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O presente trabalho teve como objetivo investigar o efeito do dibenzotiofeno DBT (derivado do petróleo) no crescimento e na morfologia de Rhizopus arrhizus UCP 402. Com o aumento da utilização de combustíveis fósseis por vários países industrializados, um sério problema ambiental se agrava devido à qualidade das reservas do petróleo, tendo em vista a quantidade de enxofre, e conseqüentemente, maior toxicidade para o meio ambiente. Por sua vez, o dibenzotiofeno (DBT) é um composto organossulfurado heterocíclico presente no óleo diesel, sendo considerado um combustível que causa grandes problemas ambientais. A utilização do DBT por R. arrhizus UCP 402 foi avaliada a partir de diferentes concentrações desse composto, observando-se os efeitos na cinética de crescimento e morfologia do fungo, na produção de quitina e quitosana, como também na formação de metabólitos tóxicos. Os resultados obtidos indicam que R. arrhizus apresenta habilidade para crescer nas diferentes concentrações do composto, porém apenas, em co-metabolismo, isto é, na presença de glicose. Observou-se uma variação no teor de proteínas totais, em quitina e quitina pela ação do DBT. A microscopia ótica evidenciou variações na morfologia de R. arrhizus, causando também alterações no padrão de ramificação das hifas. Os produtos do metabolismo do DBT indicam que ocorreu degradação do composto por R. arrhizus, cujos metabólitos apresentaram uma acentuada inibição da germinação de Repolho das 4 estações (Brassica oleracea var. capitata). Os estudos realizados demonstram um grande potencial biotecnológico de R. arrhizus no processo de degradação de DBT, podendo ser empregado no futuro em processos de biorremediação / The present work had as objective investigate the effect of dibenzotiofene - DBT (derived from oil) in the growth and morphology of Rhizopus arrhizus UCP 402. Had the increase of the use of the fossil fuels used for some industrialized countries, a serious environment problem aggravates due to quality of the reserves of the oil, in view of the amount of sulphur, and consequently, greater toxicity for the environment. In turn, the dibenzothiofene (DBT) is a composition heterociclic organossulfurated in the oil diesel, being considered a great fuel that cause ambient problems. The use of the DBT for R. arrhizus UCP 402 was evaluated from different concentrations of this composition, having observed itself the effect in kinetic of growth and the morphology, in the production of quitina and quitosana, as well as in the formation of toxic methabolits. The gotten results had indicated that R. arrhizus presents ability to grow in the different concentrations of the composition, however only, in co-metabolism, that is, in the glucose presence. A variation in the total protein text, quitina and quitosana for the action of the DBT was observed. The óptic microscopy evidenced variations in the morphology of R. arrhizus, also causing alterations in the ramification of hifas on standard. The products of the metabolism of the DBT had indicated that degradation of the composition for R. arrhizus occurred, whose methabolits had presented one accented inhibition of the germination of Cabbage of the 4 stations (oleracea Brassica to var. capitata). The carried through studies had demonstrated a great biotechnological potential of R. arrhizus in the process of degradation of DBT, being able to be used in the future in biorremediation processes

Tématem diplomové práce je imobilizace lipáz a konkrétně studium interakcí mezi lipázou a nosičem na bázi uhlíku. Lipáza izolovaná z kmene Rhizopus arrhizus byla adsorbována přímo na různé typy grafen oxidu (a1, a, b, c a d) a na grafen oxid typu a1 modifikovaný poly(ethylenglykolem), PEG-a1. Enzym adsorbovaný na nosič a1 byl následně sesítěn pomocí glutaraldehydu a vzorek byl označen jako GA(RA-a1). Vliv hydrofobního charakteru povrchu nosiče na účinnost imobilizace byl potvrzen vyšší úchovou počáteční aktivity enzymu imobilizovaného na více hydrofobním nosiči (nižší koncentrace polárních skupin) i při vyšší koncentraci rozpustného enzymu v roztoku. Stanovení enzymové aktivity bylo provedeno spektrofotometricky za použití p-nitrofenyl laurátu (p-NPL) jako substrátu. Pro imobilizovaný a volný enzym byly stanoveny základní biochemické a kinetické parametry. Optimální pH kovalentně imobilizovaného enzymu bylo posunuto do více kyselého oblasti (pH 7-8) ve srovnání s volným enzymem, kdy bylo optimum dosaženo při pH 9. Tepelná stabilita imobilizovaného enzymu byla výrazně zlepšena v případě vzorku GA(RA-a1), kde bylo aplikován glutaraldehyd po adsorpci enzymu na nosič. Sesíťování adsorbovaného enzymu pomocí glutaraldehydu vedlo ke zlepšení tepelné stability vzorku, a to pravděpodobně v důsledku intermolekulárních kovalentních vazeb. Na základě měření stability enzymu při teplotě 4 C ve fosfátovém pufru bylo prokázáno značné zlepšení úchovy lipolytické aktivity imobilizovaného vzorku oproti volnému enzymu. Volný enzym ztratil více než 84 % své původní aktivity za 42 dní, zatímco imobilizovaný enzym na nosiči c si zachoval 100 % své původní aktivity. Nejlepší stabilitu enzymu při úchově měl nosič c, když si po 180 dnech stále zachoval 87 % své původní aktivity.

Submitted by Luiz Felipe Barbosa (luiz.fbabreu2@ufpe.br) on 2015-04-17T14:59:22Z No. of bitstreams: 2 Tese Lucia Raquel Ramos Berger.pdf: 4046628 bytes, checksum: 24c661f71d38a470b0140037dc947dcd (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-04-17T14:59:22Z (GMT). No. of bitstreams: 2 Tese Lucia Raquel Ramos Berger.pdf: 4046628 bytes, checksum: 24c661f71d38a470b0140037dc947dcd (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2013-12-19 / CAPES CNPq FACEPE / Nos últimos anos, muitos estudos têm demonstrado o interesse em relação à produção e aplicação dos biopolímeros quitina e quitosana como materiais funcionais na medicina, farmácia, alimentação, biologia e engenharia. Especialmente devido a suas características únicas como biocompatibilidade, biodegradabilidade, não- toxicidade, atividade antimicrobiana, capacidade quelante e fácil produção. Após a celulose, a quitina é o segundo composto orgânico mais abundante na Terra, e é encontrado naturalmente como elemento estrutural em invertebrados e paredes celulares de fungos, principalmente na ordem Mucorales (classe Zygomycetes). A quitosana é obtida a partir da desacetilação da quitina (poli-(1-4)-2-acetamida-2-desoxi-β-D-glicosamina). A produção simultânea desses polímeros por via microbiológica mostrou ser vantajosa quando comparada com a extração tradicional a partir de crustáceos. A produção controlada por fermentação têm permitido o uso de resíduos agroindustriais como substratos de baixo custo, tais como: milhocina, manipueira, melaço e casca de mamão, o que corresponde a fontes de nutrientes alternativas para as culturas de fungos.Entre as várias aplicações da quitosana , destaca-se a sua capacidade de formar membranas, que podem ser aplicadas durante a remoção de metais pesados em ambientes aquosos . Esse método poderia ser considerado alternativa promissora, tendo em vista a inviabilidade operacional e o alto custo dos procedimentos habituais para remoção de metais. O objetivo deste estudo foi selecionar as melhores condições de cultivo para a produção satisfatória de quitina e quitosana a partir de Cunninghamella elegans e Rhizopus arrhizus e posterior aplicação de membranas de quitosana para a remoção de cádmio em soluções aquosas. Inicialmente, a produção de biomassa e de biopolímeros por estes fungos foi realizada através dos seguintes meios de cultura: meio específico modificado para Mucorales, e meios alternativos enriquecidos com milhocina (como fonte de nitrogênio) e suplementados manipueira, melaço ou suco de casca de mamão (como fontes de carbono). As caracterizações físico-químicas realizadas foram: infravermelho, difração de raios X, microscopia eletrônica de varredura, viscosidade, massa molecular, termogravimetria e densidade calorimétrica. Foi realizada a comparação entre as membranas obtidas utilizando quitosana fúngica com baixa massa molecular e quitosana comercial com massas moleculares baixa e média. Essas membranas de quitosana foram aplicadas para a remoção de Cd ( II ) a partir de soluções aquosas. E a remoção de cádmio foi estimada utilizando as análises de voltametria de redissolução anódica e voltametria de onda quadrada. Os resultados mostraram claramente as maiores produções de biomassa entre 16,00-24,60 g/L e produção de quitosana entre 77,78-77,76 mg/g obtidas utilizando os meios de cultura alternativos para R. arrhizus e C. elegans, respectivamente. Por outro lado, as produções mais elevadas de quitina de 137,17 e 235,00 mg/g foram obtidas usando os mesmos fungos e o meio de cultura padrão para Mucorales. A caracterização físico-química dos polímeros foi semelhante à encontrada para a quitina e quitosana comerciais utilizadas como padrões. A remoção de aproximadamente 100% de cádmio utilizando a membrana de quitosana de baixo custo sugere uma alternativa para complementar, ou até mesmo substituir, procedimentos de alto custo e baixa eficiência na descontaminação de efluentes industriais por metais pesados.

Während der letzten Jahrzehnte ist eine steigende Inzidenz von Infektionen durch Pilze der Ordnung Mucorales zu beobachten. Rhizopus arrhizus ist der häufigste Erreger dieser lebensbedrohlichen Infektionen, die vor allem immunsupprimierte Patienten betreffen. Aufgrund der oft schwierigen Diagnosestellung und limitierter therapeutischer Optionen liegt derzeit die Letalität von Mucormykosen zwischen 50 bis 100 %. Eine Voraussetzung für die Etablierung neuer Biomarker oder immuntherapeutischer Strategien ist ein verbessertes Verständnis der immunpathologischen Prozesse bei der Abwehr von Mucorales. In dieser Arbeit wurden daher verschiedene Immunzellpopulationen durch ruhende und ausgekeimte Stadien von R. arrhizus stimuliert und anschließend deren proinflammatorische Immunantwort gemessen. Als Vergleich diente die proinflammatorische Immunantwort der untersuchten Immunzellen nach Stimulation mit Aspergillus fumigatus. Darüber hinaus war es Gegenstand dieser Arbeit, zu charakterisieren welche Pattern Recognition Receptors (PRRs) an der Erkennung von Mucorales durch verschiedene innate Immunzellen beteiligt sind. Zugleich wurde untersucht, ob unterschiedliche Morphotypen der Pilzspezies Auswirkungen auf die Stimulation der jeweiligen PRRs haben. Hierfür wurden Koinkubations-Experimente mit neutrophilen Granulozyten sowie Peripheral Blood Mononuclear Cells (PBMCs), Monozyten und monocyte derived dendritic cells (moDCs) mit verschiedenen Morphotypen von R. arrhizus durchgeführt. Die Rezeptoren TLR2, TLR4 und/oder Dectin-1 wurden dabei durch neutralisierende Antikörper oder RNA-Interferenz blockiert. Ausgekeimte Stadien von A. fumigatus sowie R. arrhizus induzierten eine erhöhte ROS-Freisetzung in Neutrophilen, die durch isolierte oder kombinierte Blockade von TLR2, TLR4 und Dectin-1 abgeschwächt wurde. Ebenso wurde die Phagozytoseaktivität neutrophiler Granulozyten gegenüber R. arrhizus-Konidien durch Blockade von TLR4 und Dectin-1 deutlich reduziert. Im Gegensatz zu A. fumigatus induzierten sowohl ruhende Konidien als auch ausgekeimte Stadien (Keimschläuche und Hyphen) von R. arrhizus eine robuste pro-inflammatorische Zytokinantwort durch moDCs. Nach Inhibition der Dectin-1 Expression durch RNA-Interferenz zeigte sich die Transkription und Sekretion von Interleukin-1β in Gegenwart aller drei untersuchten Morphotypen von R. arrhizus deutlich vermindert (Transkription um 46 bis 68 % und Sekretion um 75 bis 79 % vermindert). Diese Ergebnisse legen nahe, dass Dectin-1 ein wichtiger Mediator bei der Einleitung der innaten Immunantwort verschiedener Zelltypen gegen R. arrhizus ist. Diese Beobachtung sollte in weiteren Studien eingehender untersucht werden, z. B. um die Eignung von Dectin-1 als Rezeptor für zelltherapeutische Ansätze wie T-Zell-Konstrukte mit chimären Antigen-Rezeptoren zu evaluieren. / While Aspergillus species remain the most common cause of opportunistic mold infections, emerging pathogens such as Mucorales account for an increasing share of invasive mycoses in immunocompromised patients. Unspecific clinical presentation, lack of reliable biomarkers, and limited therapeutic options contribute to poor outcomes of this devastating infection. Depending on the site of infection, mortality rates of mucormycoses reach up to 100%, highlighting an unmet need for advances in diagnostic and therapeutic strategies. To facilitate improvements in immune biomarker development and immunotherapy, better understanding of host defense and host-pathogen interplay is warranted. Therefore, this project sought to assess the activation of proinflammatory immune responses by resting and germinated stages of Rhizopus arrhizus, the most common pathogenic Mucorales species. Furthermore, we aimed to characterize the pattern recognition receptors (PRRs) which are involved in the detection of R. arrhizus. To that end, we co-incubated polymorphonuclear neutrophils (PMNs), peripheral blood mononuclear cells (PBMCs), monocytes, and monocyte derived dendritic cells (moDCs) from healthy donors with different morphotypes of R. arrhizus and compared immune responses to A. fumigatus stimulation. Oxidative burst of PMNs was only stimulated by germinated morphotypes of both fungi but not by resting conidia. Inhibition of TLR2, TLR4 and Dectin-1 by specific blocking antibodies significantly reduced the secretion of reactive oxygen species in response to R. arrhizus germlings and hyphae. Similarly, phagocytosis of R. arrhizus spores by PMNs was significantly reduced by blockade of TLR4 and Dectin-1, further corroborating a functional role of these receptors in the recognition of Mucorales. Co-culture studies with PBMCs, monocytes, and moDCs revealed that both resting and germinated stages of R. arrhizus induce a proinflammatory cytokine response of mononuclear phagocytes, whereas conidia of A. fumigatus were largely inert. To determine a functional role of Dectin-1 in the recognition of R. arrhizus morphotypes by mononuclear cells, moDCs were transfected with CLEC7A-siRNA, resulting in a knockdown of the Dectin-1 gene. The transfected moDCs were stimulated with different A. fumigatus and R. arrhizus morphotypes and mRNA expression and secretion of IL-1β were determined by qPCR and ELISA. Significantly weaker induction of IL-1β by germinated stages of both fungi and R. arrhizus spores was seen in siCLEC7A-transfected moDCs. Collectively, these results indicate that major pattern recognition pathways with known roles in the detection of A. fumigatus are also pivotal to mount a proinflammatory immune response to R. arrhizus.

Submitted by Biblioteca Central (biblioteca@unicap.br) on 2017-10-18T18:19:59Z No. of bitstreams: 1 dissertacao_renata_goncalves_santos_inacio.pdf: 1768781 bytes, checksum: 5fdfcec3f8878393182377f7f1dc5564 (MD5) / Made available in DSpace on 2017-10-18T18:19:59Z (GMT). No. of bitstreams: 1 dissertacao_renata_goncalves_santos_inacio.pdf: 1768781 bytes, checksum: 5fdfcec3f8878393182377f7f1dc5564 (MD5) Previous issue date: 2010-06-14 / The present work had as objective investigate the effect of dibenzotiofene - DBT (derived from oil) in the growth and morphology of Rhizopus arrhizus UCP 402. Had the increase of the use of the fossil fuels used for some industrialized countries, a serious environment problem aggravates due to quality of the reserves of the oil, in view of the amount of sulphur, and consequently, greater toxicity for the environment. In turn, the dibenzothiofene (DBT) is a composition heterociclic organossulfurated in the oil diesel, being considered a great fuel that cause ambient problems. The use of the DBT for R. arrhizus UCP 402 was evaluated from different concentrations of this composition, having observed itself the effect in kinetic of growth and the morphology, in the production of quitina and quitosana, as well as in the formation of toxic methabolits. The gotten results had indicated that R. arrhizus presents ability to grow in the different concentrations of the composition, however only, in co-metabolism, that is, in the glucose presence. A variation in the total protein text, quitina and quitosana for the action of the DBT was observed. The óptic microscopy evidenced variations in the morphology of R. arrhizus, also causing alterations in the ramification of hifas on standard. The products of the metabolism of the DBT had indicated that degradation of the composition for R. arrhizus occurred, whose methabolits had presented one accented inhibition of the germination of Cabbage of the 4 stations (oleracea Brassica to var. capitata). The carried through studies had demonstrated a great biotechnological potential of R. arrhizus in the process of degradation of DBT, being able to be used in the future in biorremediation processes. / O presente trabalho teve como objetivo investigar o efeito do dibenzotiofeno – DBT (derivado do petróleo) no crescimento e na morfologia de Rhizopus arrhizus UCP 402. Com o aumento da utilização de combustíveis fósseis por vários países industrializados, um sério problema ambiental se agrava devido à qualidade das reservas do petróleo, tendo em vista a quantidade de enxofre, e conseqüentemente, maior toxicidade para o meio ambiente. Por sua vez, o dibenzotiofeno (DBT) é um composto organossulfurado heterocíclico presente no óleo diesel, sendo considerado um combustível que causa grandes problemas ambientais. A utilização do DBT por R. arrhizus UCP 402 foi avaliada a partir de diferentes concentrações desse composto, observando-se os efeitos na cinética de crescimento e morfologia do fungo, na produção de quitina e quitosana, como também na formação de metabólitos tóxicos. Os resultados obtidos indicam que R. arrhizus apresenta habilidade para crescer nas diferentes concentrações do composto, porém apenas, em co-metabolismo, isto é, na presença de glicose. Observou-se uma variação no teor de proteínas totais, em quitina e quitina pela ação do DBT. A microscopia ótica evidenciou variações na morfologia de R. arrhizus, causando também alterações no padrão de ramificação das hifas. Os produtos do metabolismo do DBT indicam que ocorreu degradação do composto por R. arrhizus, cujos metabólitos apresentaram uma acentuada inibição da germinação de Repolho das 4 estações (Brassica oleracea var. capitata). Os estudos realizados demonstram um grande potencial biotecnológico de R. arrhizus no processo de degradação de DBT, podendo ser empregado no futuro em processos de biorremediação.

Predmetom tejto diplomovej práce bolo štúdium vplyvu detergentov na aktivitu, termostabilitu a agregáciu voľnej a imobilizovanej formy komerčného preparátu lipázy izolovanej z mikroskopickej huby Rhizopus arrhizus. Teoretická časť obsahuje ucelenú rešerš popisujúcu štruktúru, mechanizmus účinku a priemyselný význam spomínanej hydrolázy spolu s popisom chemických účinkov detergentov, pričom dôraz bol kladený predovšetkým na skupinu neionogénnych detergentov s názvom tweeny. V experimentálnej časti bol študovaný efekt tweenov na rozpustnej a imobilizovanej forme RA lipázy. Imobilizácia spočívala v priamej adsorpcii enzýmu na neupravený nosič. Ako nosič bol použitý oxidovaný grafén ošetrený tweenom (tween 20, 60, 80). Aktivita enzýmu bola stanovená spektrofotometricky za pomoci substrátu p-nitrofenyl laurátu. Zvýšenie aktivity voľnej lipázy (104 % oproti maximálnej hodnote) bolo zaznamenané pri použití tweenu 20 o koncentrácii vysoko nad hodnotou kritickej micelárnej koncentrácie (10 mmol•dm-3). Na základe štúdie imobilizačných podmienok, boli nastavené ideálne parametre pre dosiahnutie účinnej imobilizácie v spojení s čo najvyššou lipolytickou aktivitou (koncentrácia enzýmu 0,1 mg•ml-1, fosfátový tlmivý roztok pH 7,2, koncentrácia tweenu 10,8 mmol•dm-3, čas imobilizácie 1 hodina). Obe formy lipázy vykazovali maximálnu aktivitu pri 35 °C. Optimálne pH sa u imobilizovanej lipázy posunulo na hodnotu 8, v porovnaní s voľnou formou, ktorej pH optimum bolo stanovené na 9. Tepelná stabilita vykazovala približne rovnaký priebeh u oboch foriem skúmanej hydrolázy. Avšak v prípade štúdia stability enzýmu pri dlhodobej úschove bolo po imobilizácii zistené výrazné zlepšenie tohto parametru.

L(+)-Lactic acid is a commonly occurring organic acid, which is valuable due to its wide use in food and food-related industries, and its potential for the production of biodegradable and biocompatible polylactate polymers. The aim of this study was to optimize and scale-up a biotechnological process of L(+)-lactic acid production by suspended cells of R. arrhizus DAR 36017 with waste potato starch as the substrate. Commonly used inorganic and organic nitrogen sources, including ammonium sulphate, ammonium nitrate, urea, yeast extract and peptone, were assessed in conjunction with various ratios of carbon to nitrogen (C:N). Fermentation media with a low C:N ratio enhanced the production of lactic acid, biomass and ethanol, while a high C:N ratio led to production of more fumaric acid as a by-product. The use of organic nitrogen sources (yeast extract, peptone and urea) resulted in a significant reduction of lactic acid yields by 15% - 34% with a decrease of C:N from 168 to 28. The use of inorganic nitrogen sources (ammonium nitrate and ammonium sulphate) led to a high lactic acid yield of 84% - 91% at a C:N below 168. Therefore, ammonium nitrate and ammonium sulphate were considered to be better nitrogen sources for lactic acid production. Small pellets are the favoured morphological form for many fermentation processes by filamentous fungi. However, to control filamentous Rhizopus sp in the pellet form in a submerged fermentation system is difficult due to its filamentous characteristics. An acidadapted preculture technique was developed to induce the formation of the pellet form in bioreactors. Using the acid-adapted precultures, the fungal biomass can be controlled in small dispersed pellets as a dominant morphological form. With these small pellets, a lactic acid yield of 86-89%, corresponding to a concentration of 86-89g/L, was obtained in a laboratory scale process using a stirred tank reactor (STR) and a bubble column reactor (BCR). A batch bioprocess for lactic acid production was successfully scaled-up from shake flasks to laboratory scale bioreactors. Results from a simulated scale-up process revealed that the concentration and productivity of lactic acid decreased with the increase of the scale-up steps because of increased pellet size. This suggested that a one-step scale-up process using the acid-adapted preculture may be feasible in an industrial-scale bioreactor system. A comprehensive investigation of the impact of cultivation parameters on the morphology of R. arrhizus and lactic acid production was carried out in the BCR. The results showed that the fungal morphology was significantly influenced by carbon sources, pH, starch concentrations, sparger designs and aeration rates. The favoured morphology for lactic acid production was freely dispersed small pellets, which could be retained as a dominant morphology under operation conditions at pH 5.0 – 6.0, starch concentrations of 60 – 120 g/L and aeration rates of 0.2 – 0.8 vvm, using a sintered stainless steel disc sparger. The optimal cultivation conditions at pH 6.0 and aeration rate of 0.4 vvm resulted in the formation of the freely dispersed small pellets and production of 103.8 g/L lactic acid, with a yield of 87%, from 120 g/L liquefied potato starch in 48 h. This study shows a technically feasible and economically promising process for the production of lactic acid from waste potato starch. The use of waste potato starch instead of pure glucose or starch as substrate can significantly reduce the production cost, making this technology environmentally and economically attractive. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1339122 / Thesis (Ph.D.) -- University of Adelaide, School of Chemical Engineering, 2008