A produção de enzimas do complexo celulases e de xilanases se destaca no cenário mundial, pois estas são importantes para a geração de biocombustíveis, resinas, álcoois, xilitol, sorbitol, e para a indústria de detergentes e ração animal e processos de biobranqueamento, extração de óleos, pigmentos e essências a partir de biomassa lignocelulósica. A secreção e produção destas enzimas são afetadas pela repressão catabólica, devido à presença da glicose liberada. O objetivo do presente estudo foi verificar o efeito sobre a produção enzimática de Penicillium echinulatum de metabólitos e açúcares durante o cultivo submerso. As estratégias utilizadas para a remoção de substâncias do meio de cultivo incluíram filtração e uso de pellets com adsorventes e uso de filmes adsorventes. O preparo de membranas de suporte cerâmico e polimérico, impregnadas com os polímeros poli(fluoreto de vinilideno) (PVDF) e poliamida (PA), foi realizado por inversão de fases, sendo que a membrana cerâmica impregnada com solução 10% (m/v) de PVDF (C1) reteve 88% da proteínas totais da solução enzimática. A remoção de 10 ou 20% do permeado durante a filtração com membranas planas, em períodos de tempo entre 8h e 24h, resultou em aumento da atividade de β- glicosidase e endoglicanase em frascos Erlenmeyer. Com relação à atividade de xilanases, os títulos observados no 4º dia foram entre 40% e 30% superiores quando houve a filtração em 42 e 44 h, respectivamente. Quando adsorventes foram utilizados em biorreator, a atividade de endoglicanase, CBH e xilanase apresentaram um aumento de 50%, 43% e 12%, respectivamente, em meio concentrado utilizando carvão ativado quando comparado ao padrão. Os ensaios com membranas de nanofiltração permitiram retenção de 95% das enzimas do complexo e, quando utilizadas na remoção de 10% e 20% da fase líquida em frascos, resultaram em aumento na atividade enzimática. Os ensaios 18/20, 24/10, 24/20, 44/10 e 44/20 (tempo de processo, em horas, em que foi feita a filtração / volume percentual de permeado removido) apresentaram, em média, em 72 h, um incremento de 33% na atividade de endoglicanase, enquanto o ensaio 44/20, em 96h, apresentou atividade superior a 40% do padrão. Para os cultivos com Trichoderma reesei Rut C30, os ensaios com filtração de 10 % do volume dos frascos, em 12 h e 20 h apresentaram aumento da atividade de FPA, em 60 h de cultivo, de 15% e 11%, respectivamente. Nos mesmos ensaios, a atividade de endoglicanase apresentou incremento de 40,7% e 52,2%, respectivamente, demonstrando o efeito positivo da remoção de 10% de permeado sobre a produção enzimática. Já para FPA, o aumento representou 15% e 11 % para as mesmas condições / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES. / Cellulases and xylanases enzymes complex production stands out in the world because these enzymes are important for the generation of biofuels, resins, alcohols, xylitol, sorbitol, and for the detergents industry, animal feed, pulp bleaching, and in the extraction of oils, pigments and essences from lignocellulose biomass. Secretion and production of cellulases and xylanases are affected under catabolic repression conditions due to glucose release. The purpose of the present study was to verify the effect on enzymatic production of Penicillium echinulatum compared to metabolites and sugars during submerged cultivation. The strategies used for the substances removal from the medium included filtration application, the use of pellets with adsorbents, and the use of adsorbents films. Membranes preparation from both ceramic and polymeric supports impregnated with polymers PVDF and PA were performed by phase inversion method, and eventually the ceramic membrane impregnated with solution 10% (w/v) of PVDF (C1) held 88% of the total proteins from the enzymatic solution. The removal consisting in 10% or 20% from the permeate during filtration with flat membranes, in periods between 8h and 24h resulted in a rise in the activity regarding β-glucosidase and endoglucanase in Erlenmeyer flasks. In relation to xylanases activity, the titles observed on the fourth day were between 40% and 30% superiors when filtration was made in 42 and 44h, respectively. Subsequently, when adsorbents were used in the bioreactor, activity involving endoglucanase, CBH and xylonase increased 50%, 43% and 12% respectively, in concentration applying activated charcoal comparable to standard source. Moreover, tests performed with nanofiltration membranes allowed 95% retention of the enzymes from the complex, and when they were used in the removal consisted of 10% and 20% in liquid phase in flasks, results showed a rise on the enzymatic activity. The tests 18/20, 24/10, 24/20, 44/10 and 44/20 ( in relation to filtration day and volume permeated) presented, on average, in 72 hours, a 33% increment in the endoglucanase activity and, in 96 hours, the test 44/20 presented activity superior to 40% from standard value. Therefore, for tests with Trichoderma reesei Rut C30, filtration experiments with 10% of the volume of the flasks in 12h and 20 h presented an increase in FPA activity, and in 60 h, the amount found was 15% and 11%, respectively. Subsequently, for endoglucanase enzyme, the activity presented increment levels evaluated in 40,7% and 52, 2% respectively for the mentioned tests, demonstrating the positive factor of the permeate 10% removal from the enzymatic production. Lastly, for FPA the rise represented 15% and 11% in the same test conditions.

O estudo de fungos celulolíticos tem-se mostrado relevante, tendo em vista o interesse econômico do complexo celulases, especialmente na indústria têxtil e, mais recentemente, para propósitos energéticos. No presente trabalho, a fusão de protoplastos foi utilizada para combinar genótipos de mutantes parcialmente desreprimidos para produção de celulases de Penicillium echinulatum (9A02S1B9) e richoderma harzianum (AS5CH3), utilizando a técnica do doador morto, buscando-se obter recombinantes com maior produção de celulases. Nesta estratégia, ambas as linhagens tiveram seu micélio tratado com Glucanex 0,01 g/mL, para quebra da parede celular. Os protoplastos resultantes da linhagem portadora de marca de resistência ao benomil (9A02S1B9) foram inativados por calor (técnica do doador morto) de 60oC antes da etapa de fusão, a qual após foi induzida por PEG4000 e Ca2+, com protoplastos da linhagem sensível ao benomil (AS5CH3). A partir de um produto de fusão, foram selecionados 24 sub-clones, após estratégias de estabilização e seleção para precocidade e eficiência na formação de halo de hidrólise de celulose em placas de Petri. Os produtos de fusão apresentaram morfologia e esporulação semelhantes a um dos parentais, sendo treze semelhantes à Penicillium, nove semelhantes à Trichoderma e dois mostrando formas alteradas. Os produtos de fusão que segregaram para morfologia de T. harzianum apresentaram a característica de resistência ao benomil, sendo capazes de crescer e esporular em meios contendo até 100 μg/mL deste inibidor. A morfologia, o perfil de bandas, obtidos por RAPD, e o padrão de secreção de celulases dos produtos de fusão foram sempre mais semelhantes a um dos parentais. Os clones apresentaram variação quanto ao halo de hidrólise de celulose em placas de Petri e na atividade sobre papel filtro FPAases, -glicosidase ou endoglicanase, quando crescidas em cultivo submerso ou em estado sólido. Desta variabilidade, verificaram-se aumentos significativos para algumas das linhagens em relação aos parentais. A aplicação da metodologia de fusão de protoplastos para obter recombinantes entre P. echinulatum e T. harzianum, empregando a técnica do doador morto, mostrou-se adequada na geração de variabilidade para produção de celulases. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The study of cellulolytic fungi has proved to be important, considering economic interest of the cellulase complex, especially in the textile industry and, more recently, for energy purposes. In this work, the protoplast fusion was used to combine genotypes of mutants partially non repressed for cellulases production of Penicillium echinulatum (9A02S1B9) and Trichoderma harzianum (AS5CH3) using the technique dead donor, intending to obtain recombinants with higher cellulases production. In this strategy, both strains had their mycelium treated with Glucanex  0,01 g/mL, to lyse the cell wall. The protoplast obtained from the benomyl-resistant (9A02S1B9) were heat-inactivated (technique of dead donor) at 60ºC, before the step of fusion, induced by PEG4000 and Ca2+, with protoplast of the sensitive-benomyl strain (AS5CH3). Twenty four sub-clones were selected from one fusion product, after stabilization and selection strategies for precocity and efficiency in the formation clearing zones of by cellulose hydrolysis in Petri plates. The fusion products showed similar morphology and sporulation to one of parents, thirteen similar to Penicillium, nine similar to Trichoderma and two showed altered forms. The fusion products which segregate to the morphology of T. harzianum resistance to benomyl, being able to grow and sporulate in media containing up to 100 μg/mL of this inhibitor. The morphology, the profile of bands, obtained by RAPD, and the pattern of cellulase secretion by fusion products were ever more similar to one of parents. The fusants presented variation in the halo of cellulose hydrolysis in Petri plates, and in the activity on filter paper (FPAases), - glicosidase or endoglicanase, when grown submerged cultivation or solid state. From this variability, significant improvement was verified for some of the parental strains. The application of the protoplast fusion methodology to obtain recombinant between P. echinulatum and T. harzianum, using the technique of dead donor, has proved to be adequate to generate variability in the production of cellulases.

O emprego de enzimas lignocelulolíticas secretadas por microrganismos para a produção de etanol de segunda geração tem motivado pesquisas na área da engenharia de processos fermentativos, genômica e secretômica. Entre os microrganismos com potencial para a produção de celulases destacam-se variantes genéticos do fungo filamentoso Penicillium echinulatum caracterizados por produzirem altos títulos enzimáticos. Neste trabalho, estudouse a secretômica da linhagem selvagem 2HH e do mutante S1M29 de P. echinulatum, em cultivo submerso, empregando diferentes fontes de carbono: glicose, glicerol, celulose e bagaço de cana-de-açúcar pré-tratado por explosão a vapor. Análises enzimáticas possibilitaram identificar que P. echinulatum produz celulases, hemicelulases, esterases e, em menor proporção, pectinases e amilases. Outrossim, os maiores títulos enzimáticos para a maioria das enzimas foram verificados na linhagem mutante. Nos meios formulados com bagaço de cana-de-açúcar ou celulose verificou-se a indução das maiores produções enzimáticas para ambas as linhagens. A análise do secretoma por 1D-PAGE seguido de LCMS/ MS das amostras de 96 horas de cultivo permitiu identificar que em ambas as linhagens há predominância de enzimas CAZy, sendo celulases, hemicelulases e enzimas degradadoras de parede celular fúngica as mais predominantes. Celobiohidrolases, endoglicanases, β-glicosidases, xilanases, β-xilosidases e mananases foram identificadas e, em quantidades menores, ligninases, pectinases, amilases, esterases e solenina, entre outras proteínas (adesão, chaperonas, oxidoredutases, proteases, peptidases, lipases, glutaminases e hipotéticas). Os meios elaborados com glicose ou glicerol foram utilizados pelo fungo para a produção de amilases, ligninases e enzimas degradadoras da parede celular fúngica. Destaca-se a secreção 2 a 3 vezes maior de celulases pela linhagem mutante, sendo que o meio de cultivo elaborado com bagaço de cana-de-açúcar proporcionou a secreção de maiores quantidades de celulases para o mutante. Nesta condição, o complexo celulolítico da linhagem S1M29 constitui-se de 55% de celobiohidrolases, 38% de endoglicanases e 1% de β-glicosidases. Estes dados sugerem que durante o melhoramento genético do fungo ocorreram mudanças, embora não direcionais, possivelmente em nível da regulação da expressão gênica, modificações póstraducionais e alterações na capacidade para secretar proteínas extracelulares que tornaram a linhagem mutante S1M29 com potencial para ser empregada na hidrólise de lignocelulósicos. / The use of lignocellulolytic enzymes secreting by microorganisms for the production of second-generation ethanol has motivated research in the field of fermentation processes engineering, genomics and secretomics. Among the microorganisms with the potential for cellulases production are genetic variants of the filamentous fungus Penicillium echinulatum characterized by produce high enzymatic titers. In this work, it was studied the secretome of the wild type 2HH and mutant S1M29 of P. echinulatum in submerged cultivation on different carbon sources: glucose, glycerol, cellulose and sugar cane bagasse pretreated by steam explosion). Enzymatic analysis allowed verifying that P. echinulatum produces cellulases, hemicellulases, esterases, and minor proportion, pectinases and amylases. Furthermore, the major enzymes titers for most enzymes dosed were verified in the mutant strain. It was verified in the media formulated with sugar cane bagasse or cellulose the induction of the highest enzyme production for both strains. The analysis of secretome by 1DPAGE followed by LC-MS/MS, of samples collected at 96 hours of cultivation, showed that in both strains there is a predominance of CAZy enzymes, being cellulases, hemicellulases and fungal cell wall degrading enzymes the most prevalent. Cellobiohydrolases, endoglucanases, β-glucosidase, xylanase, endoxylanase, β-mannanases and xylosidases were identified and, in smaller amounts, ligninases, pectinases, amylases, esterases and swollenin, among other proteins (adhesion, chaperones, oxidoreductases, proteases, peptidases, lipases, glutaminases and hypothetical). The media elaborated with glucose or glycerol were used for producing of amylases, ligninases and fungal cell wall degrading enzymes. Highlights the secretion of 2-3 times more cellulases by the mutant, being the medium prepared with sugar cane bagasse afforded the secretion of large cellulases quantities for the mutant. In this condition, the cellulolytic complex of S1M29 strain consists of 55% cellobiohydrolases, 38% endoglucanases and 1% β-glucosidase. These data suggest that during the genetic improvement of the fungus changes occurred, although not directional, possibly at the level of regulation of gene expression, post-translational modifications and changes in the ability to secrete extracellular proteins, that have made the mutant S1M29 a potential strain to be employed in hydrolysis of lignocellulose.

No description available.

Orientadores: Sérgio Marangoni, Saulo Luis da Silva / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-10-31T14:37:34Z (GMT). No. of bitstreams: 1 GasparJunior_PascoalJose_D.pdf: 6862970 bytes, checksum: 60c120eb2e9159656d5c493ee05eca1a (MD5) Previous issue date: 2014 / Resumo: A triagem de fungos produtores de holocelulases é uma estratégia para a obtenção de enzimas capazes de hidrolisar o material lignocelulósico da biomassa vegetal, contribuindo para aumentar a viabilidade da produção de etanol celulósico Este trabalho avaliou o potencial enzimático dos fungos Penicillium corylophilum e Penicillium simplicissimum com relação às enzimas (endoglicanase, exoglicanase, ?-glicosidase, FPase, xilanase, pectinase e mananase) sobre substratos lignocelulósicos e comerciais como fonte de carbono. Além disso, avaliou-se também a influência da adição de diferentes fontes orgânicas e inorgânicas de nitrogênio sobre a atividade enzimática. Os fungos foram cultivados em triplicata em meio líquido suplementar com 1% de substrato lignocelulósico como fonte de carbono, em pH 7,0. A inoculação foi feita por suspensão de esporos (108 esporos/mL). O cultivo foi feito sob agitação a 120 rpm, a 28°C e os filtrados resultantes em 12, 24, 72 e 120 horas foram utilizados como fontes enzimáticas pelo método do DNS (ácido-3,5- dinitrosalicílico) em triplicata. De todas as atividades enzimáticas analisadas nas fontes lignocelulósicas, a atividade de xilanase do P. simplicissimum sobre a linhaça foi a mais expressiva (3,87 e 3,97 UI/ml), cultivados em 72 e 120 horas, respectivamente, e selecionada para os testes de purificação proteica. A atividade xilanásica específica aumentou consideravelmente após os passos cromatográficos de gel filtração e troca iônica (CTI), sendo inicialmente 3x10-3 atv/ µg de proteína no liofilizado e 19,2 atv/ µg de proteína na fração proveniente da CTI. Nos testes de pH, observou-se que a atividade de xilanase foi maior em pH=4,0 e temperatura de 50°C. Com relação à adição de substratos comerciais, a celulose microcristalina e a xilana apresentaram os resultados mais expressivos da indução da produção de xilanase, sendo que a concentração de 0,5% de xilana mostrou a melhor atividade enzimática em ambos os fungos estudados. A xilose apresentou uma concentração indutora mínima de 0,04% que foi suficiente para aumentar a atividade de xilanase do P. simplicissimum. Com relação à suplementação de fontes de nitrogênio no meio de cultivo para a produção de holocelulases, a adição de (NH4)2SO4 e caseína é uma alternativa importante para a potencialização das atividades de pectinase e de endoglicanase respectivamente pois foram fontes de nitrogênio que proporcionaram um aumento da atividade enzimática em ambos os fungos estudados como também nas duas fontes de carbono testadas. As frações contendo xilanases após cromatografia de gel filtração seguida de fase reversa, após análises por espectrometria de massas apresentam relação massa/carga de 18831,26 Da. A utilização do resíduo lignocelulósico da linhaça, como fonte de carbono para o cultivo submerso do Penicillium simplicissimum é uma opção ecologicamente correta, exequível e de baixo custo para a produção de xilanases. Diversas aplicações biotecnológicas como a utilização na ração animal, na indústria do papel e no etanol de segunda geração, dentre outros, possibilitam um acréscimo substancial do valor agregado desse substrato, permitindo uma ampliação da utilização da linhaça, além do aproveitamento do óleo, sem aumentar a área plantada / Abstract: Screening for producing fungi holocelulases is a strategy for obtaining enzymes that hydrolyze the lignocellulosic material from plant biomass, helping to increase the viability of cellulosic ethanol production This study evaluated the enzymatic potential of Penicillium simplicissimum and Penicillium corylophilum regarding enzymes (endoglicanase, exoglicanase, ?-glucosidase, FPase, xylanase, pectinase and mannanase) for commercial and lignocellulosic substrates as a carbon source. Furthermore, it was also evaluated the influence of the addition of different organic and inorganic nitrogen sources on enzyme activity. The fungus was grown in triplicate in a liquid medium supplement with 1% lignocellulosic substrate as carbon source, pH 7.0. The inoculation was done by the spore suspension (108 spores/ml). The cultivation was done with stirring at 120 rpm at 28 °C and the resulting filtered in 12, 24, 72 and 120 hours were used as enzyme sources for DNS (acid-3,5- dinitrosalicilic) method in triplicates. All enzymatic activities analyzed in lignocellulosic sources, the xylanase activity of P. simplicissimum about flaxseed was greater (3.87 and 3.97 IU/ml), grown at 72 and 120 h of cultivation, respectively, and selected for testing for protein purification. The specific xylanase activity increased considerably after the chromatographic steps of gel filtration and ion exchange (CTI), initially 3x10-3 atv/ mg of protein in lyophilized and 19.2 atv/ mg of protein in the fraction from the CTI. In pH testing, it was noted that the xylanase activity was higher at pH 4.0 and 50 °C. With respect to the addition of commercial substrates, microcrystalline cellulose and xylan showed the most significant results of induction of xylanase production, and the concentration of 0.5% xylan showed the best enzyme activity in both fungi studied. The xylose showed a minimal inducing concentration of 0.04% which was sufficient to increase the activity of xylanase from P. simplicissimum. With respect to supplemental nitrogen sources in the culture medium for the production of holocelulases, the addition of (NH4)2SO4 and casein can be an important tool for the enhancement of pectinase and endoglicanase activities respectively as alternative nitrogen sources that were provided an increase in enzyme activity in both fungi studied as well as the two carbon sources tested. The use of lignocellulosic residue of flaxseed as a source of carbon for submerged cultivation of Penicillium simplicissimum is an environmentally friendly, feasible and cost effective for the production of xylanases option. The fractions containing xylanases after gel filtration chromatography followed by reverse fase after analysis by mass spectrometry are related mass/charge of 18831 26 Da. The use of lignocellulosic waste of flaxseed as a source of carbon for submerged cultivation of Penicillium simplicissimum is an environmentally friendly, feasible and cost effective for the production of xylanases option. Various biotechnological applications such as use in animal feed, in the paper industry and in second generation ethanol, among others, allow a substantial increase in the value of this substrate, allowing an expansion of the use of flaxseed, plus the use of oil, without increasing acreage / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

<p>During the last ten years the cultivation of maize in Sweden has increased and is expected to grow further. Most of the maize in Sweden becomes silage which is used to feed animals at farms. Maize has in other countries been shown to be a substrate for growth of mould and especially <em>Aspergillus </em>spp., <em>Fusarium </em>spp. and <em>Pencillium </em>spp. has been reported. Members of all three of these species can, during favorable conditions, produce mycotoxins which can cause a number of different health problems in both animals and man. The aim of this study was to investigate the occurrence of mould and mycotoxins to increase our knowledge of the hygienic quality of Swedish maize silage. Microbiological analyses were made to study the growth of fungi. To analyze for fumonisin B₁, B₂and zearalenone, HPLC with fluorescence detection was made. The mycotoxins mycophenolic acid, roquefortine C, gliotoxin, penicillic acid, penitrem A, fumitremorgen C and verrucologen were analyzed with LC-MS/MS. The results showed that 47 % of the samples were contaminated with <em>Penicillium </em>spp. and 6 % had growth of <em>Aspergillus fumigatus</em>. A small amount of zearalenone was found in one sample and 0.01ppm of roquefortine C was detected in one sample. The data obtained indicate that Swedish maize silage has a moderate growth of fungi with a very low production of mycotoxins. More studies have to be performed to make more decisive conclusions.</p>

During the last ten years the cultivation of maize in Sweden has increased and is expected to grow further. Most of the maize in Sweden becomes silage which is used to feed animals at farms. Maize has in other countries been shown to be a substrate for growth of mould and especially Aspergillus spp., Fusarium spp. and Pencillium spp. has been reported. Members of all three of these species can, during favorable conditions, produce mycotoxins which can cause a number of different health problems in both animals and man. The aim of this study was to investigate the occurrence of mould and mycotoxins to increase our knowledge of the hygienic quality of Swedish maize silage. Microbiological analyses were made to study the growth of fungi. To analyze for fumonisin B1, B2 and zearalenone, HPLC with fluorescence detection was made. The mycotoxins mycophenolic acid, roquefortine C, gliotoxin, penicillic acid, penitrem A, fumitremorgen C and verrucologen were analyzed with LC-MS/MS. The results showed that 47 % of the samples were contaminated with Penicillium spp. and 6 % had growth of Aspergillus fumigatus. A small amount of zearalenone was found in one sample and 0.01ppm of roquefortine C was detected in one sample. The data obtained indicate that Swedish maize silage has a moderate growth of fungi with a very low production of mycotoxins. More studies have to be performed to make more decisive conclusions.

Age-related changes in the pH of the medium, fatty acid composition and biosynthesis of fatty acids in various subcellular fractions have been studied in submerged cultures of P. chrysogenum harvested at pH's 7.2, 4.8, 4.4 and 3.8. 14C - labeled lauric and myristic acids were each incubated with cell fractions prepared by sonification and fractional centrifugation. Products were analyzed by thin layer chromatography, gas liquid scintillation spectrometry. Conversion of labeled precursors into longer chain saturated and unsaturated fatty acids was employed as a measure of the activity of the various cell fractions.It was found that between pH 4.8 and 4.4 of the age of the culture a period of maximum fatty acid synthesis occured at the time when glucose was being taken up to a greatest extent from the medium and when cells start rapidly increasing in growth. This correlated to the increased rate of de novo synthesis in the soluble cytoplasmic fraction.The fermentation products, formic, acetic, proprionic, butyric and gluconic acid, produced at different time points of the age of the culture, accounted for the drop in the pH of the medium.

The synthesis of lipids and long chain fatty acids in young and aging cultures of Penicillium chrysogenum was studied by identifying the intracellular location of radioactively labeled intermediates in the vegetative mycelium and identifying the lipids and fatty acids into which the label from 1-14C-palmitic acid was incorporated.Previous work has indicated that the pH of the growth medium of submerged cultures of Penicillium chrysogenum dropped from pH 7.4 in newly inoculated cultures to 3.2-3.8 during the 20-40 hour growth period. Young cultures were defined as those harvested before the pH began to drop and aging cultures were those harvested after the pH stabilized at near 3.4. Changes in the fatty acid composition of various cell fractions of the mycelium harvested as the pH of the medium declined suggested that a change in utilization of lipids employed in the synthesis of structural components in the cell may have occurred as a result of a shift in fatty acid metabolism as the culture aged.Young and aging cultures were incubated with l-14C-palmitic acid and harvested after 2, 10, 60, and 120 minutes. Samples of mycelia from each harvest were examined by light and electron microscopy and were prepared for autoradiography. In addition lipids were extracted from sonified mycelial samples, analyzed for total lipids, lipid classes, and phospholipid components by thin layer chromatography. Fatty acids were identified and quantified by gas liquid chromatography with percent distribution of label in fatty acids determined by liquid scintillation spectrometry. Results of biochemical analysis of some of the major lipid components were compared with electron microscope autoradiographs and related to changes in the location of labeled fatty acid in cell organelles or cell parts as the cells aged.In young cultures 70% of the radioactive label was recovered in phospholipids while about 15% was recovered in the free fatty acid component. By contrast, in aging cultures approximately 20% of the label was recovered in the phospholipids and 80f was recovered from the free fatty acid component. Electron autoradiographic data tend to support these biochemical findings in that numerous grains occur over the membranal components of the young cells and over cytoplasmic areas of lipid depots in aging cells. Phospholipids differed markedly also with large amounts of an unidentified phospholipid type found in aging cells not seen in young cells.Light and electron microscope observations of hyphal cells showed significant alterations in cell morphology over the forty hour growth period. Young cells were long, slender, with dense cytoplasm and thin cell walls. As the culture aged, cells became progressively shorter, thicker and more clubby in appearance with prominent lipid inclusions. The vacuolar lipid depots of aging cells were ,determined to be composed of free fatty acids with 20% of label being incorpoated into C20 and C21 long chain fatty acids. The synthesis of fatty acid chains longerthan C18 has not been previously reported in intact cultures of Penicillium chrysogenum.The lipid metabolism of young cells of Penicillium chrysogenum differed from that of aging cells in many aspects. Young cells incorporated label from precursor palmitic acid into membranes. However, as the cells aged, lipids were diverted to storage. Young and aging cells differed in the amounts and composition of total lipids, lipid classes, phospholipid components and fatty acids. Differences in morphology between young and aging cells could be demonstrated by light and electron microscopy. These structural changes paralleled the biochemical changes indicating a functional dissimilarity existed between the young and aging cells.

MP1, a gene previously identified in P. marneffei by cDNA library screening, encodes a secreted cell wall mannoprotein Mp1p. Thirteen MP1 homologues named MPLP1 to 13 were previously identified in P. marneffei by BLAST analysis. Two MP1 homologues namely AFMP1 and AFMP2 which encodes Afmp1p and Afmp2p were previously identified by expressed sequence tag library screening in Aspergillus fumigatus – an important fungal pathogen closely related to P. marneffei. Mp1p, Afmp1p and Afmp2p have previously been reported to be immunogenic. Mp1p was also reported to bind fatty acid and was suggested to contribute to virulence in a MP1 knockout P. marneffei strain in a mouse model and a cell line model although the Koch’s postulates has yet been met to establish MP1 as a novel virulence factor. With reference to sequence identity of Afmp proteins to Mp1p, Afmp proteins were speculated to have functions similar to Mp1p. BLAST searches against the A. fumigatus genome identified two novel AFMPs namely AFMP3 and AFMP4. Sequence analysis of Afmp3p and Afmp4p revealed the presence of putative N-terminal signal peptide and substantial sequence identity to Mp1p, Afmp1p and Afmp2p. Two MP1 knockdown P. marneffei mutants were constructed to demonstrate suppression of MP1 expression alone can result in loss of virulence and also the dosage effect of MP1 expression on P. marneffei virulence towards mice. Subsequent mice challenge experiments using MP1 like protein (MPLP) knockdown strains suggested MP1 to be the most important virulence factor among all its homologues in P. marneffei. Histopathology examinations of organs from challenged mice suggested survival disadvantages in mice for P. marneffei mutants with knockdown of MP1 and effect of MP1 on granuloma formation in infected mice. Mice challenge experiments using AFMP1 to 4 knockdown A. fumgiatus mutants suggested significant decrease in virulence of A. fumigatus upon AFMP4 knockdown and complete protection of challenged mice upon knockdown of AFMP1 to 4. Histopathology examinations of organs from challenged mice suggested survival disadvantages in mice for A. fumigatus mutants with knockdown of AFMPs and effect of AFMPs on granuloma formation in infected mice. Mice experiments using Pichia pastoris expressing MP1 or AFMP4 suggested the effects of MP1 and AFMP4 on virulence are not caused by factors specific to P. marneffei or A. fumigatus. It was shown using a human peripheral blood mononuclear cells model that Mp1p and Afmp4p confer intracellular survival advantage to P. marneffei and A. fumigatus upon infection. Expression of Mp1p or Afmp4p in P. pastoris also confers survival advantage to this nonpathogenic yeast in human peripheral blood mononuclear cells. Reduction in proinflammatory prostaglandin E2 production were noticed in human peripheral blood mononuclear cells infected by P. marneffei, A. fumigatus or P. pastoris strains that expressed Mp1p or Afmp4p. Such reduction in eicosanoids production also coincides with the inhibition of apoptosis as shown by enzyme activity of caspase-8, caspase-9 and caspase-3 in human peripheral blood mononuclear cells. These findings suggest two novel virulence factors – Mp1p and Afmp4p, which confer survival advantages to P. marneffei and A. fumigates respectively. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy