A autólise acinar post mortem em glândulas sublinguais humanas é um fenômeno que prejudica a sua análise microscópica. Com o objetivo de esclarecer e prevenir tal ocorrência, a presente investigação foi planejada no sentido de analisar morfometricamente as possíveis influências do intervalo post mortem (IPM) e do volume de fixador histológico (VF) na ocorrência de autólise de ácinos em glândulas sublinguais de ratos. Dos sessenta animais utilizados na investigação, cinqüenta deles o foram no estudo do intervalo post mortem, sendo divididos em 2 grupos: o grupo I (25 animais) foi destinado para as avaliações morfométricas e o grupo II (25 animais) para determinação do fator de retração e densidade da glândula. Os grupos I e II foram subdivididos nos subgrupos: A e A1 (controle - 0 hora), B e B1 (3 horas post mortem), C e C1 (6 horas), D e D1 (12 horas) e E e E1 (24 horas), com 5 animais em cada um. A fixação foi realizada com 20 mL de solução de formol a 10% em tampão fosfato. Os 10 animais remanescentes foram destinados ao estudo da variação de volume do fixador, e foram divididos em 2 grupos iguais: no grupo 2mL, as glândulas dos 5 animais foram fixadas com 2 mL de solução de formol a 10% tamponada, e no grupo 20mL, as glândulas dos outros 5 animais foram fixadas com 20 mL da mesma solução. O tempo de fixação foi de 7 dias para todos. As glândulas foram processadas histologicamente, sendo os cortes histológicos corados com H.E. A análise morfométrica foi realizada em 50 campos histológicos por glândula, selecionados por amostragem sistemática, usando objetiva de 100x e ocular Kpl 8x contendo um retículo de integração constituído por 100 pontos simetricamente distribuídos. A densidade de volume dos ácinos íntegros e autolisados foi avaliada pelo método morfométrico de volumetria relativa de contagem de pontos. Houve diferença estatisticamente significante entre o IPM e a autólise acinar (p< 0,05), enquanto que não houve diferença significante quanto ao VF (p= 0,690). A autólise acinar aumentou significantemente com o aumento do período post mortem (p<0,05). Baseado nos resultados obtidos foi possível concluir que a autólise acinar em glândulas sublinguais de ratos está diretamente relacionada ao intervalo post mortem, não sendo influenciada pelo volume de fixador histológico testado no experimento. / Acinar post mortem autolysis is a phenomenon that difficult the microscopic analysis in human sublingual glands. The aim of the present study is to evaluate the influence of the post mortem interval (PMI) and formalin volume (FV) in the occurrence of acinar autolysis in sublingual glands of rats. Sixty animals were used in this study. Out of them fifty animals were divided in 2 groups for PMI investigation: group I (25 animals) for morphometric quantifications and group II (25 animals) to calculate the retraction factor and the density of the glands. The groups I and II were subdivided in subgroups with 5 animals each: A and A1 (control - 0 hour), B and B1 (3 hours post mortem), C and C1 (6 hours), D and D1 (12 hours) and E and E1 (24 hours). The remaining 10 animals were used for the FV study and were divided in two groups with different volume of formalin, 2mL and 20mL respectively. The fixation period was 7 days. The glands were processed and stained with HE. The morphometric analysis was performed in 50 histological fields, selected by systematic sampling, using lens of 100x and ocular Kpl 8x containing a Zeiss II integration grid with 100 points symmetrically distributed. The volume density of intact and autolysed acini was evaluated by the morphometric method of relative volume of counting of points. There was a statiscally significant difference between volume density acinar autolysis and PMI for all group tested (p=0,0001). The difference was not significant for FV (p = 0,690). We concluded that acinar autolysis in rat sublingual glands increased significantly with the PMI, not being influenced by the FV, as tested.

An extracellular bacteriocin-like substance produced by Clostridium acetobutylicum was detected during studies on an industrial fermentation process. The bacteriocin-like substance was not inducible by either ultraviolet light or mitomycin C, and its production was not associated with the induction of a protease. Studies on the mode of action of the bacteriocin-like substance indicated that it had no significant effect on DNA, RNA, or protein synthesis, and it did not cause the loss of intracellular ATP. However, the bacteriocin-like substance was able to lyse SDS-treated cells and cell walls of C. acetobutylicum and was identified as an autolysin. Some of the characteristics of this extracellular autolysin were determined, and after purification it was shown to be a glycoprotein with a molecular weight of 28 000.

As leveduras são de relevante importância dentro da agroindústria sucroalcooleira devido sua participação no processo fermentativo de produção de álcool. Deste modo, faz-se necessário o conhecimento deste agente fermentativo com destaque para Saccharomyces cerevisiae, principal gênero. O objetivo deste trabalho foi estudar a linhagem de levedura S. cerevisiae Y904, exposta a condições não proliferantes, após fermentação em meio que sofreu adição de óleo vegetal e sua possível correlação com manutenção da viabilidade celular. Foram realizadas análises para contagem de unidades formadoras de colônias, viabilidade celular, concentração celular, nitrogênio total na levedura e no meio, carboidratos totais, trealose e glicogênio. As leveduras submetidas a condições não proliferantes apresentaram menores teores de carboidratos totais, com destaque para trealose e glicogênio, em relação às leveduras comerciais. Saccharomyces cerevisiae sofreu queda de viabilidade acentuada após 24 h em solução fisiológica, em condições não proliferantes, sob agitação de 90 rpm e temperatura de 30 ± 1°C, seguida de uma acentuada autólise a partir de 120 h (5°dia), provavelmente, devido ao teor de carboidratos de reserva da célula que se encontravam em valores extremamente baixos, da ordem de 0,15 mg de trealose em 100 mg da matéria seca e 4 mg de glicogênio em 100 mg da matéria seca. A partir desse ponto entraram em total desorganização celular. / Yeast is highly important in sugar and alcohol agroindustry due to its role in the fermentative process of alcohol production. Thus, it is necessary to know this microorganism, most specially the Saccharomyces cerevisiae, the main species. The objective of this work was to study the strain Y904 of the yeast Saccharomyces cerevisiae under non-proliferating conditions after fermentation in a medium in which it was added vegetable oil and verify its possible correlation with the maintenance of the cellular viability. Analyses were performed in order to determine colony forming units, cellular viability, cellular concentration, total nitrogen in yeast and in medium, total carbohydrates and trehalose and glycogen contents. The yeast submitted to non-proliferating conditions presented a lower content of total carbohydrates, specially trehalose and glycogen, when compared to commercial yeasts. The viability of the yeast Saccharomyces cerevisiae Y904 markedly decreased after 24 hours in physiological solution under non-proliferating conditions in a shaker for 90 rpm at 30 ± 1°C. It was observed an accentuated autolysis from the 120 th hour (5 th day) on. This was probably because of the very low content of the carbohydrates of reserve in the cells, 0.15 mg of trehalose and 4.0 mg of glycogen in 100 mg of dry weight. From this point the cells began a total cellular disorganization.

A functional water-conducting system, the tracheary elements of the xylem, is required to sustain plant growth and development. Tracheary element formation is dependent on many biological processes terminated by programmed cell death and cellular autolysis. The final two processes are probably dependent on the activity of hydrolytic enzymes such as XCP1 and XCP2 known to be expressed in tracheary elements during these final two processes. Thus, the transcriptional regulation of XCP1 and the function of XCP2 were investigated. Qualitative and quantitative assessments of GUS activity as directed by various fragments of the XCP1 promoter showed that a 237-bp internal region was able to drive GUS expression in a tracheary element-specific manner in Arabidopsis. A 25-bp deletion at the 3' end of this region abolished GUS expression. The 237-bp region served as bait in a yeast one-hybrid analysis. Screening of yeast colonies retrieved 109 putative positive interactions, which included a potential transcriptional regulator, indole acetic acid-induced protein 8 (IAA8). An auxin responsive element that potentially binds auxin responsive transcription factors was found within the 25-bp deletion. Cis-elements were predicted by Genomatix and Athamap computer programs. The cis-elements form pyrimidine and gibberellic acid responsive elements that can potentially bind Dof and Myb transcription factors, respectively. In an independent effort, attempts to develop a mapping population to isolate upstream regulators of XCP1 expression did not succeed. Functionally, tracheary element-specific expression of XCP2 in Arabidopsis suggested a specialized role for XCP2 in final phases of tracheary element differentiation. The function of XCP2 was assessed using T-DNA insertional mutants, post-transcriptional gene silencing, and through tracheary element-specific expression of the cysteine protease inhibitor, soyacystatin N in Arabidopsis. Our findings revealed that the absence of XCP2 expression due to T-DNA insertional mutagenesis did not affect plant growth and development in the laboratory. Soyacystatin N was an effective in vitro inhibitor of cysteine proteases. Plants expressing 35S-driven cytosolic form of soyacystatin exhibited stunting and reduced apical dominance. Plants expressing pXCP1-driven cytosolic soyacystatin did not differ from wild type plants. Additionally, transgenic plants expressing pXCP1- and 35S-directed XCP2-double-stranded RNA for the silencing of XCP2 showed no unusual phenotypes compared to their wild type counterparts / Ph. D.

Doctor of Philosophy / Department of Biology / Lynn E. Hancock / The enterococci are gaining much notoriety as common nosocomial pathogens. One aspect of their pathogenesis, especially characteristic to infectious endocarditis and urinary tract infections, involves their ability to transition from the sessile state of existence to surface adherent structured communities called biofilms. Existence as biofilms, affords enterococci protection against a number of growth limiting challenges including antibiotic therapy and host immunity. In the current study a mechanistic role for two Fsr quorum-regulated extracellular proteases- gelatinase (GelE) and its cotranscribed serine protease (SprE), were explored in biofilm development of E. faecalis V583. Confocal imaging of biofilms suggested that GelE[superscript]– mutants were significantly reduced in biofilm biomass compared to V583, whereas the absence of SprE appeared to accelerate the progression of biofilm development. Culture supernatant and biofilm analysis confirmed that decreased biofilms observed in GelE[superscript]– mutants resulted from their inability to undergo autolysis and release extracellular DNA (eDNA) in planktonic and biofilm cultures, whereas SprE[superscript]– mutants produced significantly more eDNA as components of the biofilm matrix. The governing principle behind GelE mediated autolysis and eDNA release in E. faecalis V583 was demonstrated to be fratricide. GFP reporter assays of V583 populations confirmed that GBAP (gelatinase biosynthesis-activating pheromone encoded by fsrD) quorum non-responders (GelE[superscript]–SprE[superscript]–) were a minority subpopulation of prey cells susceptible to the targeted fratricidal action of the quorum responsive predatorial majority (GelE[superscript]+SprE[superscript]+). The killing action is dependent on GelE, and the GelE producer population is protected from self-destruction by the co-production of SprE as an immunity protein. Targeted gene inactivation and protein interaction studies demonstrate that extracellular proteases execute their characteristic effects following downstream interactions with the primary autolysin, AtlA. Finally, comparison of virulence effects of isogenic extracellular protease mutants (∆gelE, ∆sprE and ∆gelEsprE) relative to parental strain (V583) in a rabbit model of enterococcal endocarditis confirmed a critical role for GelE in the infection process. In conclusion, the data presented in this thesis are consistent with significant roles for GelE and SprE in biofilm mediated pathogenesis of enterococcal infections.

Metaloproteases/desintegrinas (MD) isoladas de venenos de serpentes são potentes inibidores de agregação plaquetária e de adesão celular, processos envolvidos em doenças como trombose e câncer. As MD pertencem a classe PIII das SVMPs (´snake venom metalloproteinases´) que são constituídas por três domínios: metaloprotease (M), tipo-desintegrina (D) e rico em cisteína (C). A função dos três domínios nas atividades das moléculas ainda não é totalmente conhecida, e estudos com o objetivo de esclarecer suas funções são importantes para o desenvolvimento de novos fármacos. Algumas proteínas da classe PIII apresentam elevada atividade auto-proteolítica (liberando peptídeo constituído dos domínios D e C) enquanto que em outras PIII esta atividade não é menos evidente. Neste trabalho nós estudamos MD isoladas de veneno de Bothrops jararaca (bothropasina) e Bothrops alternatus (alternagina) em relação aos seus processos de autólise. Alternagina e bothropasina apresentaram diferentes comportamentos em relação à auto-proteólise, apesar do elevado grau de identidade entre as duas moléculas. Nesse trabalho, caracterizamos a estabilidade química e o comportamento de desenovelamento da proteína alternagina nativa pela guanídina HCl usando emissão de fluorescência intrínseca em combinação com espectroscopia de dicroísmo circular ´far´-UV. As amostras de alternagina, purificadas do veneno liofilizado, foram monitoradas por dicroísmo em comprimento de onda de 220nm e os resultados mostraram estruturas intermediárias no processo de desnaturação da proteína. Estudos semelhantes foram feitos com a bothropasina, com o objetivo de relacionar seus diferentes comportamentos auto-proteolíticos com os processos de desnaturação. Nas duas proteínas ocorreu uma alta correlação entre os estudos de auto-proteolise e de desnaturação. As MD isoladas de B. jararaca, jararagina e bothropasina, são isoformas descritas na literatura como isoláveis através de diferentes cromatografias. Neste trabalho, utilizamos os diferentes protocolos descritos para verificar a porção da isoforma majoritária no veneno. As amostras de proteína serão analisadas por espectrometria de massas, uma vez que a região N-terminal das proteínas está bloqueada. / Metalloproteinases/disintegrin (MD) isolated from snake venom are potent inhibitors of platelet aggregation and cell adhesion, processes involved in illnesses as cancer and thrombosis. MD belong to the PIII class of the metalloproteinase/disintegrin gene family and they are constituted by three domains: the catalytic domain, metalloprotease; disintegrin-like (D) and cysteine-rich (R). Some MD proteins are rapidly processed (producing the disintegrin-like/ cysteine-rich domains), while others MD are processed slowly. In this work, we studied the autolysis process of the MD isolated from the venom of Bothrops jararaca (bothropasin) and Bothrops alternatus (alternagin). Despite high sequence identity, alternagin and bothropasin showed different autolysis processes. The processing of the alternagin produces an intermediate with molecular mass of 43kDa whereas in the processing of the bothropasin this intermediate is almost not observable. In this work we studied alternagin and bothropasin under the viewpoint of chemical stability and the unfolding process to guanidine hydrochloride (Gnd-HCl) using dichroism circular and fluorescence spectrometry. The CD spectra (220nm) of the alternagin purified from the lyophilized venom showed an intermediate structure in the unfolding process. These studies were performed on bothropasin with the goal to relate the autolysis and unfolding process. These studies revealed a high correlation between both proteins . The MD isolated from B. jararaca, jararagin and bothropasin, are isophorms purified from different chromatograph processes reported in the literature. In this work, different purification processes were used to check the major isophorm. Due to the blocked N-terminal region, these proteins will be assessed by mass spectrometry.

Made available in DSpace on 2014-06-11T19:32:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-12-16Bitstream added on 2014-06-13T20:24:24Z : No. of bitstreams: 1 oliveira_am_dr_rcla.pdf: 1319026 bytes, checksum: b6ad03694a53b696678fbabde842876e (MD5) / O presente trabalho teve por objetivo otimizar a autólise de levedura fresca de cervejaria (Saccharomyces cerevisiae), visando a extração máxima de ácido ribonucléico da biomassa na produção do extrato de levedura. As variáveis estudadas foram pH, temperatura, % de NaCl, % de NH3, tempo de processo e, métodos de recuperação de RNA do autolisado. Os experimentos foram realizados por meio de quatro ensaios delineados segundo Box & Benken (1989) e avaliado pela Metodologia da Superfície de Resposta, utilizando-se o Software Statística 5.1 e a análise estatística ANOVA. A otimização foi concluída por meio do quinto ensaio com a produção do extrato nas condições otimizadas (55,2ºC, 9,8% de NaCl em pH=5,1 por 24 horas e, 12,2% de NH3 a 60ºC sob agitação a 200 rpm/15minutos. Três métodos foram avaliados para recuperação do RNA e das frações de extrato e parede celular: 1) autólise/plasmólise; 2) choque térmico por 1 minuto a 68ºC seguido da autólise/plasmólise 3) hidrólise química alcalina. Pelo processo de autólise em combinação com 9,8% de NaCl, a taxa de extração de RNA em 24 horas foi de 89,7%, com um rendimento de 51,3% em massa de extrato com 57,9% de proteína e, 48,7% de parede celular desidratada com 21,7 % de proteína. A utilização de 12,2% de NH3 em base seca de levedura permitiu o aumento na taxa de extração de RNA de 89,7 para 93,6%, mas um forte escurecimento foi verificado no extrato obtido. Na recuperação do RNA após precipitação protéica em pH 4,3 com posterior uso de 2 volumes de etanol em pH=2, recuperou-se 15,47%, 13,80% e 7,42% de RNA respectivamente com purezas de 49,85%, 51,70% e 38,70%. As taxas de extração de RNA da biomassa foram de 87,45% para o método 1; 91,40% para o método 2 e 78,80% para o terceiro método, indicando uma boa alternativa para redução do teor de RNA da biomassa e produção do extrato rico... / The present work had for objective to optimize the autolysis of fresh brewery’s yeast (Saccharomyces cerevisiae), aiming the maximum extraction of ribonucleic acid of biomass in the yeast extract production. The studied variables were pH, temperature, % of NaCl, % NH3, processing time and RNA recovering methods from autolysed. The experiments were accomplished by mean of four delineated assays according to Box & Benken (1989) and evaluated by Surface Methodology of Answer, utilizing the software Statistica 5.1. and the analysis statistics “ANOVA”. The optimization was concluded by mean of the fifth assay with an extract production in the optimized conditions (55.2ºC, 9.8% of NaCl in pH 5.1 for 24 hours and, 12.2% of NH3 at 60ºC under agitation at 200 rpm/15 minutes. Tree methods were evaluated for RNA recovering and of the extract fractions and cell wall: 1) autolysis/plasmolysis; 2) thermic shock during 1 minute at 68ºC followed of autolysis/plasmolysis; 3) alkaline chemical hydrolysis. The process of autolysis in combination with 9.8% of NaCl, the RNA extraction yield in 24 hours was of 89.7%, with a yield of 51.3% in extract mass with 57.9% of protein and, 48.7% of dehydrated cell wall with 21.7% of protein. The utilization of 12.2% of NH3 in dried base of yeast allowed the increase in the RNA yield extraction from 89.7 to 93.6%, but a strong darkness was observed in the obtained extract. The RNA recovering after 4.3 pH proteic precipitation with posterior use of 2 ethanol volumes in pH 2.0, it was recovered 15.47%, 13.8% and 7.42% of RNA respectively with purities of 49.85%, 51.70% and 38.70%. The RNA extraction yields of biomass were of 87.45% for the method 1; 91.40% for the method 2 and 78.80% for the third method, indicating a good alternative for RNA content reduction of biomass and rich extract production in nucleotides. The extract fractions were evaluated... (Complete abstract click electronic access below)

Lignocellulose, mostly from agricultural and forestry resources, is a potential renewable material for sustainable development of biorefineries. From previous studies, reducing sugar production through biological pretreatment involves two steps: solid-state fermentation (SSF) for delignification, followed by enzymatic hydrolysis by adding celluloytic enzymes (cellulase and xylanase etc.). In the process described in this thesis, the necessary enzymes are produced in-situ and the hydrolysis proceeds directly after the solid-state fermentation. Enzyme hydrolysis releases free amino nitrogen (FAN), reducing sugar and many other potential nutrients from the fermented materials. This method additionally avoids the need for removal of inhibitors compared with conventional chemical pretreatment processes. A range of solid-state fermentations were carried out to investigate the effect of washing procedure, particle size and nitrogen supplement on Trichoderma longibrachiatum growth. From these preliminary studies it was concluded that nitrogen supplementation is a crucial factor to improve significantly the fungi growth and production of feedstock using sugarcane bagasse as raw material. In order to evaluate the influence of environmental humidity on petri dish experiments, moist environments were investigated, with over 75% relative humidity to limit water evaporation from solid-state fermentation. The results showed that moist environments gave approximately 1.85 times the reducing sugar yield than dry environments. The process of simultaneous enzymatic hydrolysis of substrates and fungal autolysis were also studied. The degree of hydrolysis was affected by initial fermented solid to liquid ratio, temperature and pH range. The optimal conditions for subsequent hydrolysis of fermented solids were determined. The optimal solid to liquid ratio, 4% (w/w), temperature 50°C and pH 7 were established. The highest final reducing sugar, 8.9 g/L and FAN, 560 mg/L, were measured after 48 h. The fungal autolysis was identified by image analysis as well as by the consumption of nutrient and the release of free amino nitrogen and phosphorous. Solid state fermentation in a multi-layer tray bioreactor and a packed-bed bioreactor were also developed, with moist air supply for oxygen provision and heat removal. Fermented solids in the multi-layer bioreactor led to the highest subsequent hydrolysis yield on reducing sugar, FAN and Inorganic Phosphorous (IP), 222.85 mg/g, 11.56 mg/g and 19.9 mg/g, respectively. These series of fermentation experiments illustrate the feasibility for the application of consolidated bioprocessing, through simultaneous pretreatment and enzyme production as a more economic and environment-friendly process compared with those reported for chemical pretreatment followed by commercial enzyme process. A growth kinetic model regarding both growth and respiration is also proposed. Ethanol production was studied using the generic feedstock produced from sugarcane bagasse and soybean hulls. Total ethanol yield reached 0.31 mg g-1 (61.4% of theoretical yield) after 30 h of submerged fermentation. The result of subsequent fermentation has already shown the potential of the generic microbial feedstock to be used to produce varied products depending on the microorganism utilised.

Orientador: Pedro de Oliva Neto / Banca: Iolanda Cristina Silveira Duarte / Banca: Maria das Graças de Almeida Felipe / Banca: Eleonora Cano Carmona / Banca: Crispin Humberto Garcia Cruz / Resumo: O presente trabalho teve por objetivo otimizar a autólise de levedura fresca de cervejaria (Saccharomyces cerevisiae), visando a extração máxima de ácido ribonucléico da biomassa na produção do extrato de levedura. As variáveis estudadas foram pH, temperatura, % de NaCl, % de NH3, tempo de processo e, métodos de recuperação de RNA do autolisado. Os experimentos foram realizados por meio de quatro ensaios delineados segundo Box & Benken (1989) e avaliado pela Metodologia da Superfície de Resposta, utilizando-se o Software Statística 5.1 e a análise estatística ANOVA. A otimização foi concluída por meio do quinto ensaio com a produção do extrato nas condições otimizadas (55,2ºC, 9,8% de NaCl em pH=5,1 por 24 horas e, 12,2% de NH3 a 60ºC sob agitação a 200 rpm/15minutos. Três métodos foram avaliados para recuperação do RNA e das frações de extrato e parede celular: 1) autólise/plasmólise; 2) choque térmico por 1 minuto a 68ºC seguido da autólise/plasmólise 3) hidrólise química alcalina. Pelo processo de autólise em combinação com 9,8% de NaCl, a taxa de extração de RNA em 24 horas foi de 89,7%, com um rendimento de 51,3% em massa de extrato com 57,9% de proteína e, 48,7% de parede celular desidratada com 21,7 % de proteína. A utilização de 12,2% de NH3 em base seca de levedura permitiu o aumento na taxa de extração de RNA de 89,7 para 93,6%, mas um forte escurecimento foi verificado no extrato obtido. Na recuperação do RNA após precipitação protéica em pH 4,3 com posterior uso de 2 volumes de etanol em pH=2, recuperou-se 15,47%, 13,80% e 7,42% de RNA respectivamente com purezas de 49,85%, 51,70% e 38,70%. As taxas de extração de RNA da biomassa foram de 87,45% para o método 1; 91,40% para o método 2 e 78,80% para o terceiro método, indicando uma boa alternativa para redução do teor de RNA da biomassa e produção do extrato rico... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The present work had for objective to optimize the autolysis of fresh brewery's yeast (Saccharomyces cerevisiae), aiming the maximum extraction of ribonucleic acid of biomass in the yeast extract production. The studied variables were pH, temperature, % of NaCl, % NH3, processing time and RNA recovering methods from autolysed. The experiments were accomplished by mean of four delineated assays according to Box & Benken (1989) and evaluated by Surface Methodology of Answer, utilizing the software Statistica 5.1. and the analysis statistics "ANOVA". The optimization was concluded by mean of the fifth assay with an extract production in the optimized conditions (55.2ºC, 9.8% of NaCl in pH 5.1 for 24 hours and, 12.2% of NH3 at 60ºC under agitation at 200 rpm/15 minutes. Tree methods were evaluated for RNA recovering and of the extract fractions and cell wall: 1) autolysis/plasmolysis; 2) thermic shock during 1 minute at 68ºC followed of autolysis/plasmolysis; 3) alkaline chemical hydrolysis. The process of autolysis in combination with 9.8% of NaCl, the RNA extraction yield in 24 hours was of 89.7%, with a yield of 51.3% in extract mass with 57.9% of protein and, 48.7% of dehydrated cell wall with 21.7% of protein. The utilization of 12.2% of NH3 in dried base of yeast allowed the increase in the RNA yield extraction from 89.7 to 93.6%, but a strong darkness was observed in the obtained extract. The RNA recovering after 4.3 pH proteic precipitation with posterior use of 2 ethanol volumes in pH 2.0, it was recovered 15.47%, 13.8% and 7.42% of RNA respectively with purities of 49.85%, 51.70% and 38.70%. The RNA extraction yields of biomass were of 87.45% for the method 1; 91.40% for the method 2 and 78.80% for the third method, indicating a good alternative for RNA content reduction of biomass and rich extract production in nucleotides. The extract fractions were evaluated... (Complete abstract click electronic access below) / Doutor

Metaloproteases/desintegrinas (MD) isoladas de venenos de serpentes são potentes inibidores de agregação plaquetária e de adesão celular, processos envolvidos em doenças como trombose e câncer. As MD pertencem a classe PIII das SVMPs (´snake venom metalloproteinases´) que são constituídas por três domínios: metaloprotease (M), tipo-desintegrina (D) e rico em cisteína (C). A função dos três domínios nas atividades das moléculas ainda não é totalmente conhecida, e estudos com o objetivo de esclarecer suas funções são importantes para o desenvolvimento de novos fármacos. Algumas proteínas da classe PIII apresentam elevada atividade auto-proteolítica (liberando peptídeo constituído dos domínios D e C) enquanto que em outras PIII esta atividade não é menos evidente. Neste trabalho nós estudamos MD isoladas de veneno de Bothrops jararaca (bothropasina) e Bothrops alternatus (alternagina) em relação aos seus processos de autólise. Alternagina e bothropasina apresentaram diferentes comportamentos em relação à auto-proteólise, apesar do elevado grau de identidade entre as duas moléculas. Nesse trabalho, caracterizamos a estabilidade química e o comportamento de desenovelamento da proteína alternagina nativa pela guanídina HCl usando emissão de fluorescência intrínseca em combinação com espectroscopia de dicroísmo circular ´far´-UV. As amostras de alternagina, purificadas do veneno liofilizado, foram monitoradas por dicroísmo em comprimento de onda de 220nm e os resultados mostraram estruturas intermediárias no processo de desnaturação da proteína. Estudos semelhantes foram feitos com a bothropasina, com o objetivo de relacionar seus diferentes comportamentos auto-proteolíticos com os processos de desnaturação. Nas duas proteínas ocorreu uma alta correlação entre os estudos de auto-proteolise e de desnaturação. As MD isoladas de B. jararaca, jararagina e bothropasina, são isoformas descritas na literatura como isoláveis através de diferentes cromatografias. Neste trabalho, utilizamos os diferentes protocolos descritos para verificar a porção da isoforma majoritária no veneno. As amostras de proteína serão analisadas por espectrometria de massas, uma vez que a região N-terminal das proteínas está bloqueada. / Metalloproteinases/disintegrin (MD) isolated from snake venom are potent inhibitors of platelet aggregation and cell adhesion, processes involved in illnesses as cancer and thrombosis. MD belong to the PIII class of the metalloproteinase/disintegrin gene family and they are constituted by three domains: the catalytic domain, metalloprotease; disintegrin-like (D) and cysteine-rich (R). Some MD proteins are rapidly processed (producing the disintegrin-like/ cysteine-rich domains), while others MD are processed slowly. In this work, we studied the autolysis process of the MD isolated from the venom of Bothrops jararaca (bothropasin) and Bothrops alternatus (alternagin). Despite high sequence identity, alternagin and bothropasin showed different autolysis processes. The processing of the alternagin produces an intermediate with molecular mass of 43kDa whereas in the processing of the bothropasin this intermediate is almost not observable. In this work we studied alternagin and bothropasin under the viewpoint of chemical stability and the unfolding process to guanidine hydrochloride (Gnd-HCl) using dichroism circular and fluorescence spectrometry. The CD spectra (220nm) of the alternagin purified from the lyophilized venom showed an intermediate structure in the unfolding process. These studies were performed on bothropasin with the goal to relate the autolysis and unfolding process. These studies revealed a high correlation between both proteins . The MD isolated from B. jararaca, jararagin and bothropasin, are isophorms purified from different chromatograph processes reported in the literature. In this work, different purification processes were used to check the major isophorm. Due to the blocked N-terminal region, these proteins will be assessed by mass spectrometry.