham rats (p <0.05), and no difference was detected comparing the colectomy and colectomy+hepatectomy groups. Red blood cells morphology did not differ among groups. Serum levels of AST, ALT and alkaline fosfatase were significantly higher in colectomy+hepatectomy than in colectomy rats (p<0.001). CONCLUSION: Hepatectomy associated with colectomy lowered the uptake of radiopharmaceutical in liver and in red blood cells in rats, coinciding with changes in liver enzymatic activity.

Applications of functional nucleic acids in imaging, sensing and drug delivery and investigations of DNAzyme-metal interactions

Functional nucleic acids (FNA), including nucleic acids catalysts (ribozymes and DNAzymes) and ligands (aptamers), have been discovered in nature or isolated in a laboratory through a process called in vitro selection. They are nucleic acids with functions similar to protein enzymes or antibodies. They have been developed into sensors with high sensitivity and selectivity; it is realized by converting the reaction catalyzed by a DNAzyme/ribozyme or the binding event of an aptamer to a fluorescent, colorimetric or electrochemical signal. While a number of studies have been reported for in vitro sensing using DNAzymes or aptamers, there are few reports on in vivo sensing or imaging. MRI is a non-invasive imaging technique; smart MRI contrast agents were synthesized for molecular imaging purposes. However, their rational design remains a challenge due to the difficulty to predict molecular interactions. Chapter 2 focuses on rational design of smart T1-weighted MRI contrast agents with high specificity based on DNAzymes and aptamers. It was realized by changing the molecular weight of the gadolinium conjugated DNA strand with the analytes, which lead to analyte-specific water proton relaxation responses and contrast changes on an MRI image. The designs are general; the high selectivity of FNA was retained. Most FNA-based fluorescent sensors require covalent labeling of fluorophore/quencher to FNAs, which incurrs extra expenses and could interfere the function of FNAs. Chapter 3 describes a new sensor design avoiding the covalent labeling of fluorophore and quencher. The fluorescence of malachite green (MG) was regulated by the presence of adenosine. Conjugate of aptamers of MG and adenosine and a bridge strand were annealed in a solution containing MG. The MG aptamer did not bind MG because of its hybridization to the bridge strand, resulting in low fluorescence signal of MG. The hybridization was weakened in the presence of adenosine, leading to the binding of MG to its aptamer and a fluorescence increase. The sensor has comparable detection limit (20 micromolar) and specificity to its labeled derivatives. Enzymatic activity of most DNAzymes requires metal cations. The research on the metal-DNAzyme interaction is of interest and challenge to scientists because of the lack of structural information. Chapters 4 presents the research on the characterization of the interaction between a Cu2+-dependent DNAzyme and Cu2+. Electron paramagnetic resonance (EPR) and UV-Vis spectroscopy were used to probe the binding of Cu2+ to the DNAzyme; circular dichroism was used to probe the conformational change of the DNAzyme induced by Cu2+. It was proposed that the conformational change by the Cu2+ binding is important for the activity of the DNAzyme. Chapter 5 reports the dependence of the activity of 8-17 DNAzyme on the presence of both Pb2+ and other metal cations including Zn2+, Cd2+ and Mg2+. It was discovered that presence of those metal cations can be cooperative or inhibitive to 8-17 activity. It is hypothesized that the 8-17 DNAzyme had multiple binding sites for metal cations based on the results. Cisplatin is effective killing tumor cells, but with significant side effects, which can be minimized by its targeted delivery. Chapter 6 focuses on the effort to functionalize liposomes encapsulating cisplatin by an aptamer that selectively bind nucleolin, an overexpressed protein by breast cancer cells. The study proved the selective cytotoxicity to breast cancer cells of the aptamer-functionalized liposome.

Atividade β-D-N-Acetilglucosaminidásica de enzimas imobilizadas extraídas da Artemia franciscana e possíveis aplicações biotecnológicas

A β-D-N-acetilglucosaminidase extracted and partially isolated from crustacean Artemia franciscana by ammonium sulfate precipitation and filtration gel chromatography Bio Gel A 1.5m. the enzyme was immobilized on ferromagnetic Dacron yielding a insoluble active derivative with 5.0 units/mg protein and 10.35% of the soluble enzyme activity. β-D-N-acetilglucosaminidase-ferromagnetic Dacron was easily removed from the reaction mixture by a magnetic field, it was reused for ten times without loss in its activity. The ferromagnetic Dacron was better activated at pH 5.0. The particles visualized at scanning electron microscope (SEM) had presented different sizes, varying between 721nm and 100µm. Infra red confirmed immobilization on support, as showed by primary amino peaks at 1640 and 1560 cm-1 . The immobilize enzyme presented Km of 2.32 ± 0.48 mM and optimum temperature of 50°C. Bought presented the same thermal stable of the soluble enzyme and larger enzymatic activity at pH 5.5. β-D-N-acetilglucosaminidase-Dacron ferromagnético showed sensible for some íons as the silver (AgNO3), with loss of activity. The β-D-N acetilglucosaminidase activity for mercury chloride (HgCl2), whom is one of the most toxic substance joined in nature, it was presented activity already diminished at 0,01mM and lost total activity at 4mM, indicating sensitivity for this type of metal. β-D-N-acetilglucosaminidase-ferromagnetic Dacron showed degradative capacity on heparan sulfate, the enzyme still demonstrated degradative capacity on heparan sulphate, suggesting a possible application to produce fractions of this glycosaminoglycan

Avaliação do potencial probiótico de leveduras isoladas de azeitonas de mesa da cultivar Negrinha de Freixo

Probióticos são definidos como microrganismos vivos, que quando administrados em quantidades adequadas, conferem benefícios à saúde do hospedeiro. Atualmente a pesquisa de microrganismos probióticos a partir da fermentação da azeitona tem-se centrado nas bactérias ácido-lácticas, sendo escassos os estudos envolvendo leveduras. No presente trabalho avaliou-se o potencial probiótico de estirpes de leveduras previamente isoladas durante o processo de fermentação natural de azeitona de mesada cultivar Negrinha de Freixo. Foram avaliadas 16 estirpes em relação à atividade enzimática (catalase, amilase, xilanase, protease e β-glucosidase); ao crescimento a 37ºC; ação inibitória frente a microrganismos patogénicos; capacidade de autoagregação; atividade antioxidante (utilizando o método de DPPH); e resistência ao aparelho digestivo humano, a partir de uma simulação in vitro da digestão gástrica e pancreática. Os resultados apresentados para a atividade enzimática indicaram que em alguns isolados foi detetado fraca atividade das enzimas protease, xilanase e amilase. Já uma atividade forte de lipase foi observada nas estirpes Pichia manshurica e Saccharomyces cerevisiae (15A e 15B). Para a enzima β-glucosidase, identificou-se atividade forte em Rhodotorula graminis, Rhodotorula glutinis, Candida norvegica, Pichia guilliermondii e Galactomyces reessii. Relativamente à capacidade de crescimento à temperatura corporal (37ºC), três estirpes (Saccharomyces cerevisiae 15B; Candida tropicalis 1A; e Pichia membranifaciens 29A) destacaram-se por apresentar maior taxa específica de crescimento. A capacidade bloqueadora dos radicais livres DPPH foi verificada em 10 estirpes, sendo as estirpes de S. cerevisiae as que mais se destacaram dentre as outras. As estirpes C. norvegica e G. reessii (34A) apresentaram capacidade antifúngica frente ao microrganismo patogénico Cryptococcus neoformans. Em relação à capacidade de autoagregação avaliada, as estirpes S. cerevisiae (15A), Candida tropicalis (1A) e C. norvegica (7A) apresentaram ao fim de 24 horas percentagens superiores a 80%. Relativamenteà resistência frente às condições presentes no trato gastrointestinal in vitro, a estirpe P. guilliermondii (25A), destacou-se dentre as demais, por apresentar maior capacidade de sobrevivência em todo o processo digestivo simulado. As estirpes Candida boidinii (37A) e S. cerevisiae (15A) apresentaram menor capacidade de sobrevivência nestas condições. Contudo, serão necessários testes adicionais para complementar estes resultados.

Isolamento, seleção e cultivo de leveduras com potencia biotecnológico para a produção de endo-xilaneses

Xilanases são enzimas que catalisam a hidrólise das xilanas e têm sido em grande parte, obtidas a partir de bolores e bactérias. No entanto poucos estudos têm sido relatados sobre a produção destas enzimas por leveduras. O presente trabalho teve como objetivo isolar leveduras de diferentes fontes vegetais visando à produção de xilanases, além de maximizar sua produção, estudar o uso de diferentes fontes de nitrogênio e cultivar as leveduras em meios contendo coprodutos agroindustriais. As amostras de alimentos e resíduos foram enriquecidas em caldo extrato de malte e levedura e isoladas em Ágar Nutriente Wallerstein, as leveduras isoladas foram, a seguir, avaliadas quanto à capacidade de degradar xilana presente no meio e produzir halos de hidrólise, os quais foram visualizados através do uso do corante vermelho congo. Os micro-organismos selecionados como potenciais produtores de xilanase foram crescidos em meio complexo líquido e as atividades enzimáticas de endoxilanase, β-xilosidase, carboximetilcelulase, celulase total, pH e concentração de biomassa foram avaliadas ao longo de 96 h de cultivo. Dentre as leveduras isoladas, sete foram selecionadas, e a 18Y foi a que apresentou a maior atividade de endo- xilanase (2,7 U.mL-1 ), sendo esta isolada de chicória e identificada como Cryptococcus laurentii. Esta estirpe apresentou capacidade de produzir xilanase com baixos níveis de celulase, sendo assim selecionada neste trabalho. A maximização de endo-xilanase foi avaliada fazendo uso de planejamento experimental onde primeiramente foi realizado um planejamento fracionário 2 6-2 para verificar os efeitos do pH inicial e as concentrações de xilana, peptona, (NH4)2SO4, extrato de levedura e KH2PO4 sobre a atividade enzimática. Após selecionar as variáveis xilana, peptona, pH e extrato de levedura foi realizado um delineamento composto central rotacional (24 ) onde todos os cultivos foram mantidos a 30°C, 150 rpm durante 96 h sendo retiradas alíquotas para determinação das atividades, pH e biomassa. A produção máxima foi de 6,9 U.mL-1 usando 10,0 g.L-1 de extrato de levedura, 10,0 g.L-1 de peptona, 10,0 g.L-1 de xilana, 1,0 g.L-1 de (NH4)2SO4 em pH 6,5 o que permitiu um incremento de mais de 250% sobre a atividade. Posteriormente foram realizados ensaios avaliando diferentes fontes e concentrações de nitrogênio orgânico e inorgânico. A presença de NH4NO3 e (NH₄)₂SO₄ usados na concentração de 3% proporcionaram as maiores atividades de endo-xilanase (6,2 e 6,0 U.mL-1 respectivamente). O sulfato de amônio foi selecionado e fixado em 1 g.L-1 e logo após um planejamento completo 22 foi realizado onde as variáveis xilana e extrato de levedura foram estudadas e as demais fixadas. As condições ótimas estabelecidas para a produção da enzima foram: concentração de xilana de 18,6 g.L-1 , concentração de extrato de levedura de 10 g.L-1 atingindo 14 U.mL-1 . Após a maximização enzimática estudou-se o crescimento de Pichia pastoris NRRL Y-1603 e Cryptococcus laurentti em cinco substratos agroindustriais visando a possibilidade estes substratos substituírem a xilana em cultivos para a produção de endo-xilanase. Os ensaios foram realizados utilizando os subtratos pré-tratados com NaOH 4% e não tratados. Para inserção dos mesmos aos meios de cultivo, estes foram moídos e adicionados na concentração de 2%. O pré-tratamento para todos as fontes de hemicelulose foi eficiente e promoveu aumento nas atividades produzidas. Cryptococcus laurentti apresentou maior atividade enzimática (8,7 U.mL-1 ) em farelo de arroz desengordurado e pré- tratado enquanto que a levedura Pichia pastoris NRRL Y-1603 apresentou sua melhor condição para produção de endo-xilanase quando cultivada em meio contendo casca de aveia e o farelo de arroz pré-tratados, alcançando atividades máximas de 7,6 e 7,5 U.mL-1 .

Obtenção e caracterização de queratinase de bacillus sp. P45 a partir de coprodutos e aplicação na produção de queijos cremosos enriquecidos com chia e quinoa

As proteases constituem 60-65% do mercado global das enzimas industriais e são utilizadas na indústria de alimentos no processo de amaciamento de carne, na síntese de peptídeos, preparo de fórmulas infantis, panificação, cervejarias, produtos farmacêuticos, diagnósticos médicos, como aditivos na indústria de detergentes e na indústria têxtil no processo de depilação e transformação do couro. Proteases específicas produzidas por micro-organismos queratinolíticos são chamadas de queratinases e distinguem-se de outras proteases pela maior capacidade de degradação de substratos compactos e insolúveis como a queratina. Atualmente, processos que apontem o uso total das matérias-primas e que não resultem em impactos negativos ao meio ambiente tem ganhado destaque. Dentro desta temática, destacam-se a reutilização da farinha de penas residual durante o cultivo do Bacillus sp. P45 para produção de proteases e a biomassa residual de levedura, ambas com elevados teores de proteínas, podendo ser utilizadas no cultivo do Bacillus sp. P45 para obtenção de proteases. O objetivo deste trabalho foi obter a enzima queratinase purificada em grandes quantidades, sua caracterização, bem como a sua aplicação em processos de coagulação enzimática do leite para o desenvolvimento de um queijo cremoso enriquecido com farinha de chia e quinoa. Além disso, aplicar diferentes coprodutos para produção de enzimas proteolíticas e queratinolíticas. A presente tese foi dividida em quatro artigos: no primeiro foi realizado a obtenção da queratinase purificada em maiores quantidades e a determinação dos parâmetros de estabilidade térmica e a influência de componentes químicos na atividade enzimática. A obtenção da enzima em maiores quantidades alcançou fatores de purificação de 2,6, 6,7 e 4,0 vezes, paras 1º SAB, 2º SAB e diafiltração, respectivamente. A recuperação enzimática alcançou valores de 75,3% para o 1º SAB, 75,1% no 2º sistema e 84,3% na diafiltração. A temperatura de 55ºC e o pH 7,5 foram determinados como ótimos para atividade da enzima queratinase. O valor da energia de desativação (Ed) médio foi de 118,0 kJ/mol e os valores de z e D variaram de 13,6 a 18,8ºC, e 6,9 a 237,3 min, respectivamente. Além disso a adição de sais (CaCl2, CaO, C8H5KO4 e MgSO4) elevou a atividade da enzima na presença destes compostos. O segundo artigo apresenta a aplicação da queratinase como coagulante de leite bovino e sua aplicação na obtenção de queijo cremoso enriquecido com chia e quinoa. A enzima mostrou atividade de coagulação semelhante ao coagulante comercial, na concentração de 30mg/mL. A enzima purificada foi empregada de forma eficiente na fabricação do queijo cremoso, que apresentou valores de pH de 5,3 e acidez de 0,06 a 0,1 mol/L, com elevação durante os 25 dias de armazenamento. O terceiro artigo apresenta o perfil do queijo cremoso enriquecido com farinha de chia e quinoa, o qual apresentou alto índice de retenção de água (>99,0%) e baixos valores de sinérese (<0,72%). Elevados teores de fibras foi verificado (3,0 a 5,0%), sugerindo seu consumo como fonte de fibras. As análises microbiológicas foram de acordo com a legislação vigente. Na análise sensorial foi verificado altos valores de suavidade ao paladar e verificado maiores valores de consistência e untabilidade nas amostras com maiores concentrações de nata e quinoa. O quarto artigo traz a extração de β-galactosidase por ultrassom e o uso da biomassa residual da levedura, bem como o uso de farinha de penas residuais como substrato para obtenção de proteases. O ultrassom foi eficiente para ruptura celular e extração de β-galactosidase, apresentando alta atividade (35,0 U/mL) e rendimento (876,0 U/g de biomassa). A maior atividade proteolítica (1300 U/mL em 32 h) e queratinolítica (89,2 U/mL) verificadas ocorreram utilizando-se a biomassa e a farinha de penas residuais, respectivamente. Maior produtividade proteolítica (40,8 U/mL/h) foi verificado no meio utilizando biomassa residual como substrato. Já a maior produtividade queratinolítica (2,8 U/mL/h) foi alcançada utilizando farinha de penas reutilizada.

Aspectos estruturais, farmacológicos e biológicos de fucanas da alga marrom sargassum vulgare

The present study examines the chemical composition and their effects on free radicals, inflammation, angiogenesis, coagulation, VEGF effects and cellular proliferation of a polysaccharides from alga Sargassum vulgare. The sulfated polysaccharide was extracted from brown seaweed by proteolysis with enzymes maxataze. The presence of proteins and sugars were observed in crude polysaccharides. Fractionation of this crude extract was made with growing concentration of acetone (0.3-1.5 v) and produced four groups of polysaccharides. Anionic polysaccharides from brown seaweed Sargassum vulgare, SV1and PSV1 were fractionated (SV1) and purified (PSV1), and displayed with high total sugars and sulfate content and very low level of protein. This fucan SV1 contains low levels of protein and high carbohydrate and sulfate content. This polysaccharides prolonged activated partial thromboplastin time (aPTT) at 50 μg (>240 s). SV1 was found to have no effect on prothrombin time (PT), corresponding to the extrinsic pathway of coagulation. SV1 exhibits high antithrombotic action in vivo, with a concentration ten times higher than heparin. Polysaccharides from S. vulgare promoted direct inhibition enzymatic activity of thrombin and stimulated enzymatic activity of FXa. SV1 showed optimal inhibitory activity of thrombin (50.2±0.28%) at a concentration of 25 μg/mL. Its antioxidant action on scavenging radicals by DPPH was (22%), indicating the polymer has no cytotoxic action (hemolytic) on ABO and Rh blood types in different erythrocyte groups and displays strong anti-inflammatory action on all concentrations tested in the carrageenan-induced paw edema model, demonstrated by reduced edema and cellular infiltration. Angiogenesis is a dynamic process of proliferation and differentiation. It requires endothelial proliferation, migration, and tube formation. In this context, endothelial cells are a preferred target for several studies and therapies. The antiangiogenic efficacy of polysaccharides was examined in vivo in the chick chorioallantoic membrane (CAM) model by using fertilized eggs. Decreases in the density of the capillaries were assessed and scored. The results showed that SV1 and PSV1 have an inhibitory effect on angiogenesis. These results were also confirmed by inhibition tubulogenesis in rabbit aorta endothelial cell (RAEC) in matrigel. These compounds were assessed in Apoptosis assay (Annexin V - FITC / PI) and cell viability by MTT assay of RAEC. These polysaccharides do not affect the viability and do not have apoptotic or necrotic action. RAEC cell when incubated with SV1 and PSV1showed inhibition of VEGF secretion, observed when compounds were incubated at 25, 50 and 100 μg/μL. The VEGF secretion with the RAEC cell line for 24 h, was more effective for PSV1 at 50 μg/μL(71.4%) than SV1 100 μg/μL (75.9%). SV1 and PSV1 had an antiproliferative action (47%) against tumor cell line HeLa. Our results indicate that these sulfated polysaccharides have antiangiogenic and antitumoral actions

Plejotropowe działanie neuropeptydów allatostatynowych w regulacji procesów fizjologicznych u chrząszczy

Wydział Biologii