Com o intuito de gerar subsídios para melhoria do processo de produção de Metarhizium anisopliae, o presente estudo teve como principais objetivos, determinar os efeitos de inibidores de proteinases de soja no crescimento vegetativo, esporulação e virulência do fungo e comparar a produção, viabilidade e virulência dos conídios produzidos em diferentes tipos de arroz e aditivos. A adição de 5 g.L-1 de inibidores de proteinases semi-purificados de soja ou 0,5 g.L-1 de inibidor purificado do tipo Kunitz no meio de cultura ME resultou em grande aumento na esporulação (de duas a 75 vezes) sem afetar a viabilidade dos conídios de quatro isolados (ESALQ-1037, IBCB348, E9, F20) de M. anisopliae. A presença destes inibidores de proteinases também alterou a morfologia dos conídios produzidos em ME. Os mecanismos responsáveis por estas alterações fisiológicas não foram determinados, mas provavelmente estejam associados à ação anti-nutricional, diminuindo a absorção protéica e estimulando a esporulação. Os conídios produzidos no meio com adição de 0,5 g.L-1 de inibidor de proteinase do tipo Kunitz ou com 2,5 g.L-1 de albumina de soro bovino e no meio BDA apresentaram virulência superior aos conídios produzido no meio ME sem inibidores. Quando o inibidor de proteinase do tipo Kunitz foi adicionado à suspensão de conídios do fungo antes da pulverização de lagartas de Diatraea saccharalis, a eficiência de controle foi 35,1%, inferior ao apresentado pelos demais tratamentos sem a presença deste inibidor. Nos estudos para determinação dos melhores tipos de arroz para produção de M. anisopliae, tentou-se correlacionar à produção de conídios com características destes substratos como valor nutricional, teor de resíduos de agrotóxicos e densidade de microorganismos. O arroz parboilizado foi responsável pela maior produção de conídios (4,38 x 109 conídios.g-1). Este tipo de arroz apresentou teor de proteína bruta menor do que a maioria dos arrozes e o maior teor de umidade (41,3% após a autoclavagem). Além disto, os grãos após autoclavagem ficaram menos gelatinosos e mais soltos, o que facilita o processo de produção do fungo. Enquanto que os arrozes dos tipos brancos polidos, canjicão e integral ficaram pegajosos e formaram grumos, o que provavelmente deve acarretar em menor superfície de desenvolvimento para o fungo. O segundo melhor arroz, o canjicão, com produção de 3,42 x 109 conídios.g-1, teve a maior quantidade de fungos contaminantes nos grãos crus. Quantidades intermediárias de conídios foram produzidas pelos arrozes branco polido irrigado, de terras altas e orgânico. O integral foi o que resultou na menor quantidade de conídios (1,53 x 109 conídios.g-1), sendo o mais rico em minerais, proteína bruta e extrato etéreo. Nenhum dos aditivos (farelo de soja, grãos de soja partidos, extrato de soja, peptona de soja, inibidores de proteinases de soja semipurificados e purificado do tipo Kunitz, polpa cítrica e levedura) resultou em aumento na produção de conídios em comparação com o arroz parboilizado sem aditivos. Os conídios produzidos em todos os arrozes e aditivos apresentam viabilidade superior a 99%. As vantagens da utilização do arroz parboilizado levando-se em consideração custo, facilidade de manuseio e produtividade são discutidas. / In order to optimize the production process of Metarhizium anisopliae, the present study aimed to determine the effects of soybean proteinase inhibitors on growth, sporulation and virulence of the fungus and to compare yield, viability and virulence of M. anisopliae conidia produced in different types of rice and additives. The addition of 5 g.L-1 of semi-purified soybean proteinase inhibitor or 0.5 g.L-1 of Kunitz-type inhibitor purified on the culture medium ME resulted in large increases in sporulation (two to 75 times) without affecting the viability of conidia of four M. anisopliae isolates (ESALQ-1037, IBCB348, E9, F20). The presence of proteinase inhibitors altered morphology of conidia produced in ME. Mechanisms responsible for these physiological changes in the fungus have not been determined, but it is probably associated to an anti-nutritional action, reducing the absorption of protein and stimulating sporulation. Spores produced in the medium with the addition of 0.5 g.L-1 Kunitz-type inhibitor purified or 2.5 g.L-1 of bovine serum albumin (BSA) and PDA medium showed higher virulence of conidia produced in ME without inhibitors. When the Kunitz-type inhibitor was added to conidial suspension of the fungus before spraying larvae of Diatraea saccharalis, control efficiency was 35.1% lower than that presented in other inhibitor-free treatments. In the studies aiming to determine the best types of rice for production of M. anisopliae, we tried to correlate production of conidia with characteristics of these substrates such as nutritional content, pesticide residues and density of microorganisms. The parboiled rice was responsible for greater production of conidia (4.38 x 109 conidia.g-1). This type of rice showed crude protein content lower than most rice and the highest moisture content (41.3% after autoclaving). Besides that, grains became less gelatinous and loose after autoclaving, and these feature favored fungus production. While types of polished white, brown rice and course (broken) rice grain were sticky and formed clumps, providing a smaller area for fungus development. The second best rice, course rice grain, with production of 3.42 x 109 conidia.g-1, had the highest amount of fungal contaminants in raw grains. Intermediate amounts of conidia were produced by white irrigated polished rice, upland rice and organic rice. The brown rice was the kind that resulted in fewer conidia (1.53 x 109 conidia.g-1), being the richest in minerals, protein and lipids. None of the additives (soybean meal, soybean parties, soy extract, soy peptone, semi-purified soybean proteinase inhibitor, Kunitz-type inhibitor purified, citrus pulp and yeast) resulted in increased production of conidia compared to parboiled rice without additives. Conidia produced in all types of rice and additives presented viability greater than 99%. The advantages of the use of parboiled rice taking into consideration the cost, easy handling and productivity are discussed.

PDI ("potato cathepsin D inhibitor ") and NID ("novel inhibitor of cathepsin D ") from potato (Solanum tuberosum) belong to the protein family of Kunitz inhibitors (I3 family, Merops database). These 20 kDa isoinhibitors with the typical β-trefoil architecture inhibit aspartic and serine peptidases. In this thesis, the constructs for recombinant expression of PDI and NID in the yeast Pichia pastoris system were prepared and high-producing colonies were selected. Both proteins were identified in the cultivation media by mass spectrometry and N-terminal sequencing. A purification protocol for PDI with three chromatographic steps was designed. Analogous functional properties were demonstrated for the purified recombinant PDI and the native PDI isolated from a natural source. Analysis of the inhibitory specificity showed that PDI is a potent inhibitor of selected aspartic peptidases from the A1 family and serine peptidases from the S1 family, including a relevant enzyme of insect origin. This finding supports the hypothesis that Kunitz inhibitors are involved in plant defense against herbivorous insects. The inhibitors prepared within the project will be used for analysis of the reactive centers against target peptidases by protein crystallography. (In Czech) Key words: proteolytic enzymes, activity...

Neun Jahre nach der russischen und französischen Erstveröffentlichung erschien 1922 im Russischen Musikverlag neben der englischen schließlich auch eine deutsche Übersetzung von Rimsky-Korsakows berühmtem Traktat. Exemplarisch wird die Rezeption von Rimsky-Korsakows Schrift untersucht 1. in nach 1922 geschriebene deutschsprachigen Instrumentationslehren 2. in Dokumenten des hochschulischen Unterrichts 3. in der deutschsprachigen Historiographie der fraglichen musiktheoretischen Teildisziplinen 4. anhand Kompositionen aus der Zeit um 1930. Die mit dem Orchester verbundenen Klangideale hatten sich gewandelt. Man legte Wert auf Trennschärfe und erstrebte einen Klang, der ein gespaltener genannt und als Gegensatz zu einem verschmelzenden konstruiert wurde. Man suchte eine rohe, holzschnitthafte Orchestration. Den tiefen klangtechnischen Einsichten Rimsky-Korsakows und dem Raffinement des russischen Orchestrators konnten und wollten sich die Musiker dennoch nicht entziehen. Im Konflikt zwischen der Faszination durch seine Schrift und der neuen Klangmode zeichneten sich alsbald mehrere Lösungen ab. Besonders eine war erfolgreich: Rimsky-Korsakows ausdrückliche Empfehlungen ließen sich gegen den Strich bürsten oder auch allzu wörtlich nehmen; genau das verbürgte in den Dreißiger Jahren Expressivität. / Nine years after the Russian and eight after the French first publications, the German, alongside the English translation of Rimsky-Korsakov’s famous treatise was eventually published by the Edition Russe de musique in 1922. In answering this question, the following has been taken into account: i) orchestration guides written in German after 1922; ii) documents from education institutions; iii) German historiography of the theoretical discipline in question; and iv) works by German composers who are known to have reacted against Rimsky-Korsakov’s model of orchestration. The sound ideal associated with the orchestra had changed. Selectivity was emphasized and composers sought to create an orchestral sound that was seen as ‘split up’ and thought of as the opposite to something merging and melting. The new composers sought a raw, woodcut-like orchestration. Some musicians nonetheless could not or did not want to escape from the deep technical insights of Rimsky-Korsakov, from the sophistication and raffinement of the Russian orchestrator. The conflict between the fascination by his writing and the new sound fashion soon offered multiple solutions. Especially one of them was successful: Rimsky-Korsakov\\\'s explicit recommendations could be understood against the grain, or could be taken too literally: in the thirties just this guaranteed expressivity.

Neun Jahre nach der russischen und französischen Erstveröffentlichung erschien 1922 im Russischen Musikverlag neben der englischen schließlich auch eine deutsche Übersetzung von Rimsky-Korsakows berühmtem Traktat. Exemplarisch wird die Rezeption von Rimsky-Korsakows Schrift untersucht 1. in nach 1922 geschriebene deutschsprachigen Instrumentationslehren 2. in Dokumenten des hochschulischen Unterrichts 3. in der deutschsprachigen Historiographie der fraglichen musiktheoretischen Teildisziplinen 4. anhand Kompositionen aus der Zeit um 1930. Die mit dem Orchester verbundenen Klangideale hatten sich gewandelt. Man legte Wert auf Trennschärfe und erstrebte einen Klang, der ein gespaltener genannt und als Gegensatz zu einem verschmelzenden konstruiert wurde. Man suchte eine rohe, holzschnitthafte Orchestration. Den tiefen klangtechnischen Einsichten Rimsky-Korsakows und dem Raffinement des russischen Orchestrators konnten und wollten sich die Musiker dennoch nicht entziehen. Im Konflikt zwischen der Faszination durch seine Schrift und der neuen Klangmode zeichneten sich alsbald mehrere Lösungen ab. Besonders eine war erfolgreich: Rimsky-Korsakows ausdrückliche Empfehlungen ließen sich gegen den Strich bürsten oder auch allzu wörtlich nehmen; genau das verbürgte in den Dreißiger Jahren Expressivität. / Nine years after the Russian and eight after the French first publications, the German, alongside the English translation of Rimsky-Korsakov’s famous treatise was eventually published by the Edition Russe de musique in 1922. In answering this question, the following has been taken into account: i) orchestration guides written in German after 1922; ii) documents from education institutions; iii) German historiography of the theoretical discipline in question; and iv) works by German composers who are known to have reacted against Rimsky-Korsakov’s model of orchestration. The sound ideal associated with the orchestra had changed. Selectivity was emphasized and composers sought to create an orchestral sound that was seen as ‘split up’ and thought of as the opposite to something merging and melting. The new composers sought a raw, woodcut-like orchestration. Some musicians nonetheless could not or did not want to escape from the deep technical insights of Rimsky-Korsakov, from the sophistication and raffinement of the Russian orchestrator. The conflict between the fascination by his writing and the new sound fashion soon offered multiple solutions. Especially one of them was successful: Rimsky-Korsakov\\\''s explicit recommendations could be understood against the grain, or could be taken too literally: in the thirties just this guaranteed expressivity.