Les glicogen i midó sintases són glicosiltransferases que catalitzen la transferència de residus glucosil a l'extrem no reductor d'una cadena creixent d'un glucà α-1,4, retenint la configuració del carboni anomèric del sucre transferit. Aquest procès és central en el metabolisme energètic de la majoria d'èssers vius.En aquest treball presentem l'estructura cristal·logràfica de la glicogen sintasa de Pyrococcus abyssi (PaGS). Aquest enzim és termoestable i presenta una activitat màxima a 80ºC i és capaç d'utilitzar indistintament ADPG i UDPG com a donadors de glucosil. La PaGS forma de cossos d'inclusió en totes les condicions d'expressió en E.coli assajades. Amb el mètode de renaturalització en columna hem aconseguit que la PaGS es replegui correctament i ens ha permès obtenir la quantitat de proteïna pura necessària per encetar estudis de cristal·lització i de caracterització en solució.D'altra banda, el domini C-terminal de la PaGS s'expressa de forma soluble i la seva purificació ens ha permès disposar de quantitat suficient de proteïna per a realitzar estudis de cristal·lització. Aquest domini és monomèric tant en solució com en el cristall. La resolució de l'estructura del domini C-terminal a 1.7 Å a partir de reemplaçament molecular utilitzant el domini homòleg de la GS d'Agrobacterium tumefaciens (AtGS) ha permès utilitzar-lo com a model inicial per a resoldre l'estructura de la PaGS a 2.8 Å.La PaGS és un trímer tant en solució com en el cristall, amb una disposició triangular plana. Cadascuna de les seves subunitats està formada per dos dominis αβα tipus plegament de Rossmann separats per un solc molt profund on es troba el centre catalític de l'enzim. L'estructura global és molt similar a la de la resta d'estructures resoltes del grup GT-B que actuen amb retenció de la configuració anomèrica. La trimerització de la PaGS implica exclusivament els dominis N-terminals de l'enzim, quedant els dominis C-terminals lliures per a poder realitzar la transició d'obertura i tancament necessària per a la catàlisi.El domini C-teminal conté la cavitat d'unió a nucleòtid, mentre que la majoria d'interaccions amb l'oligosacàrid acceptor es produeixen a través de residus del domini N-terminal. L'elevada similitud del centre actiu entre les GS i les GP suggereix que ambdues operen a través d'un mecanisme catalític similar, si be no idèntic.L'estructura de la PaGS i la seva comparació amb l'estructura de l'AtGS ofereixen les bases per a entendre l'especificitat de les GS eucariotes per UDPG, com a substrat donador de glucosil, i la promiscuïtat de les GS d'arqueons per unir tant UDPG com ADPG.La localització d'una molècula de glucosa en la superfície de la PaGS i la comparació amb les GP eucariotes permeten suggerir un possible lloc d'emmagatzematge del glicogen en les GS.

La estructura molecular de diversos componentes celulares contiene, entre otros elementos, nitrógeno. La fuente de nitrógeno preferida por las bacterias y la mayoría de microorganismos es el amonio, el cual no siempre se encuentra disponible en el medio. Por ello, las bacterias han desarrollado mecanismos moleculares que les permiten obtener nitrógeno de fuentes alternativas como pueden ser las purinas. Este trabajo se centra en el metabolismo de las purinas como fuentes de nitrógeno en la enterobacteria Klebsiella pneumoniae. En este trabajo hemos identificado y caracterizado el sistema génico de K. Pneumoniae implicado en la asimilación de hipoxantina como fuente de nitrógeno, al cual hemos denominado hpx. El sistema hpx está formado por cuatro unidades transcripcionales. Las unidades hpxDE, hpxR y hpxO se transcriben a partir de promotores dependientes de la subunidad (sigma-70) 70 de la RNA polimerasa, mientras que 9hpxPQT presenta un promotor dependiente de (sigma-54) 54. El operón hpxDE está implicado en la oxidación de hipoxantina a ácido úrico. Sin embargo, los productos de los genes hpxD y hpxE no se asemejan a las xantina deshidrogenasas descritas hasta el presente. En base a la similitud de HpxD y HpxE con distintos componentes de la familia de las dioxigenasas, proponemos que el operón hpxDEcodifica una dioxigenasa formada por una oxidorreductasa (HpxE) y una oxigenasa (HpxD). El producto génico del gen hpxO está implicado en la oxidación de ácido úrico a alantoína. Sin embargo, dicha proteína no presenta similitud a uricasas sino a monooxigenasas de compuestos aromáticos dependientes de FAD. Proponemos que HpxO es una FAD-monooxigenasa que catalizaría la oxidación de ácido úrico al intermediario 5-hidroxiisourato, el cual sería transformado a alantoína por la acción secuencial de las proteínas HpxT y HpxQ. El gen hpxP codifica un transportador de purinas cuyo sustrato muy probablemente es ácido úrico. El gen hpxR codifica una proteína de la familia de reguladores LysR que actúa como represor de su propia transcripción y como activador del operón hpxDE El sistema hpx está sometido a una doble regulación, la llevada a cabo por el sistema global del nitrógeno y la llevada a cabo por la regulación específica de la vía. Mientras que el gen hpxR se expresa de manera constitutiva y el gen hpxO no está fuertemente regulado, los operones hpxDE y hpxPQT son inducidos tanto por limitación de nitrógeno como por la presencia de los inductores hipoxantina (hpxDE) o ácido úrico (hpxPQT). La regulación por nitrógeno del operón hpxPQT tiene lugar a través del sistema NTR de K. Pneumoniae, el cual se activa en condiciones de nitrógeno limitantes. El promotor PhpxP depende de la subunidad (sigma-54) 54 de la RNA polimerasa y es reconocido por el activador NtrC (NiTrogen Regulatory protein C). El regulador que reconoce el ácido úrico como molécula inductora todavía no ha sido identificado. Es de destacar que la regulación por nitrógeno del operón hpxDE no tiene lugar a través del sistema NTR. Resultados preliminares sugieren la existencia de un regulador no caracterizado hasta el presente al que proponemos denominar NR (Nitrogen Repressor). Este regulador reprimiría la expresión del operón hpxDE en condiciones de exceso de nitrógeno. Esta es la primera vez que se describe en K. Pneumoniae un sistema de regulación por nitrógeno distinto de NTR, hecho que evidencia la importancia del estudio del sistema génico hpx en esta enterobacteria. La regulación específica está llevada a cabo por el regulador HpxR, el cual reconoce a la hipoxantina como molécula inductora del operón hpxDE. / Gene organization and regulation of the genetic system hpx involved in utilization of hypoxanthine as nitrogen source in Klebsiella pneumoniae"Purines play a key role in nucleic acid and nucleotide metabolism of all cells. In addition, they can be used as nitrogen sources by many microorganisms when ammonium, the preferred nitrogen source, is limiting. Purine catabolism in enterobacteria has been poorly characterized at the genetic level. In this study we identify and characterize the gene cluster (named hpx)responsible for the oxidation of hypoxanthine to allantoin in K. pneumoniae. This genetic system is essential fot the aerobic assimilation of hypoxanthine as nitrogen source under nitrogen limiting conditions.This system is composed by four transcriptional units: hpxDE, hpxR, hpxO and hpxPQT. hpxP gene encodes a purine transporter. The hpxDE operon encodes subunits of the enzyme that catalyzes the oxidation of hypoxanthine to uric acid. The structure of this enzyme is more related to class IB dioxygenases than to xanthine dehydrogenases. Genes hpxO, hpxQ and hpxT are involved in the oxidation of uric acid to allantoin. The hpxO gene product seems to be more related to FAD containing monooxygenases than to uricases. Finally, hpxR encodes a LysR-type regulator that activates hpxDE expression and represses its own gene.Metabolism of hypoxanthine as nitrogen source is regulated by nitrogen conditions and by purines. hpxDE and hpxPQT operons are the units regulated at both levels. Transcription of hpxPQT operon is induced under nitrogen starvation and by uric acid through a regulatory protein other than HpxR. Nitrogen control of this unit is mediated by the NTR system.Expression of hpxDE operon is induced under nitrogen limiting conditions, independently fromthe NTR system, and by hypoxanthine through HpxR.

Lipases are carboxylic ester hydrolases which act on acylglycerols to liberate fatty acids and glycerol. These enzymes are currently attracting an enormous attention because they are among the most versatile and widely used enzymes in biotechnological applications and due to their unique properties. Moreover, these enzymes and their inhibitors have a high pharmacological interest because some microbial lipases can act as virulence factors in several infectious diseases. Therefore, the general objective of the present work was the isolation, cloning, characterization and inhibition by natural substances of microbial lipases with interest in biotechnology and infectious diseases.The first chapter was focused on the isolation and characterization of new Bacillus lipases to evaluate their biotechnological potential. The lipolytic system of several very active strains with an unknown lipolytic system was analyzed, and then, the lipases LipA from Bacillus megaterium CECT 370 and LipA from Bacillus sp. BP-6 were isolated, cloned and characterized. Both enzymes are family I.4 carboxylesterases closely related to Bacillus subtilis LipB, and have a high biotechnological potential due to their high stability and due to their molecular and biochemical properties.Chapter 2 consisted in the isolation of 724 microorganisms from soil samples collected from a subtropical forest of Puerto Iguazú (Argentina). Among them, 449 showed one or more of the biotechnologically-interesting enzymatic activities "true lipase", carboxylesterase, cellulase, xylanase and pectinase. CR-53 and CR-179, two very active isolates, were subsequently identified as two strains closely related to the species Rhodococcus erythropolis and Bacillus subtilis, respectively. Further analysis revealed that strain CR-53 produces a cell-bound carboxylesterase of 60 kDa, one of the first lipases known in the genus Rhodococcus, whereas strain CR-179 possesses a lipolytic system related to that of other Bacillus.Chapter 3 was focused in the development of a new, fast, simple and more sensitive colorimetric microassay with a low cost and suitable for high-throughput analysis of purified or non-purified lipolytic enzymes. The assay was subsequently used to evaluate the effect of several saturated fatty acids on five cell-bound or secreted (Paeni)Bacillus lipases. These lipids inhibited all the enzymes analyzed, although secreted lipases were activated by low concentrations of these compounds. Activation of microbial lipases by fatty acids is a phenomenon detected here for the first time, and could be related to the properties and biological function of these secreted enzymes.Chapter 4 consisted in the analysis of the inhibitory effect of several natural substances (saponins, flavonoids and alkaloids) on the model lipase from Candida rugosa (CRL) to evaluate their potential as antilipase drugs. beta-aescin, digitonin, kaempferol and (±)-catechin were selected as the best candidates for the treatment or prevention of lipase-related diseases due to the strong inhibition they produced on CRL, as well as due to their other beneficial effects and their low toxicity.The aim of chapter 5 was to isolate, clone, characterize and evaluate the inhibition of lipases from the pathogens Propionibacterium acnes and Helicobacter pylori. The analysis of GehA from Propionibacterium acnes P-37, a lipase considered as a major etiological agent in the pathogenesis of acne, revealed that this enzyme is very adapted to the skin conditions. EstV (HP0739), a family V carboxylesterase which was identified by analyzing Helicobacter pylori 26695 proteome, is the first lipase from this pathogen that has been cloned, purified and characterized. The evaluation of the effect of several natural substances on GehA and EstV revealed that beta-aescin, glycyrrhizic acid, (±)-catechin and kaempferol are promising candidates for the treatment of acne and/or ulcer due to their strong inhibitory activity on these lipases, as well as due to their other anctiacne or antiulcer effects and their low toxicity.

Actualment, es pot considerar que vivim a l'era dels plàstics. En els últims 50 anys els plàstics d'origen petroquímic han esdevingut un dels materials més emprats. La seva versatilitat, propietats tècniques i un cost relativament baix (0,70$US per Kg) són alguns dels factors que han influït en la proliferació del seu ús. Fins al moment han estat descrites moltes i molt diverses aplicacions per aquest material (Zinn et al., 2001).El principal desavantatge dels plàstics convencionals és la seva baixa biodegradabilitat. Els residus plàstics no són biodegradables i els costos dels processos de reciclatge són molt elevats (Yu et al., 1998). D'altra banda, la disponibilitat, l'accessibilitat i l'explotació de les fonts de carboni d'origen fòssil són cada vegada més limitades. El fet que sigui una font d'energia no renovable, conjuntament amb l'augment del seu cost ha fet necessari buscar altres fonts d'energia alternatives, per la qual cosa l'aplicació de processos biotecnològics ha despertat gran interès pel seu potencial.Per aquest motiu, al començament dels anys 80s, es començaren a realitzar diferents aproximacions en el desenvolupament i en la producció de plàstics biodegradables com per exemple: la cel·lulosa, el midó, la quitina, la poliamida, l'àcid polilàctic (PLA) i els polihidroxialcanoats (PHAs), ja que són considerats com una alternativa als "petroplàstics".Els Polihidroxialcanoats (PHAs) són polièsters biològics sintetitzats per nombrosos microorganismes i acumulats intracel·lularment en forma de grànuls com a reservori d'energia i de font de carboni. Aquest fenomen, sovint, està associat a un excés de la font de carboni i a la limitació d'algun altre nutrient (p.e: el nitrogen, el fòsfor, etc.). Els PHAs són materials biodegradables, biocompatibles i totalment estereoespecífics (tots els estereocentres tenen configuració [R]). Aquests biopolímers presenten propietats mecàniques semblants a alguns termoplàstics sintètics (p.e. el polipropilè), per altra banda el fet de ser materials biocompatibles fa que siguin bons canditats en el camp de la biomedicina. D'altra banda els PHAs tenen una gran versatilitat en termes de composició, de tal manera que es poden obtenir mcl-PHAs funcionalitzats susceptibles de ser modificats químicament a posteriori per tal de modular les propietats del polímer original.Els objectius d'aquesta tesi són:1. L'obtenció de nous polihidroxialcanoats (PHAs) funcionalitzats a partir de cultius de Pseudomonas aeruginosa 42A2 emprant com a substrats fonts de carboni definides i olis vegetals.2. La determinació estructural, caracterització físico-química i l'estudi microscòpic dels polièsters obtinguts.3. La derivatització dels PHAs funcionalitzats.En el present treball s'ha demostrat que Pseudomonas aeruginosa 42A2 es capaç de créixer i acumular PHA a partir dels següents substrats: àcid nonanoic, àcid undecanoic, àcid undecenoic, extracte monodi, àcid linoleic i oli de llinosa. Tots els PHAs obtinguts han estat caracteritzats mitjançant RMN, CG-EM i FTIR, de la mateixa manera també s'han analitzat les propietats tèrmiques (DSC i TGA) i s'han determinat els pesos moleculars (GPC). Quan s'utilitza l'oli de llinosa com substrat s'obté un PHA altament funcionalitzat que conté un 16% d'unitats monomèriques poliinsaturades (8,3% de C14:3 i 7,6% de (C14:2+C12:2). Aquest polímer funcionalitzat s'ha derivatitzat mitjançant tractament fotoquímic amb radiació UV per tal d'induir l'entrecreuament de les cadenes laterals poliinsaturades i generant així un nou polímer.Els canvis estructurals deguts a la reacció d'entrecreuament han estat seguits mitjançant diverses tècniques analítiques (RMN, FTIR, CG-EM, DSC i TGA). En aquest treball, també es presenta un estudi tant del bacteri com del PHA com a biomaterial emprant diverses tècniques microscòpiques (TEM i AFM), essent la primera vegada que es recullen imatges d'un polihidroxialcanoat reticulat.Paral·lelament, es va realitzar l'escalat a bioreactor dels cultius amb àcid undecanoic i àcid undecenoic, com a co-substrats. En els diferents experiments realitzats en bioreactor s'han obtingut polímers amb diferent contingut de monòmers insaturats (15-70%), indicant així que, el règim d'alimentació continu en els cultius en bioreactor permet modular la composició del polímer i augmentar el percentatge d'acumulació de PHA fins a un 50% en front del 20% que s'obtenia en els cultius amb matrassos. / Nowadays one may consider that we are living in the age of the plastics. The global dependence on petroleum-derived plastics has increased over the last 50 years. However the rapid depletion of crude oil, the increasing problems with the biodegradation of these plastics and the environmental effects has prompted much interest in biologically derived polymers, particularly of the biodegradable class, produced by biotechnological processes. Among the different types of biodegradable polymers, a class that is drawing considerable attention is the polyhydroxyalkanoates (PHAs). Polyhydroxyalkanoates are optically active biopolyesters synthesized by a large number of microorganisms using a wide range of substrates such as fatty acids, sugars, oily wastes, etc. These polymers exhibit thermoplastic and elastomeric properties, depending on their composition. Moreover their high biocompatibility allows these polymers to be good candidates for medical applications.This doctoral thesis deals in a first part with the biosynthetic production and characterization of functionalized PHA, using defined substrates (nonanoic, undecanoic and undecenoic acid) and blends of fatty acids (linoleic acid (60%), linseed oil and an extract of hydroxylated fatty acids) as well as the undecanoic and undenoic acid has been used as a cosubstrates to obtained a block copolymer. The scale up to a 10L bioreactor of this process allows to improve the yield of PHA-accumulation reaching up to 50% versus the 20% obtained in the flasks experiments. Furthermore, the continuous feeding strategy let us to improve and to modulated the final composition of the biopolymers. Several experiments have been carried out rendering in polymers with different content of unsaturated monomers (15-70%). In the second part of this work, the functionalized PHA obtained from linseed oil, which contain up to 16% of polyunsaturated monomers (8,3% C14:3 i 7,6% (C14:2+C12:2), were modified by UV-irradiation to enhance the crosslinking reaction among the polyunsaturated side chains, in order to obtain a novel polymer with new and improved properties. The structural modifications as well as the thermal properties have been followed during the reaction by various analytical techniques (NMR, FTIR, GC, GC-MS, HPLC-MS, GPC, DSC and TGA). Besides the analytical techniques used for the characterization of these biopolymers, in this work is also showed a study of the bacterial cells as well as the PHA as a biomaterial using microscopical techniques (TEM and AFM), being the first time that a crosslinked PHA micrograph is acquired.

Mestre -- Escola Nacional de Saude Publica, Rio de Janeiro, 2000.

Made available in DSpace on 2014-08-13T14:50:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-10-16Bitstream added on 2014-08-13T18:01:30Z : No. of bitstreams: 1 000747021_20151016.pdf: 607081 bytes, checksum: bbdf4b1c6ca753edb9c6aef714441073 (MD5) Bitstreams deleted on 2015-10-16T13:54:03Z: 000747021_20151016.pdf,. Added 1 bitstream(s) on 2015-10-16T13:54:46Z : No. of bitstreams: 1 000747021.pdf: 964954 bytes, checksum: 60f92abc05445fbbd653fbb32d487c4c (MD5) / A família Trypanosomatidae compreende um grande número de protozoários parasitas, incluindo importantes agentes etiológicos de doenças humanas. Entre os causadores de doenças, destacam-se Trypanosoma brucei (responsável pela doença do sono), Trypanosoma cruzi (agente causador da doença de Chagas) e Leishmanias sp (causadoras de leishmanioses). Esses parasitas possuem como mecanismo para formação de seus mRNAs, a reação de trans-splicing, que envolve a excisão de íntrons (regiões intergênicas) e a união dos éxons de dois transcritos independentes, SL RNA (spliced leader) e pré-mRNA aceptor. Em outro trabalho realizado no laboratório, a reação de trans-splicing in vitro foi estabelecida utilizando extratos nucleares (livre de células) de formas epimastigotas de T. cruzi e/ou formas procíclicas de T. brucei e como pré-mRNA aceptor, a sequência parcial de alfa tubulina (contendo a região intrônica, splice site e parte do éxon da alfa tubulina) de T. cruzi marcada radioativamente (dados ainda não publicados). O uso de material radioativo é fator limitante, devido a sua alta periculosidade (extremamente nocivo ao homem e ao meio ambiente) e, também, pelo alto custo relativo aos trâmites burocráticos atuais para sua importação. Portanto, neste trabalho reproduziu-se a reação sem o uso de material radioativo, utilizando-se a transcrição reversa/ reação em cadeia da polimerase (RT-PCR) e gel de agarose corado por brometo de etídeo para a análise dos produtos formados. A metodologia permitirá a avaliação da interferência de substâncias tripanocidas, buscando potenciais alvos terapêuticos nas ribonucleoproteínas de parasitas, preservando as células do hospedeiro. / The Trypanosomatidae family includes a large number of protozoan parasites, with importants etiological agents of human diseases. Among the causes of diseases stand out Trypanosoma brucei (responsible for sleeping sickness), Trypanosoma cruzi (Chagas disease causative agent) and Leishmania sp (causing leishmaniasis). These parasites have a mechanism that form their mRNAs, the trans-splicing reaction, which involves the introns (intergenic regions) excision and two exons union of the independent transcripts, RNA SL (spliced leader) and pre-mRNA acceptor. In another laboratory study, the trans-splicing in vitro reaction was established using nuclear extracts (cell-free) from epimastigotes forms of T. cruzi and / or procyclic forms of T. brucei, and as pre-mRNA aceptor was used the alpha tubulin partial sequence (containing the intronic region, splice site and a radiolabelled part of alpha tubulin exon from T. cruzi (unpublished data). The use of radioactive material is a limiting factor, due to its high hazard (extremely harmful to humans and environment), and also by the high cost relative to the current bureaucratic procedures for importation. Therefore, in this work the reaction was reproduced without the use of radioactive material, using the reverse transcription / polymerase chain reaction (RT-PCR) and agarose gel stained with ethidium bromide for analysis of the formed products. The method allows the evaluation of trypanocidal substances interference searching potential therapeutic targets in the parasite ribonucleoprotein, preserving the host cells.

Resumo: A principal meta deste trabalho foi a de realizar estudos com leveduras industriais brasileiras utilizadas em usinas, na tentativa de viabilizar o emprego da Tecnologia de Fermentação de Mostos com Altos Teores de Açúcares Fermentecíveis, na produção de etanol combustível. Inicialmente, foram realizados experimentos para obtenção de informações relativas as linhagens, na fermentação de sacarose, maltose e glicose, em meios contendo uma base nitrogenada e suplementado com fontes de nitrogênio com diferentes complexidades estruturais. Os estudos mostraram que as linhagens industriais apresentam diferentes perfis de crescimento nos meios suplementados com diferentes fontes de nitrogênio, que variaram de um simples sal de amônio, a um hidrolisado ácido de proteínas (casaminoácidos) e hidrolisado enzimático de proteínas (peptona). Entretanto, a maior capacidade de acúmulo de biomassa pode não ter um reflexo direto na capacidade de utilização de açúcares e a consequente produção de etanol pelas leveduras industriais. Além da natureza estrutural da fonte de nitrogênio e do tipo de açúcar, a presença do oxigênio, em maior quantidade nos cultivos agitados, interferiu sobremaneira no desempenho fermentativo das leveduras industriais, e até no acúmulo de trealose. No geral, foi a suplementação com peptona que propiciou maior produção de biomassa, preservação da viabilidade celular e consumo mais eficiente da fonte de carbono, em cultivos agitados e não agitados. Os estudos com alta densidade celular tiveram como objetivo definir condições experimentais para a condução da Fermentação de Mosto com Altos Teores de Açúcares Fermentescíveis, e que pudessem propiciar ao final do processo a completa utilização da sacarose, associada á preservação da viabilidade... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In this work studies with Brazilian yeast for ethanol production were conducted in order to verify the possibility of utilization of brazilian yeasts for ethanol production in an industrial process known as Very High Gravity Sucrose Fermentation Technology, to produce wine with high ethanol contends. This technology has the advantage to reduce production costs by increasing fermentation yields. Initially, studies were conducted with sucrose, maltose and glucose fermetation in medium containing YNB, supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Data suggest that yeast strains vary in their response to nitrogen source complex structure, kind of sugar and to oxygen availabity. In general, under peptone supplementation all strains, in shaking and static conditions, showed higher biomass accumulation, efficient sugar utilization and yeast viability was preserved. Sugar utilization by industrial strains not always was directly correlated with higher biomass accumulation. Trehalose accumulation was also influenced by the structural complexity of nitrogen sources, the kind of sugar and the presence of oxygen. Studies with high cell density were conducted to define experimental parameters for Very High Gravity Sucrose Fermentation, in order to induce at the end of process full sugar exhaustion together cell viability preservation. Complete sucrose utilization was detected only in media with 22 and 25% (w/v) sucrose. In the presence of higher sucrose contends (30% (w/v)), total sucrose exhaustion was obtained in a sugar cane based medium, supplemented with 2% (w/v) peptone. The results described in this thesis suggest that industrial yeasts show differing nitrogen demand, and the utilization of Very High Gravity Sucrose Fermentation technology could be carried only after finding the appropriated nutritional and fermentation conditions / Orientador: José Roberto Ernandes / Banca: João Atilio Jorge / Banca: Jonas Contiero / Banca: Sandra Helena da Cruz / Banca: Luis Henrique Souza Guimarães / Doutor

Made available in DSpace on 2014-06-11T19:30:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-02-28Bitstream added on 2014-06-13T20:40:28Z : No. of bitstreams: 1 paes_mcf_dr_jabo.pdf: 652606 bytes, checksum: 36fc9416b423c16aa4b65e4486ef48e2 (MD5) / O gênero Brycon tem despertado grande interesse nas instituições de pesquisa, não só pela excelente qualidade e sabor da carne, mas também por características adequadas à pesca esportiva, rápido crescimento e ganho de peso. A piracanjuba tem sofrido drástica redução dos estoques em decorrência da ação antrópica estando na lista do IBAMA de espécies ameaçadas de extinção. A criopreservação de gametas e embriões de espécies migradoras e espécies raras surge como uma alternativa para a reprodução, repovoamento, criação de banco genético, transporte e possível obtenção de larvas ao longo do ano. Assim, este trabalho teve por objetivo ampliar os conhecimentos de criogenia utilizando embriões de piracanjuba (Brycon orbignyanus), com o intuito de estabelecer um protocolo de resfriamento para ser utilizado em projetos de reprodução e conservação desta espécie. Após a obtenção dos embriões por meio de reprodução induzida, estes, quando na fase de fechamento do blastóporo, foram contados e subdivididos em parcelas, considerando como unidade experimental um tubo (vacutainer) contendo 100 embriões. Cada tratamento teve oito repetições. Com exceção dos grupos controles, os demais tratamentos foram submetidos a diferentes combinações e concentrações de crioprotetores (sacarose, metanol, etilenoglicol e DMSO), diferentes temperaturas (0,0±2,0°C e 8,0±2,0ºC) e tempos de estocagem em refrigerador (6h, 10h, 24h, 72h, 168h). Transcorridos os tempos de estocagem, os embriões foram aclimatados, rehidratados e transferidos para incubadoras para monitoramento do desenvolvimento e aferições das taxas de eclosão, sobrevivência e deformidade. Para avaliar o desenvolvimento de tecidos e células após a realização do experimento, amostras foram fixadas, observadas em estereomicroscópio e... / The genus Brycon have aroused great interest of research institutions, not only for the excellent quality and flavor of the meat, but also for characteristics suited to sport fishing, quick growth and weight gain. Piracanjuba suffers drastic reduction in natural environment as a result of human action and is in the list of endangered species of IBAMA. The cryopreservation of gametes and embryos of migratory and rare species is an alternative to reproduction, repopulation, creating genetic database, transport of larvae and obtainment of larvae throughout the year. The aim of this study is expand the knowledge of cryogenics using embryos of piracanjuba (Brycon orbignyanus) and establishing a cooling protocol to be used in projects of reproduction and conservation of this species. After obtaining embryos from induced reproduction, those ones that were in this phase of blastopore closuring, were colected, counted and divided into groups, considering as experimental unit each tube (vacutainer) containing 100 embryos. Each treatment had eight replications. Except for the control groups, the other treatments were subjected to different combinations and concentrations of cryoprotectants (sucrose, methanol, ethylene glycol and DMSO), different temperatures (0,0 ± 2,0ºC and 8,0 ± 2,0°C) and storage time on refrigerator (6h, 10h, 24h, 72h, 168h). Elapsed times of storage, the embryos were acclimated, washed and transferred to incubators for monitoring the development of measurements and hatching rates, survival and deformity. To evaluate the development of tissues and structures after the experiment, samples were put on fixative solution and observed under stereomicroscope, and after that were processed and analyzed by light microscopy and transmission electron microscopy. It was observed that... (Complete abstract click electronic access below)

Made available in DSpace on 2014-06-11T19:32:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-02-15Bitstream added on 2014-06-13T18:44:34Z : No. of bitstreams: 1 dayoowoyemi_i_dr_rcla.pdf: 2213436 bytes, checksum: 57ef54bb4924dae1b326ff6c146c0802 (MD5) / Atualmente, existe um crescente interesse em explorar diversos habitats, a fim de revelar a biodiversidade microbiana, incluindo as leveduras. Tal diversidade ainda não acessada guarda a descoberta de novas espécies para ciência, provavelmente muitas das quais com potencial para aproveitamento em processos biotecnológicos. Com o objetivo de explorar e conservar a diversidade de fungos, o Central de Recursos Microbianos da UNESP (CRM – UNESP) mantém em seu acervo várias estirpes de leveduras isoladas de ecossistemas diversos, sendo alguns deles pouco explorados. No início deste trabalho sabíamos que muitas das leveduras depositadas no acervo do CRM – UNESP não estavam totalmente caracterizadas tanto em nível taxonômico, quanto em relação ao potencial biotecnológico que poderiam apresentar. Portanto, o presente estudo foi desenhado para caracterizar e identificar taxonomicamente leveduras depositadas no CRM – UNESP, bem como selecionar estirpes que produzem enzimas extracelulares degradadoras de polissacarídeos como amilase, celulase, xilanase, pectinase e ligninase. Usando uma abordagem polifásica, um total de 340 isolados de leveduras foi identificado, sendo que 71,2% compreendem 43 taxa de ascomicetos e os restantes 28,8% foram classificados em 27 taxa de basidiomicetos. O estudo também levou à descoberta de 8 prováveis novas espécies. Baseado nesta constatação, a classificação taxonômica e análise filogenética foi realizada para duas espécies anamórficas de ascomicetos e uma espécie teleomórfica de basidiomiceto. A descrição destas três espécies é apresentada neste estudo. Os resultados demonstraram que Wickerhamiella kiyanii FB1-1DASPT e W. pindamonhangabaensis H10YT pertencem à clade Wickerhamiella da ordem Saccharomycetales... / In recent time, there has been an increasing interest in exploring diverse ecological habitats in order to reveal the yeast biodiversity. The increased awareness in the biotechnological potentials of yeasts has also spurred attempts to search for new species with novel biotechnological capabilities. Aiming to explore and conserve the fungal diversity from various ecosystems, the UNESP – Central for Microbial Resources (UNESP – CMR) harbors various strains of ecologically diverse yeasts isolates, some of which were yet to be identified. Therefore, this study was designed to identify and characterize some yeasts from the UNESP – MRC and to select strains possessing extracellular plant polysaccharide degrading enzymes namely amylase, cellulase, xylanase, pectinase and ligninase. Using a polyphasic approach, a total of 340 strains were identified. Taxonomic classification grouped 71.2% of these isolates into 43 ascomycetous taxa while the remaining 28.8% were classified in 27 basidiomycetous taxa. The study also led to the discovery of 8 putative new species. As a result, we classified two anamorphic species in the Ascomycota and one teleomorphic species in the Basidiomycota. In this study we provide the description of both species. Our results demonstrated that the two ascomycetous species proposed as Wickerhamiella kiyanii FB1-1DASPT and W. pindamonhangabaensis H10YT belong to the Wickerhamiella clade of the Saccharomycetales (Saccharomycetes) while the basidiomycetous species proposed as Bulleromyces texanaensis ATT064T belong to the Bulleromyces / Papiliotrema / Auriculibuller clade of the Tremellales (Agaricomycotina). In order to show the significance of intraspecific diversity in yeasts, in one of our studies, we subjected 11 strains, (including the type strain CBS 8960T) of Hannaella kunmingensis... (Complete abstract click electronic access below)

Made available in DSpace on 2015-03-03T11:52:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-07-25Bitstream added on 2015-03-03T12:06:21Z : No. of bitstreams: 1 000806886_20160725.pdf: 581026 bytes, checksum: 4c55faecb6d2b9cbebcf0fed8076771a (MD5) Bitstreams deleted on 2016-07-25T13:17:27Z: 000806886_20160725.pdf,. Added 1 bitstream(s) on 2016-07-25T13:18:38Z : No. of bitstreams: 1 000806886.pdf: 2412685 bytes, checksum: 76a763d7dfe3317535d6f9ec19a7cd78 (MD5) / As endoglicanases EC 3.2.1.4 são enzimas capazes de hidrolisar as ligações glicosídicas ?-1,4 da celulose, resultando na liberação de glicose, celobiose e celo-oligossacarídeos. Empregadas mundialmente em diversos processos industriais, por exemplo, como amaciante de tecidos de algodão e bioestonagem de jeans, na extração de sucos de frutas, na adição em detergentes comerciais, na fabricação de cerveja, e na produção de papel e de etanol. Atualmente, há um grande interesse em enzimas que hidrolisem biomassa lignocelulósica para obtenção de etanol de segunda geração. A imobilização de enzimas em matrizes sólidas oferece muitas vantagens, entre as quais, reuso da enzima, a fácil separação do produto e o aumento da estabilidade operacional. Os objetivos deste trabalho foram: produzir CMCases secretadas por Aspergillus niger e Humicola grisea var. thermoidea em cultivo sólido e submerso; imobilizar CMCases em suporte agarose com diferentes ativações químicas; determinar a estabilidade térmica e as propriedades cinéticas dos derivados obtidos, comparando-os com as enzimas livres; avaliar a capacidade de reuso dos derivados ativos e estáveis e analisar qualitativamente o produto de hidrólise dos substratos celulósicos. O fungo A. niger secretou grandes quantidades de CMCases, 34,1 U.mg-1, em meio Vogel, utilizando pó de bagaço de cana-de-açúcar como fonte de carbono. O pH 5 foi determinado como o ótimo para a enzima, apresentando instabilidade ao pH 10. Com a utilização de aditivos estabilizante, a trealose a 10% (m/v), manteve, em pH 10, 80% de atividade residual, por 25 horas. Em relação à temperatura, apresentou uma faixa ampla de atividade entre 45°C a 75°C, tendo como temperatura ótima 65°C. O derivado enzimático que ofereceu melhor estabilidade foi aquele em que a imobilização foi conduzida juntamente com o substrato carboximeticelulose (CMC 1%), em... / The endoglucanases EC 3.2.1.4 are enzymes that hydrolyze the ?-1, 4 glycosidic linkages of the cellulose producing glucose, cellobiose and cello-oligosaccharides. Used worldwide in many industrial processes such as fabric softener and bioestonagem cotton jeans, extraction of fruit juices, in the addition of commercial detergents, in brewing, and the production of paper and ethanol. Currently, there is great interest in enzymes that hydrolyze lignocellulosic biomass to obtain second-generation ethanol. The immobilization of enzymes on solid arrays provides many advantages as the enzyme reuse, easy separation of the product and increased operational stability. The aims of this study were to produce secreted: CMCases produce secreted by Aspergillus niger and Humicola grisea var. thermoidea in solid and submerged cultivation; CMCases immobilized on agarose support with different chemical activations; determine the thermal stability and kinetic properties of the derivatives obtained comparing them with the free enzyme; evaluate the reuse capacity assets and stable derivatives and qualitatively analyze the product of hydrolysis of cellulosic substrates. The fungus A. niger secreted large amounts of CMCases, 34.1 U.mg-1 amid Vogel, using powdered sugarcane bagasse as a carbon source. The pH of 5 was determined as the optimum for the enzyme, presenting instability to pH 10. With the use of stabilizing additives, the 10% trehalose (w / v), kept at pH10, 80% residual activity for 25 hours. Regarding temperature there was a broad activity range from 45 °C to 75 °C and the optimum temperature as 65 °C. The enzyme derivative which offered better stability was one in which immobilization was conducted with the carboxymethycellulose substrate (1% CMC) in agarose support Glutaraldehyde (70% of reactive groups), preserving catalytic activity of 35% at 60 °C for 250 hours. Through qualitative TLC analysis, it...