O gênero Candida é responsável pela maioria das infecções fúngicas nosocomiais. A identificação do provável foco de origem é de extrema importância para elucidar a epidemiologia desse tipo de infecção e nesse sentido, a utilização de métodos de tipagem, que avaliam características fenotípicas e moleculares dos isolados, é essencial. Assim, o objetivo desse estudo foi tipar isolados de C. albicans, antes e após armazenamento e em diferentes ocasiões de coleta, a fim de verificar a manutenção dos biotipos apresentados, e o poder de discriminação dos métodos utilizados. Foi avaliada a microbiota leveduriforme da saliva de 73 estudantes universitários (tempo 0) sendo que após 180 dias (tempo 180) foi realizada nova coleta de saliva daqueles que apresentaram isolamento de C. albicans na primeira coleta. Os isolados foram analisados por ocasião das duas coletas, quanto à produção de exoenzimas fosfolipase e proteinase, pela morfologia das colônias, pelo perfil de suscetibilidade frente à anfotericina B, fluconazol e itraconazol e pela tipagem molecular por RAPD. As leveduras, após isoladas, foram armazenadas em ágar Sabouraud dextrose (ASD) e água destilada esterilizada. Após 180 dias, foram realizadas, novamente, as provas de tipagem. Todos isolados de C. albicans foram produtores de fosfolipase, nas duas coletas, embora tenha havido oscilação de atividade enzimática entre moderada e alta, no período. O enzimotipo prevalente nos tempos 0 e 180 foi, respectivamente, 22 e 32. Com relação à proteinase, 100% das leveduras apresentaram atividade moderada da enzima no tempo 0. No tempo 180 esse percentual foi de 85%, sendo que os demais não apresentaram atividade dessa enzima. Após armazenamento em ASD e água destilada, foi detectada alteração da atividade de ambas enzimas e conseqüente mudança de enzimotipos, em 40 e 30% dos isolados, respectivamente. Foram identificados 8 morfotipos diferentes de C.albicans no tempo 0 e apenas 50% foi mantido no tempo 180. O morfotipo mais comum foi 000-0. Após armazenamento, a maioria dos isolados apresentou alteração no morfotipo. Todos isolados mostraram-se sensíveis aos antifúngicos analisados nos tempos 0 e 180, denotando apenas um antifungotipo, o 111. Somente um isolado após estocagem em ASD, teve mudança de perfil de sensível para dose dependente ao itraconazol de modo que o antifungotipo foi alterado. A tipagem molecular por RAPD, com os primers OPA-09, OPB-11 e OPE-18, mostrou 19 tipos moleculares distintos entre os isolados obtidos na primeira coleta e permitiu identificar que um isolado de C.albicans obtido no tempo 180, não era relacionado ao obtido, do mesmo indivíduo, no tempo 0. Os demais mostraram perfil de fragmentos de DNA relacionado entre as coletas. Após estocagem, por ambos métodos, todos isolados mostraram correlação genética com o padrão obtido no tempo 0. Os resultados obtidos demonstram que os métodos de conservação aplicados neste estudo não permitem a manutenção da estabilidade das características fenotípicas avaliadas. Por outro lado, além da estabilidade dos biotipos gerados, a tipagem molecular por RAPD mostrou o melhor índice discriminatório, dentre as metodologias utilizadas, ratificando sua capacidade em diferenciar isolados, de uma mesma espécie e, portanto a sua utilidade em inquéritos epidemiológicos. / The Candida genus is responsible for most nosocomial fungal infections. The identification of the probable origin focus is very important to elucidate the epidemiology of this kind of infection and so, the usage of typing methods that evaluate phenotypic and molecular characteristics are essential. Therefore, the objective of this study was to type C. albicans isolates, before and after culture storage and in two different collection occasions to verify the biotype maintenance and the discriminatory power of the utilized methods. The salivary yeast microbiota of 73 university students (time 0) was evaluated and after 180 days (time 180) a new saliva collection of those that had presented C. albicans on the first collection was made. The isolates were analysed, in the two collection occasions on the basis of exoenzymes phospholipase and proteinase production, by colonial morphology, susceptibility profile to amphotericin B, fluconazole and itraconazole and according to molecular typing by RAPD. After isolation, the yeasts were storaged on Sabouraud dextrose agar (SDA) and in sterile distilled water. After 180 days, typing tests were carried out again. All C. albicans isolates were phospholipase productors, in both collections, even though enzyme activity had oscillated between moderate and high at that period. The prevalent enzymotype at time 0 and 180 were, respectively, 22 and 32. In relation to the proteinase, 100% of the yeasts showed moderate enzyme activity at time 0. At time 180, this percentage was 85%, and the others didn`t show enzyme activity. After storage on SDA and in distilled water, it was detected activity alteration of both enzymes and consequent enzymotype change, in 40 and 30% of isolates, respectively. Eight different C. albicans morphotypes were identified at time 0 and just 50% were maintained at time 180. The most common morphotype was 000-0. After storage most isolates presented changes in the morphotype. All isolates showed susceptibility to the analysed antifungals at times 0 and 180, showing just one antifungaltype, the 111. Only one isolate, after storage on SDA had a profile change from susceptible to dose dependent susceptible to itraconazole in a way that the antifungaltype wasn`t changed. The molecular typing by RAPD, with primers OPA-09, OPB-11 and OPE-18, showed 19 distinct molecular types among first collection isolates and allowed to identify that one C. albicans isolate from time 180 wasn`t related with the isolate obtained at time 0, from the same individual. The others showed DNA fragment profiles related between collection occasions. After storage by both methods, every isolate showed genetic relatedness with the profile obtained at time 0. The obtained results showed that the preservation methods used in this study don`t allow stability maintenance of the phenotypical characteristics evaluated. On the other hand, besides biotypes generated stability, the molecular typing by RAPD showed the best discriminatory index, between methodologies used, ratifying its ability in discriminating isolates of the same specie and so, its utility in epidemiological inquiries

Autoantikörper gegen Proteinase 3 (cANCA) stellen einen hochsensitiven und spezifischen Seromarker für das Krankheitsbild der Wegenersche Granulomatose dar. Während die Ätiologie dieser systemischen Vaskulitis unbekannt ist, weisen zahlreiche klinische und experimentelle Daten darauf hin, daß cANCA an der Entstehung und Chronifizierung dieser Erkrankung beteiligt sind. Insbesondere die Konsequenzen einer cANCA-Ligation an Proteinase 3 (PR3)-exprimierende Neutrophile wurden intensiv untersucht und man geht davon aus, daß Anti-PR3-Antikörper über die Aktivierung inflammatorischer, neutrophiler Zellfunktionen die Vaskulitis fördern. Über die Auswirkungen von cANCA auf Monozyten, die eine wichtige Rolle in der Regulation der Immunantwort spielen und weitere Zielzellen dieser Antikörper darstellen, ist hingegen bisher wenig bekannt. Aktivierte Monozyten sind nicht nur ein wesentlicher Bestandteil der Granulome, sondern sind auch entscheidend an den vaskulären Entzündungsinfiltraten beteiligt. Ziel der vorliegenden Studie war es daher, die Effekte von cANCA auf inflammatorische Zellfunktionen PR3-exprimierender Monozyten zu charakterisieren. Dabei war insbesondere ihr Einfluß auf die transendotheliale Migration und die Sekretion proinflammatorischer Mediatoren von Interesse. Die Isolation humaner Monozyten erfolgte mittels Gegenstromzentrifugation. Die monozytäre Transmigration wurde in mit humanen Endothelzellen bewachsenen Filtereinsätzen untersucht und mittels eines chemotaktischen Gradienten und/oder TNF-Stimulation der Endothelzellen gefördert. Die transendotheliale Migration von mit murinen, monoklonalen Anti-PR3-Antikörpern vorinkubierten Monozyten war, unabhängig vom Aktivierungszustand der Endothelzellen, deutlich vermindert. Dieser Effekt konnte sowohl durch vier von fünf cANCA-IgG-Fraktionen von Patienten mit aktiver WG als auch durch F(ab´)2-Fragmente der Autoantikörper reproduziert werden. Da bekannt ist, daß cANCA die proteolytische Aktivität von PR3 inhibieren und membrangebundenen Formen der leukozytären Serinproteasen PR3, humaner leukozytärer Elastase (HLE) und Cathepsin G (CathG) eine Rolle bei der Extravasion von Leukozyten zugesprochen wird, wurde daraufhin überprüft, ob PR3 für die monozytäre Transmigration von Bedeutung ist. Der physiologische Inhibitor dieser Serinproteasen, 1-Antitrypsin, und ein synthetischer Inhibitor von PR3 und HLE, CE-2072, verminderten die Anzahl migrierender Monozyten in gleichem Maße wie der vollständige Anti-PR3-Antikörper bzw. dessen F(ab´)2-Fragmente. Dahingegen hatte SLPI, ein Serpin, das lediglich CathG und HLE inhibiert, keinen Effekt auf die Anzahl migrierender Zellen. Dabei waren die Effekte von Anti-PR3-Antikörpern und 1-Antitrypsin nicht additiv, wodurch die Annahme, daß die Anti-PR3-mediierte Reduktion der monozytären Transmigration auf einer funktionellen Inhibition von PR3 beruht, untermauert wird. Die Reduktion der monozytären Transmigration ging nicht mit einer modifizierten endothelialen Adhäsion der Monozyten einher, wie unter dynamischen und statischen Bedingungen gezeigt werden konnte. Weder die rollende noch die feste Adhäsion der Monozyten wurde durch Anti-PR3-Antikörper oder Serinproteaseinhibitoren beeinträchtigt. Auch die durchflußzytometrisch quantifizierte Expression monozytärer ß1- und ß2-Integrine wurde durch die Autoantikörper nicht beeinflußt. Diese Ergebnisse zeigen erstmals, daß PR3 an der transendothelialen Migration, nicht aber der Adhäsion von Monozyten teilhat. Ein weiteres, wesentliches Ergebnis der vorliegenden Studie ist die, im Vergleich zu entsprechenden IgG-Kontrollen, massive Freisetzung proinflammatorischer Mediatoren aus Monozyten in Gegenwart muriner Anti-PR3-Antikörper bzw. humaner cANCA, die mittels ELISA ermittelt wurde. Die Sekretion war zeitabhängig, wobei die Sekretion von TNF- und IL-1ß der von Thromboxan A2, IL-6 und IL-8 vorausging. Im Gegensatz zu den Auswirkungen von cANCA auf die monozytäre Transmigration, konnten diese Effekte nicht durch die alleinige Ligation des Antikörpers an das Antigen PR3 reproduziert werden, sondern waren von einer simultanen Ligation der Autoantikörper an PR3 und FcR auf der monozytären Oberfläche abhängig. Eine cANCA-mediierte Retention adhärenter Monozyten im Gefäßbett bei gleichzeitiger Aktivierung der monozytären Freisetzung inflammatorischer Zytokine und Prostanoide durch cANCA, könnte nicht nur die Entstehung der extra- und perivaskulären, granulomatösen Entzündung, sondern auch die Aufrechterhaltung der nekrotisierenden Vaskulitis fördern. Insgesamt weisen die Ergebnisse dieser Studie erstmals auf eine funktionelle Rolle von PR3 bei der transendothelialen Migration von Monozyten hin. Außerdem liefern sie weitere wesentliche Hinweise darauf, wie die Interaktion von cANCA mit Monozyten an der Pathogenese der Wegenerschen Granulomatose beteiligt sein könnten.

"This thesis is based on the following articles, referred to in the text by the Roman numerals given below. In addition some unpublished results are presented. I. Caiyan Wu ... [et al] Improvement of the secretion of extracellular proteins and isolation and characterization of the amylase I (amyI) gene from Ophiostoma floccosum [pub. in ] Gene 384: 96-103 -- II. Caiyan Wu ... [et al.] Activity-based identification of secreted serine proteases of the filamentous fungus Ophiostoma. Accepted by Biotechnology letters DOI 10.1007/s10529-007-9333-6 -- III. Caiyan Wu ...[et al.] Expression of a thermostable bacterial xylanase in the filamentous fungus Ophiostoma floccosum. Submitted to Letters in applied microbiology in July 2007." - leaf 9. / Thesis (PhD)--Macquarie University, Division of Environmental and Life Sciences, Dept. of Chemistry & Biomolecular Sciences, 2007. / Bibliography: leaves 100-123. / Introduction -- Materials and methods -- Results and discussion -- Conclusion and future aspects -- References -- Publications I, II and III. / Ophiostoma spp. belong to the Ophiostomataceae family, a large group of ascomycetes, which are the most frequent blue stain fungi isolated from stained wood. Most Ophiostoma species do not compromise the strength properties of wood, but do reduce the aesthetic quality of timber and therefore decrease the economic value of lumber. Some albino variants of O. floccosum and O. piliferum have been used as biological control agents to prevent blue staining. This successful whole organism approach plus the added capability of extracellular protein secretion makes Ophiostoma spp. attractive for industrial application. In addition, Ophiostoma produces only a small range of abundantly secreted proteins in liquid culture, which can facilitate downstream purification of any recombinant gene product introduced into the system. Genes encoding efficiently secreted proteins provide a potential souce for strong promoters for high-level gene expression. These characteristics provide an excellent starting point for the development of a novel expression system. / In this study, UV-mutagenesis was applied to improve protein secretion in Ophiostoma floccosum. Amylase activity was used as an indicator for enhanced protein secretion after repeated rounds of mutagenic treatment. Several mutants of O. floccosum derived by UV mutagenesis were isolated and the total amount of secreted protein was increased by 4 to 6 times. The amylase activity in the culture supernatant of the best mutant (MQ.5.1) was increased by more than 240-fold compared to the initial parental strain. At the same time, the amount of total secreted protein was about six times greater to that of the parental strain. Proteinase profiles in the culture supernatants of several key mutants were characterized for the future matching of an expression host with a particular gene product. N-terminal sequencing of the five dominant proteins separated by SDS-PAGE from the culture supernatant was conducted. Two of the proteins identified were subtilisin-like proteinases and one was a pepsin-like proteinase. In addition, one protein was identified as an_-amylase and one remained unidentified. A 6.5 kb DNA fragment was isolated by Genomic Walking PCR using primers based on the _-amylase amino acid sequence. The amplified fragment contained the entire gene encoding_-amylase (amyl) and its regulatory sequences. Analysis showed that multiple transcripts were generated from the single _-amylase gene locus. / A series of expression vectors containg the _-amylase regulatory sequences and partial amyl gene were constructed. Several selection markers were screened and the hph gene conferring hygromycin resistance under the regulation of the Aspergillus nidulans gpd promoter was chosen and inserted into the amyl expression vectors. The gene encoding a red fluorescent protein DsRed-E5 was used as a reporter gene to test the expression system using mutant MQ.5.1 as host. However, no transformants were obtained by either biolistic transformation or protoplast transformation. Subsequently, an alternative strategy was developed using a thermostable xylanase B as a reporter. Thermostable xylanase activity was detected in the culture supernatants of several transformants. Production of xylanase by transformant SS41 which exhibited high secreted xylanase activity was investigated. Xylanase activity in the culture supernatant of SS41 was visualized by a zymogram gel assay. Two active proteins with molecular masses of around 27 and 30 kDA, which were larger than the predicted Mr of 25 kDA were detected. This is the first report describing successful expression of a recombinant thermostable bacterial enzyme in Ophiostoma. / Mode of access: World Wide Web. / 158 leaves col. ill

La balance entre synthèse et dégradation des collagènes joue un rôle crucial dans le développement de pathologies de cicatrisation. Les collagènes fibrillaires sont synthétisés et sécrétés sous forme de précurseurs dans l'espace extracellulaire. Ils subissent alors une maturation protéolytique par des métalloprotéases, telles que les « BMP-1/Tolloid-like » (BTPs), permettant la formation de fibres de collagène. Ce clivage protéolytique est stimulé de façon spécifique par deux glycoprotéine, les « Procollagen C-Proteinase Enhancers » (PCPE-1 et -2). Ainsi, l'objectif de mon travail de recherche a été d'étudier les rôles de PCPE-1 et -2 dans la cicatrisation cutanée. De façon intéressante, nous avons montré que les BTPs et PCPE-1 étaient exprimés au niveau du derme et que leur expression augmentait pendant la première semaine de cicatrisation. PCPE-1 est principalement exprimée par les fibroblastes alors que PCPE-2 est retrouvée dans les kératinocytes. De plus, nous avons montré que PCPE-2 était abondante dans les cellules myéloides, suggérant un rôle pendant la phase inflammatoire de cicatrisation. Nos résultats montrent que PCPE-2 ne stimule pas efficacement la maturation des procollagènes fibrillaires et inhibe même l'activité de BMP-1 sur des substrats non-collagéniques, suggérant un rôle inattendu de PCPE-2. Enfin, aucune différence majeure n'a été observée dans la peau de souris déficientes en PCPE-2 au cours de la cicatrisation, suggérant que PCPE-1 serait le principal stimulateur impliqué dans la maturation et le dépôt des fibres de collagènes. Ainsi, ce travail suggère que PCPE-1 et -2 joueraient des rôles distincts dans la peau et pendant la cicatrisation cutanée / The imbalance between collagen assembly and degradation during skin wound healing is a major factor contributing to wound healing pathologies. Fibrillar collagen precursors are synthesized by fibroblasts and processed in the extracellular space by specific metalloproteases (e.g. BMP-1/Tolloid-like Proteinases, or BTPs) to form collagen fibrils. These proteolytic maturations can be efficiently stimulated by two glycoproteins, Procollagen C-Proteinase Enhancers (PCPE-1 and -2). The main goal of our study was to analyze the possible redundant and specific roles of the two PCPE proteins during wound healing.Interestingly, both BTPs and PCPE-1 were found to be expressed in the dermis and to be significantly increased during the first week after injury. Surprisingly, PCPE-1 is mainly expressed by dermal fibroblasts, whereas PCPE-2 is lowly expressed in fibroblasts and more abundant in basal keratinocytes. Moreover, PCPE-2 appears to be expressed by myeloid cells suggesting that PCPE-2 might rather play a role during the inflammatory phase of wound healing. In addition, our results indicate that recombinant PCPE 2 does not efficiently enhance their proteolytic maturation of fibrillar procollagens and can even inhibit the action of BMP-1 on other non-collagenous substrates, suggesting a differential and unexpected role of PCPE-2. Finally, the in vivo role of PCPE-2 was investigated using a Pcolce2 knockout model; skin morphology and wound healing were not affected by PCPE-2 loss, indicating that PCPE-1 is the main enhancer involved in collagen deposition during wound healing. Thus, this work suggests that PCPE-1 and -2 play distinct roles in the skin and during wound healing

A catepsina L, uma cisteína proteinase da família da papaína, é a principal proteinase digestiva do besouro Tenebrio molitor. Estudos anteriores do nosso grupo mostraram que existem três catepsinas L no intestino médio do T. molitor, uma delas é lisossômica (CAL 1) e as outras duas são digestivas (CAL 2 e CAL 3). As estruturas 3D das enzimas digestivas foram recentemente elucidadas. Com o objetivo de estudar em detalhes as propriedades das enzimas digestivas, CAL 3 foi expressa como um zimógeno em E. coli, purificada por cromatografia de afinidade e autoativada em meio ácido. Foram realizados ensaios de atividade com 63 peptídeos FRET derivados da sequência Abz-KLRSSKQ-EDDnp em um espectrofluorímetro termostatizado a 30 ºC, monitorando-se continuamente a variação de fluorescência em 320 nm (λex) e 420 nm (λem). Os parâmetros kcat e KM obtidos foram utilizados na determinação da hidrofobicidade dos subsítios (H) e da função de cada subsítio através da razão das energias livres de ativação do complexo enzima-substrato (ΔG‡T) e de ligação da enzima com o substrato (ΔGs). Os resultados mostram que o subsítio S2 está envolvido prioritariamente em catálise e é bastante seletivo para substratos com resíduos hidrofóbicos em P2. Esse subsítio é o mais hidrofóbico dentre os analisados, encontrando-se num bolsão localizado no interior da enzima. O subsítio S\'2, por outro lado, é o que apresentou a menor especificidade dentre os analisados. Este subsítio está envolvido prioritariamente na ligação com o substrato e se localiza na superfície da enzima, o que pode facilitar a acomodação de diferentes cadeias laterais em P\'2 do substrato, não oferecendo muitas restrições espaciais. O subsítio S1, hidrofílico, não é muito seletivo, o que pode ser consequência de sua localização na superfície da enzima. Esse subsítio está prioritariamente envolvido na ligação com o substrato. O subsítio S\'1, assim como S1, está localizado na superfície da enzima, é hidrofílico e não muito seletivo. No entanto, esse subsítio tem papel na catálise além de atuar na ligação do substrato. Numa análise inicial da estrutura 3D deste subsítio, sua função catalítica foi atribuída à presença de parte da cavidade oxiânica. Uma enzima com mutação no resíduo W187, pertencente à cavidade oxiânica e a S\'1, foi produzida e purificada, no entanto essa enzima não apresentou atividade. Uma análise mais aprofundada mostrou que a falta de atividade pode ser atribuída ao fato do resíduo de aminoácido mutado fazer parte de um cluster aromático essencial à estabilização da tríade catalítica. Os dados obtidos na caracterização de S\'1 e S\'2 permitem inferir que a acilação é o passo limitante da reação da CAL 3. Além disso, os resultados deste trabalho mostram que o conceito de hidrofobicidade de subsítios proposto anteriormente pelo grupo parece ser aplicável a subsítios que apresentem especificidades mais restritas. / Cathepsin L, a cysteine proteinase of the papain family, is the major digestive proteinase in the beetle Tenebrio molitor. Previous studies of our group showed that there are three cathepsins L in T. molitor midgut, one is lysosomal (CAL1) and two are digestive (CAL2 and CAL3). The 3D structures of the digestive enzymes were recently elucidated. With the aim to study in details the digestive enzymes specificities, CAL3 was expressed in E. coli as a zymogen, purified by affinity chromatography and autoactivated in acid conditions. Activity assays were performed in a thermostated spectrofluorometer at 30 ºC with 63 FRET peptides derived from the lead sequence Abz-KLRSSKQ-EDDnp, continuously monitoring the fluorescence changes at 320 nm (λex) and 420 nm (λem). The parameters kcat and KM were used in the determination of subsite hydrophobicity (H) and subsite role based on the ratio of complex enzyme-substrate activation energy (ΔG‡T) and free energy of substrate binding (ΔGs). The data obtained suggest that the S2 is mainly involved in catalysis and is very selective to substrates with hydrophobic residues in P2. This subsite is the most hydrophobic among the analyzed and is located in a pocket in the enzyme interior. S\'2, on the other hand, is the less selective subsite and is mainly involved in substrate binding and is located on the enzyme surface, what can ease the accommodation of different side chains located in P\'2 by not imposing many spatial restrictions. S1, is hydrophilic and not very selective, what may be a consequence of its location on the enzyme surface. This subsite is mainly involved in substrate binding. S\'1, just like S1, is located on the enzyme surface, is hydrophilic and not very selective. However this subsite has a role in catalysis besides the role in substrate binding. In an initial 3D structure analysis its catalytic function was attributed to the presence of a part of the oxyanion hole. An enzyme with mutation in the residue W187, which apparently belonged both to the oxyanion hole and S\'1, was produced and purified, but this enzyme was inactive. A better analysis showed that the lack of activity can be attributed to the fact that the mutated residue belongs to an aromatic cluster that is essential to the catalytic triad stabilization. The data obtained in S\'1 and S\'2 characterization suggest that acylation is the limiting step in CAL 3 reaction. The results presented in this work support the concept of subsite hydrophobicity previously proposed by our group, which seems to be true to subsites with more restrict specificities

Os inibidores inibidores de serino proteinases do tipo Bowman-Birk (BBI) possuem dois sítios ativos e são encontrados em plantas das famílias Fabaceae e Poaceae. Neste trabalho foi apresentada a estrutura primária e o padrão de expressão de 14 seqüências expressas (EST, expressed sequence tags) de BBI putativos encontradas no banco de dados do "Projeto Transcriptoma da Cana-de-açúcar" (SUCEST). Estas quatorze seqüências foram utilizadas em conjunto com outras 87 seqüências de BBI previamente descritas e depositadas no banco de dados "GenBank" para a construção de árvores filogenéticas da família BBI. A análise filogenética mostrou que os BBI de monocotiledôneas e dicotiledôneas podem ser claramente separados em diferentes grupos e a topologia das árvores filogenéticas sugere um padrão evolutivo diferente das famílias de BBI em plantas. Os inibidores de dicotiledôneas são bem conservados e acumularam diferenças sutis durante a evolução. Em contrapartida, os inibidores de monocotiledôneas são altamente variáveis, indicando a ocorrência de um processo evolutivo interessante, baseado em eventos de duplicação intragênica e mutação. Dois inibidores de serino proteinases, um de tripsina e outro de quimotripsina, derivados do gene que codifica o inibidor Bowman-Birk de soja, foram expressos na superfície do fago filamentoso M13 e utilizados para a construção de bibliotecas de variantes. Para tal foram feitas mutações em quatro aminoácidos do sítio ativo destes inibidores e duas bibliotecas de expressão na superfície de fagos filamentosos foram construídas. Posteriormente, estas bibliotecas foram utilizadas para a seleção de variantes que melhor interagiam com a tripsina bovina e de Diatraea saccharalis e com as enzimas digestivas presentes no extrato intestinal desta praga. Os variantes selecionados foram seqüenciados, analisados e caracterizados. Os resultados mostraram que a técnica de expressão na superfície de fagos filamentosos foi eficiente para selecionar novos inibidores. Além disto, com as mutações realizadas, foi possível transformar a alça de inibição de quimotripsina em uma alça de inibição de tripsina. / The Bowman-Birk inhibitors (BBIs) are double headed inhibitors of serine proteinase found in plants from Fabaceae and Poaceae families. We describe the primary structure and the gene expression profile of 14 putative BBIs from the sugarcane expressed sequence tag (SUCEST) database and show how we used these newly discovered sequences together with 87 previously described BBI sequences from the "GenBank" database to construct phylogenetic trees for the BBI family. Phylogenetic analysis revealed that BBI-type inhibitors from monocotyledonous and dicotyledonous plants could be clearly separated into different groups, while the overall topology of the BBI tree suggests a different pattern of evolution for BBI families in flowering plants. We also found that BBI proteinase inhibitors from dicotyledonous plants were well conserved, accumulating only slight differences during their evolution. In addition, we found that BBIs from monocotyledonous plants were highly variable, indicating an interesting process of evolution based on internal gene duplications and mutation events. Two serine-type proteinase inhibitors, a trypsin and a chymotrypsin, both derived from the soybean Bowman-Birk inhibitor, were expressed on the surface of a filamentous phage. Site mutations were made in four positions of the reactive sites of these inhibitors and two phage-display libraries were constructed. Later, these libraries were used to select better ligands to the bovine and Diatraea saccharalis trypsin and to the midgut enzymes of this insect pest. The selected variants were sequenced, analyzed and characterized. The results showed that the phage-display technique is efficient to select new proteinase inhibitors. Furthermore, it was possible to modify the chymotrypsin loop into a trypsin loop using the library constructed by the insertion of a degenerated primer.

O gênero Candida é responsável pela maioria das infecções fúngicas nosocomiais. A identificação do provável foco de origem é de extrema importância para elucidar a epidemiologia desse tipo de infecção e nesse sentido, a utilização de métodos de tipagem, que avaliam características fenotípicas e moleculares dos isolados, é essencial. Assim, o objetivo desse estudo foi tipar isolados de C. albicans, antes e após armazenamento e em diferentes ocasiões de coleta, a fim de verificar a manutenção dos biotipos apresentados, e o poder de discriminação dos métodos utilizados. Foi avaliada a microbiota leveduriforme da saliva de 73 estudantes universitários (tempo 0) sendo que após 180 dias (tempo 180) foi realizada nova coleta de saliva daqueles que apresentaram isolamento de C. albicans na primeira coleta. Os isolados foram analisados por ocasião das duas coletas, quanto à produção de exoenzimas fosfolipase e proteinase, pela morfologia das colônias, pelo perfil de suscetibilidade frente à anfotericina B, fluconazol e itraconazol e pela tipagem molecular por RAPD. As leveduras, após isoladas, foram armazenadas em ágar Sabouraud dextrose (ASD) e água destilada esterilizada. Após 180 dias, foram realizadas, novamente, as provas de tipagem. Todos isolados de C. albicans foram produtores de fosfolipase, nas duas coletas, embora tenha havido oscilação de atividade enzimática entre moderada e alta, no período. O enzimotipo prevalente nos tempos 0 e 180 foi, respectivamente, 22 e 32. Com relação à proteinase, 100% das leveduras apresentaram atividade moderada da enzima no tempo 0. No tempo 180 esse percentual foi de 85%, sendo que os demais não apresentaram atividade dessa enzima. Após armazenamento em ASD e água destilada, foi detectada alteração da atividade de ambas enzimas e conseqüente mudança de enzimotipos, em 40 e 30% dos isolados, respectivamente. Foram identificados 8 morfotipos diferentes de C.albicans no tempo 0 e apenas 50% foi mantido no tempo 180. O morfotipo mais comum foi 000-0. Após armazenamento, a maioria dos isolados apresentou alteração no morfotipo. Todos isolados mostraram-se sensíveis aos antifúngicos analisados nos tempos 0 e 180, denotando apenas um antifungotipo, o 111. Somente um isolado após estocagem em ASD, teve mudança de perfil de sensível para dose dependente ao itraconazol de modo que o antifungotipo foi alterado. A tipagem molecular por RAPD, com os primers OPA-09, OPB-11 e OPE-18, mostrou 19 tipos moleculares distintos entre os isolados obtidos na primeira coleta e permitiu identificar que um isolado de C.albicans obtido no tempo 180, não era relacionado ao obtido, do mesmo indivíduo, no tempo 0. Os demais mostraram perfil de fragmentos de DNA relacionado entre as coletas. Após estocagem, por ambos métodos, todos isolados mostraram correlação genética com o padrão obtido no tempo 0. Os resultados obtidos demonstram que os métodos de conservação aplicados neste estudo não permitem a manutenção da estabilidade das características fenotípicas avaliadas. Por outro lado, além da estabilidade dos biotipos gerados, a tipagem molecular por RAPD mostrou o melhor índice discriminatório, dentre as metodologias utilizadas, ratificando sua capacidade em diferenciar isolados, de uma mesma espécie e, portanto a sua utilidade em inquéritos epidemiológicos. / The Candida genus is responsible for most nosocomial fungal infections. The identification of the probable origin focus is very important to elucidate the epidemiology of this kind of infection and so, the usage of typing methods that evaluate phenotypic and molecular characteristics are essential. Therefore, the objective of this study was to type C. albicans isolates, before and after culture storage and in two different collection occasions to verify the biotype maintenance and the discriminatory power of the utilized methods. The salivary yeast microbiota of 73 university students (time 0) was evaluated and after 180 days (time 180) a new saliva collection of those that had presented C. albicans on the first collection was made. The isolates were analysed, in the two collection occasions on the basis of exoenzymes phospholipase and proteinase production, by colonial morphology, susceptibility profile to amphotericin B, fluconazole and itraconazole and according to molecular typing by RAPD. After isolation, the yeasts were storaged on Sabouraud dextrose agar (SDA) and in sterile distilled water. After 180 days, typing tests were carried out again. All C. albicans isolates were phospholipase productors, in both collections, even though enzyme activity had oscillated between moderate and high at that period. The prevalent enzymotype at time 0 and 180 were, respectively, 22 and 32. In relation to the proteinase, 100% of the yeasts showed moderate enzyme activity at time 0. At time 180, this percentage was 85%, and the others didn`t show enzyme activity. After storage on SDA and in distilled water, it was detected activity alteration of both enzymes and consequent enzymotype change, in 40 and 30% of isolates, respectively. Eight different C. albicans morphotypes were identified at time 0 and just 50% were maintained at time 180. The most common morphotype was 000-0. After storage most isolates presented changes in the morphotype. All isolates showed susceptibility to the analysed antifungals at times 0 and 180, showing just one antifungaltype, the 111. Only one isolate, after storage on SDA had a profile change from susceptible to dose dependent susceptible to itraconazole in a way that the antifungaltype wasn`t changed. The molecular typing by RAPD, with primers OPA-09, OPB-11 and OPE-18, showed 19 distinct molecular types among first collection isolates and allowed to identify that one C. albicans isolate from time 180 wasn`t related with the isolate obtained at time 0, from the same individual. The others showed DNA fragment profiles related between collection occasions. After storage by both methods, every isolate showed genetic relatedness with the profile obtained at time 0. The obtained results showed that the preservation methods used in this study don`t allow stability maintenance of the phenotypical characteristics evaluated. On the other hand, besides biotypes generated stability, the molecular typing by RAPD showed the best discriminatory index, between methodologies used, ratifying its ability in discriminating isolates of the same specie and so, its utility in epidemiological inquiries

Both liquid and solid wastes from Pacific whiting surimi manufacturing were characterized and value-added products were recovered. A proteinase in surimi wash water (SWW) was determined to be cathepsin L with Mr 54,200 on SDS-substrate gel. Heat treatment and acidification shifted the activity zone to M [subscript r] 39,500. No evidence of calpain, cathepsin B or H activity was found. Cathepsin L from SWW was recovered by ohmic heating (55°C for 3 min), ultrafiltration, and freeze-drying with overall yield of 0.83 g protein/L SWW and 78% recovery of activity. A 5.9 purification fold was achieved by these processes. The recovered enzyme had an optimum activity at pH 4.0 and showed preferable hydrolytic activity towards casein, acid-denatured hemoglobin and myofibrils. β-Mercaptoethanol, dithiothreitol and urea enhanced the enzyme activity. The recovered proteinase showed 18.5% residual activity after 7 wk storage at 4°C. Proteolytic activity in solid waste and digestive organs from Pacific whiting was investigated. Pepsin-like proteinase predominated in solid waste, while trypsin-like proteinase was predominant in viscera. Carboxypeptidase b was found in both viscera and solid waste. Protein hydrolysate was produced from Pacific whiting solid waste (PWSW) using commercial proteinase, Alcalase, under optimum hydrolysis conditions. Enzyme concentration, reaction time and waste/buffer ratio affected the hydrolysis and nitrogen recovery (NR). Correlation between the degree of hydrolysis (DH) and NR was high (R₂=0.978). Freeze-dried hydrolysate contained 79.97% protein and showed similar amino acid composition to PWSW and Pacific whiting muscle but tryptophan was reduced. With different DH (20, 30, 40, 50, 60%), surface hydrophobicity, total and surface sulfhydryl content decreased as the DH increased. The hydrolysate showed a high solubility over a wide pH range. Fat adsorption and fat binding capacity were reduced, while foam expansion was enhanced with an increased DH. Hydrolysate with DH of 30% showed highest emulsifying activity. Low emulsion stability and high foam stability were obtained in all hydrolysates tested. Hydrolysate showed antioxidant activity, but no obvious differences in activity were found with varying DH and hydrolysate concentrations. / Graduation date: 1997

The family of human kallikrein-related peptidases (KLKs) numbers 15 serine proteinases implicated in tumour progression. Despite the wide tissue distribution of KLKs and the numerous reports of their differential expression in pathological settings, the signalling mechanism(s) whereby these enzymes regulate tissue function are not yet known. Further, knowledge of the levels of their activity, as well as of their potential endogenous targets, has only been extracted from in vitro studies and cell culture systems. We hypothesized that KLKs can trigger tumour signalling via proteinase-activated receptors (PARs), a family of G-protein-coupled receptors. To test our hypothesis, we evaluated the ability of KLKs 5, 6, and 14: to activate or prevent signalling via PARs 1, 2, and 4 in cells and tissues expressing these receptors. Further, we used a novel activity-based probe approach, coupled with conventional immunoassay (ELISA), to determine the abundance of active KLK6 relative to total immunoreactive KLK6 in cancer-related biological fluids. We concluded that KLKs can regulate multiple signalling pathways triggered by PARs 1, 2, and 4, resulting in calcium release, platelet aggregation and vascular relaxation, and they can cause murine inflammation. Further, our activity-based ELISA demonstrated the presence of active KLK6 in ovarian cancer ascites fluids and cancer cell supernatants. We, therefore, suggest that tumours can produce active KLKs, which can potentially control tumour behaviour by regulating PAR activity.

Gag polyprotein is the precursor of HIV-1 structural proteins, required for correct assembly, budding and maturation of viral particle within HIV-1 life cycle. The process of maturation into an infectious virion is dependent on Gag and GagPol cleavage at nine predefined sites by HIV-1 proteinase. Its disruption is one of the main targets of HIV treatment. HIV-1, however, develops resistance to the proteinase inhibitors by creating mutations in both the proteinase and the substrate. The Gag processing by HIV-1 proteinase is a highly sequential process, that happens in specific order and rate. Previous biochemical studies determined the kinetic data of these processes using oligopeptides representing naturally occuring cleavage sites. This thesis describes the cleavage of the Gag polyprotein itself, which is the natural substrate of HIV-1 proteinase. For this purpose, the full-length Gag polyprotein was recombinantly prepared in bacterial expression system. The cleavage was carried out and its products were analyzed via SDS-PAGE and Western blotting. The substrate specificity of the wild-type and mutant HIV-1 proteinase with respect to the full-length wild-type Gag polyprotein was compared. Substantial differences were observed between the rates of individual steps of cleavage by the wild-type and mutant...