Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2018-05-14T14:05:11Z No. of bitstreams: 2 Dissertação - Artur Ribeiro de Sá Alexandre - 2018.pdf: 1970509 bytes, checksum: a04966ec3693633b41c6a2e6328e5db3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-05-14T14:08:13Z (GMT) No. of bitstreams: 2 Dissertação - Artur Ribeiro de Sá Alexandre - 2018.pdf: 1970509 bytes, checksum: a04966ec3693633b41c6a2e6328e5db3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-05-14T14:08:13Z (GMT). No. of bitstreams: 2 Dissertação - Artur Ribeiro de Sá Alexandre - 2018.pdf: 1970509 bytes, checksum: a04966ec3693633b41c6a2e6328e5db3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-04-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Bioprospecting is defined as the search for organisms, genes, enzymes, compounds, processes and any pieces from living beings, that could have economic potential, and eventually lead to a product development. Thus, enzymes, protein catalysts, have aroused the interest of industries for its conversion mode specific biomass, low energy cost and not production of toxic waste. Chitinases are enzymes that catalyze the lysis of chitin (biopolymer composed of N-acetylglucosamine monomers), and thus have several applications, among them: obtaining N-acetylglucosamine monomers, used in the production of prebiotics; degradation of chitinous waste from fishing activities; and the use in control of fungi and insects. The present work aimed to bioprospect chitinase producing bacteria from soil samples and to characterize the enzymatic activity patterns of the best bacterial isolate. To do so, a screening with 17 soil samples collected in the states of Minas Gerais, Santa Catarina and Rio Grande do Sul, was carried out using a culture medium with colloidal chitin. Thirteen chitinase producing bacteria were obtained, among them, the isolate Q1 (identified as Paenibacillus illinoisensis), demonstrated to be a good producer of the enzyme and thus was selected for determination and optimization of its chitinase activity evaluating reaction time, temperature and pH. The chitinase produced by the isolate showed 0.098 U of activity, which was subsequently improved to 0.66 U when tested under the optimal conditions of 1 hour of reaction at 37 ºC and pH 4, an increase of 573% over the initial value. The values of chitinase activity from the isolate P. illinoisensis are close to those found in other studies, which also emphasize the potential application of the enzyme, mainly in the control of phytopathogenic pests. Bioprospecting of chitinase producing bacteria is possible and promising. / A Bioprospecção é definida como a busca por organismos, genes, enzimas, compostos, processos e partes provenientes de seres vivos, que possam ter potencial econômico, e eventualmente, levar ao desenvolvimento de um produto. Nesse âmbito, as enzimas, catalisadores biológicos de natureza proteica, têm despertado o interesse de indústrias pelo seu modo de conversão de biomassa específico, de baixo custo energético e que não produz resíduos tóxicos. As quitinases são enzimas que catalisam a quebra da quitina (biopolímero composto por monômeros de N-acetilglicosamina), possuindo assim, diversas aplicações, dentre as quais: a obtenção de monômeros de Nacetilglicosamina, usados na produção de pré-bióticos; a degradação de resíduos quitinosos oriundos da pesca e do consumo de crustáceos; e uso no combate de fungos e insetos. O presente trabalho teve como objetivo bioprospectar bactérias produtoras de quitinase a partir de amostras de solo e caracterizar a enzima do melhor isolado bacteriano quanto aos padrões de atividade enzimática. Para tal, foi realizada uma triagem com 17 amostras de solo coletadas nos estados de Minas Gerais, Santa Catarina e Rio Grande do Sul, utilizando meio de cultura com quitina coloidal. Foram obtidas 13 colônias bacterianas produtoras de quitinase, dentre elas, o isolado Q1 (identificado como Paenibacillus illinoisensis), que se mostrou bom produtor da enzima e foi selecionado para testes de determinação e otimização da atividade enzimática quanto ao tempo de reação, temperatura e pH. A quitinase produzida pelo isolado apresentou atividade de 0,098 U, sendo melhorada posteriormente para 0,66 U quando testada nas condições ótimas de 1 hora de reação, a 37 ºC e em pH 4, um aumento de 573% em relação ao valor inicial. Os valores obtidos da atividade da qutininase produzida pela P. illinoisensis são próximos aos encontrados em outras pesquisas, que destacam também, o potencial de aplicação da enzima, principalmente no combate de pragas fitopatogênicas. A bioprospecção de bactérias produtoras de quitinase é possível e promissora.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / As quitinases sÃo enzimas capazes de hidrolisar as ligaÃÃes β-(1,4)-glicosÃdicas presentes em biopolÃmeros de N-acetil-β-D-glucosamina, principalmente quitina, um polissacarÃdeo estrutural presente na parede celular de diversos fungos. No presente trabalho, uma quitinase de classe I de feijÃo-de-corda (Vigna unguiculata) foi expressa em sistemas heterÃlogos e a proteÃna recombinante (rVuChi) foi caracterizada bioquimicamente bem como em relaÃÃo ao seu efeito sobre o crescimento micelial e germinaÃÃo de esporos/conÃdios de fungos filamentosos. A seqÃÃncia de DNA codificando a proteÃna foi amplificada por PCR e clonada nos vetores de expressÃo pET32a(+) e pPICZαA, para expressÃo heterÃloga em Escherichia coli e Pichia pastoris, respectivamente. A expressÃo de rVuChi em cÃlulas de E. coli ArticExpress DE3 se deu em corpos de inclusÃo. Em seis estirpes de P. pastoris a proteÃna recombinante foi secretada, de forma solÃvel, para o meio de cultura. Na fraÃÃo extracelular da estirpe KM71H foi observada a maior atividade quitinolÃtica, apÃs 72 horas de induÃÃo. A detecÃÃo de rVuChi foi feita por SDS-PAGE e com o kit Invision His-Tag stain, onde foram identificadas duas bandas protÃicas de massas moleculares aparentes de 30 e 33 kDa. Ambas as bandas apresentaram a mesma sequÃncia N-terminal e a ausÃncia de N-glicosilaÃÃo foi verificada. A quitinase recombinante estava presente principalmente na fraÃÃo F0/40 precipitada com sulfato de amÃnio e foi purificada a homogeneidade tanto por cromatografia de afinidade em matriz de quitina (com rendimento de 18,31 mg por litro de meio de cultura), quanto por cromatografia de interaÃÃes hidrofÃbicas em coluna de Phenyl Sepharose CL-4B (rendimento de 13,2 mg/L), seguidas de ultrafiltraÃÃo em membrana com limite de exclusÃo de 50 kDa. A rVuChi apresentou atividades endo e exo-quitinolÃtica frente a quitina coloidal e hidrolisou glicol-quitina em gel de SDS-PAGE, embora nÃo tenha apresentado atividade contra substratos sintÃticos contendo p-nitrofenol. A quitinase purificada apresentou massa molecular de 32 e 33,1 kDa por cromatografia de exclusÃo molecular em colunas de Superose 12 HR e Superdex 200, respectivamente. Em gel bidimensional, rVuChi apresentou um conjunto de seis âspotsâ com pI entre 4,44 e 5,15. A quitinase mostrou-se ainda termoestÃvel em temperaturas atà 50 ÂC e sua atividade enzimÃtica mÃxima ocorreu em pH 5. Em geral, a presenÃa de Ãons metÃlicos causou uma reduÃÃo de sua atividade enzimÃtica. O agente quelante EDTA (0,5%) estimulou a atividade enzimÃtica enquanto que o detergente SDS (0,5%) a inibiu totalmente. A quitinase recombinante apresentou 37% de hÃlice alfa e 26% de folha beta, como determinado por espectroscopia de dicroÃsmo circular. A desnaturaÃÃo de 50% das molÃculas de rVuChi ocorreu a 54,41 ÂC. Os espectros de fluorescÃncia revelaram que a proteÃna produzida em P. pastoris estava em sua conformaÃÃo totalmente enovelada. A quitinase recombinante de feijÃo-de-corda foi capaz de inibir totalmente a germinaÃÃo de esporos de Penicillium herquei atà 48 horas, na dose de 100 μg e causou inibiÃÃo de 68%, nas doses de 50, 25 e 12,5 μg. Na dose de 150 μg, houve uma inibiÃÃo de 55% na germinaÃÃo dos conÃdios de Rhizoctonia solani e um leve efeito sobre a germinaÃÃo dos esporos de Colletotrichum lindemuthianum e C. musae. Nenhum efeito da rVuChi foi observado sobre a germinaÃÃo de esporos dos fungos C. gloeosporioides, Fusarium solani e F. oxysporum. AlÃm disso, a proteÃna recombinante retardou o crescimento micelial de P. herquei em aproximadamente 50% (100 μg), porÃm nÃo apresentou efeito sobre o crescimento micelial dos demais fungos. Desta forma, a quitinase classe I de V. unguiculata à uma proteÃna com atividade antifÃngica. / Chitinases are enzymes that hydrolyze the β-(1,4) glycosidic bonds present in biopolymers of N-acety-β-D-glucosamine, mainly chitin, a structural polysaccharide which is found in cell walls of several fungi. In plants, chitinases play a role as defense proteins against the attack of pests and pathogens. In this work, a class I chitinase from cowpea (Vigna unguiculata) was expressed in heterologous systems. The recombinant protein (rVuChi) was purified, and characterized biochemically and in relation to its effects on mycelial growth and germination of spores/conidia of filamentous fungi. The DNA coding sequence of the cowpea chitinase was amplified by PCR and the products cloned in the expression vectors pET32a(+) and pPICZαA, for heterologous expression in Escherichia coli and Pichia pastoris, respectively. In E. coli cells, the recombinant fusion protein occurred mainly as inclusion bodies. On the other hand, in six strains of P. pastoris, the recombinant cowpea chitinase was secreted in a soluble form into the culture medium. The highest chitinase activity was detected in the extracellular fraction of KM71H strain, 72 hours after induction. The recombinant VuChi was detected by SDS-PAGE and Invision His-Tag stain kit, which identified two protein bands with apparent molecular masses of 30 and 33 kDa. These two protein bands showed the same N-terminal sequence, and an absence of N-glycosylation. Most recombinant chitinase secreted into the culture medium was recovered in the fraction F0/40, precipitated with ammonium sulfate. The expressed protein was purified to homogeneity by affinity chromatography on chitin matrix (yield of 18.31 mg per liter of culture medium), or by hydrophobic interactions chromatography on a column of Phenyl Sepharose CL-4B (yield = 13.2 mg/L), followed by ultrafiltration in a membrane with exclusion limit of 50 kDa. The purified rVuChi was able to hydrolyze colloidal chitin (in solution) as well as glycol chitin (in SDS-PAGE), although it did not show enzymatic activity against synthetic substrates containing p-nitrophenol. The purified chitinase showed molecular masses of 32 and 33.1 kDa by size exclusion chromatography on columns of Superose 12 HR and Superdex 200, respectively. When submitted to 2D electrophoresis, rVuChi presented a set of six spots with pI values between 4.44 and 5.15. The chitinase was thermostable at temperatures up to 50  C and the enzyme activity was highest at pH 5. In general, the presence of metal ions caused a reduction of its enzymatic activity. The chelating agent EDTA (0.5%) stimulated the enzyme activity, whereas in the presence of the detergent SDS (0.5%) the rVuChi activity was completely inhibited. The recombinant chitinase showed 37% of alpha helix and 26% of beta sheet, as determined by circular dichroism spectroscopy. Denaturing of 50% of the rVuChi molecules occurs at 54.41  C. The fluorescence spectra showed that the protein produced in P. pastoris was in its fully folded conformation. The recombinant cowpea chitinase was able to completely inhibit the germination of spores of Penicillium herquei, after 48 hours, at a dose of 100 mg, and caused 68% inhibition at doses of 50, 25 and 12.5 mg. At a dose of 150 mg, there was 55% inhibition on conidial germination of Rhizoctonia solani and a slight effect on spore germination of Colletotrichum lindemuthianum and C. musae. There was no effect of rVuChi on spore germination of C. gloeosporioides, Fusarium solani and F. oxysporum. In addition, the recombinant protein delayed the mycelial growth of P. herquei in approximately 50% (at the dose of 100 mg) but had no effect on mycelial growth of the other fungi. Therefore, the cowpea class I chitinase is a protein with anti-fungal activity.

Made available in DSpace on 2014-07-29T15:16:35Z (GMT). No. of bitstreams: 1 dissertacao parte 1 lidiane biologia.pdf: 35041 bytes, checksum: c58f88ce1be4655ee71c7b5ab0bef247 (MD5) Previous issue date: 2008-04-03 / Paracoccidoides brasiliensis is a human pathogenic dimorphic fungus. The recombinant chitinase from P. brasiliensis, Pbcts1r, was overexpressed in Escherichia coli using pET-32a (+) as vector. The enzyme was produced as inclusion bodies and became soluble by Sarkosyl being purified by a single step using a Ni-NTA resin. Pbcts1r showed activity against 4-MU-(GlcNAc)3 and 4-MU-(GlcNAc)2, presenting a endochitinase activity. Immunoblot reaction with anti-Pbcts1r identified two proteins in yeast crude extract. A partial purification of P. brasiliensis yeast crude extract by cationic-exchange chromatography on HPLC revealed two different chitinases, Pbcts1 and Pbcts2, with molecular mass of 45 kDa and 34 kDa, respectively. Pbcts2 has exochitinase activity and Pbcts1 has endochitinase activities. Reactions with anti- Pbcts1r showed the presence of Pbcts1 and Pbcts2 in crude extracts of yeast and transition from mycelium to yeast. On mycelium crude extracts was found only Pbcts1 and on yeast cell wall extract only Pbcts2. Both proteins were found to be secreted by yeast parasitic phase showing their probable importance in the permanence of the fungus in the human host. Phylogenetic relationships between the orthologs Pbcts1 and the putative Pbcts2 indicated the presence of a common ancestral. During evolution, P. brasiliensis could have acquired Pbcts2 and Pbcts1 playing distinct roles in order to growth and survive in diverse environment on saprophytic and parasitic phases / Paracoccidioides brasiliensis é um fungo dimórfico patogênico humano. A quitinase recombinante de P. brasiliensis, Pbcts1r, foi superexpressa em Escherichia coli utilizando pET-32(a)+ como vetor. A enzima foi produzida em corpos de inclusão e se tornou solúvel pela adição de sarkosyl, sendo purificada em um único passo com a utilização da resina Ni-NTA. Pbcts1r mostrou atividade diante de 4-MU-(GlcNAc)3 e 4- MU-(GlcNAc)2, apresentando atividade de endoquitinase. A reação de imunoblot com anti-Pbcts1r identificou duas proteínas no extrato bruto de levedura. A purificação parcial do extrato bruto de P. brasiliensis por cromatografia de troca-catiônica em HPLC revelou duas quitinases diferentes, Pbcts1 e Pbcts2, com massas moleculares de 45 e 34 kDa, respectivamente. Pbcts2 tem atividade de exoquitinase e Pbcts1 de endoquitinase. Reações com anti-Pbcts1r mostraram a presença de Pbcts1 e Pbcts2 no extrato bruto de levedura e transição de micélio para levedura. No extrato bruto de micélio foi encontrado somente Pbcts1 e no extrato de parede celular de levedura somente Pbcts2. Ambas as proteínas foram encontradas secretadas pela fase parasitária (levedura), mostrando a provável importância dessas proteínas na permanência do fungo no hospedeiro. Relações filogenéticas entre os ortólogos Pbcts1 e a provável Pbcts2 indicam a presença de um ancestral comum. Durante a evolução, P. brasiliensis poderia ter adquirido Pbcts2 e Pbcts1 desempenhando diferentes papéis para o crescimento e sobrevivência do fungo na fase saprofítica e parasitária

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / No presente trabalho, foi realizado um estudo preliminar sobre a expressÃo de um gene de quitinase de classe I de feijÃo-de-corda (Vigna unguiculata L.Walp), a clonagem e expressÃo desse gene em cÃlulas de Escherichia coli BL21(λ)DE3 e a determinaÃÃo via modelagem por homologia da estrutura tridimensional dessa proteÃna. A expressÃo do gene da quitinase de classe I de feijÃo-de-corda foi obtida a partir de RT-PCR com oligonucleotÃdeos iniciadores especÃficos. Nessas reaÃÃes, foram usadas amostras de RNA total de sementes e vagens em diferentes estÃgios de crescimento (2, 4, 6, 8 , 10, 12, 14, 16 e 18 dias), pertencentes a dois genÃtipos contrastantes quanto a infecÃÃo pelo Callosobruchus maculatus, IT81D-1053 (resistente) e TE97-419-07F (suscetÃvel). A expressÃo foi avaliada tambÃm em folhas, raÃzes, epicÃtilo e hipocÃtilo de dois genÃtipos contrastantes quanto a infecÃÃo pelo nematÃide das galhas Meloidogyne incongnita, CE-31 (resistente) e TE97-411-1F (suscetÃvel). A clonagem do gene VuChiI foi realizada a partir do produto amplificado da RT-PCR de sementes de IT81D-1053, e do produto amplificado a partir do DNA genÃmico de MONTEIRO. Foram obtidos 11 clones confirmados, dos quais 9 foram seqÃenciados. A subclonagem do clone R7 foi realizada em pET15b e a expressÃo da proteÃna recombinante foi induzida na presenÃa de IPTG 1mM. A proteÃna recombinante, com aproximadamente 30 kDa, foi visualizada atravÃs de um SDS-PAGE. A proteÃna purificada atravÃs de uma cromatografia de afinidade em coluna de Sepharose com NÃquel imobilizado nÃo apresentou atividade quitinÃsica significativa. Os modelos gerados para os clones obtidos e para a quitinase nativa, indicam que as mutaÃÃes ocorridas nÃo alteram os sÃtios ativos das molÃculas. Dessa forma, a quitinase de classe I do feijÃo-de-corda parece apresentar expressÃo constitutiva em todas as partes da planta. A quitinase recombinante obtida foi pouco ativa. As mutaÃÃes pontuais nos clones obtidos sugerem a ocorrÃncia de isoformas dessa proteÃna, o que ainda deve ser elucidado no futuro / In this work we have made a preliminary study on the expression of a class I chitinase gene from cowpea (Vigna unguiculata L.Walp), cloning and expression of this gene in Escherichia coli BL21(λ)DE3 cells and, through homology modeling, we determined the three dimensional structure of this protein. The expression of the class I quitinase gene from cowpea was performed by RTÂPCR from the total RNA, with specific primers, from seeds and pods in distinct stages of development (2, 4, 6, 8 , 10, 12, 14, 16 and 18 days), belonging to two contrasting genotypes regarding to infection by Callosobruchus maculatus, IT81DÂ1053 (resistant) e TE97Â419Â07F (susceptible). The gene expression in leaves, roots, epicotyls and hipocotyls from two contrasting genotypes considering the infection by the nematoid Meloidogyne incongnita, CEÂ31(resistant) e TE97Â411Â1F (susceptible). The VuChiI gene cloning was accomplished from the amplified product on RTÂPCR with IT81DÂ1053 seeds, and the amplified product from the genomic DNA of MONTEIRO. Eleven clones were obtained, from which nine were sequenced. The cloning of R7 clone was accomplished in pET15b vector and the expression of the recombinant protein was induced in the presence of IPTG 1mM. The protein, with 30 kDa, was visualized through a SDSÂPAGE. The protein purified through an affinity chromatography in Sepharose column with immobilized Nickel did not had a significative hydrolytic activity. The models generated for the clones and for the native chitinase, have indicated that mutations that occurred did not changed the molecule's active sites. Thus, the class I chitinase gene from feijÃo de corda seems to present constitutive expression in all parts of the plant. The recombinant chitinase obtained was inactive. The specific mutations in the resulting clones suggests the occurrence of isoforms of this protein, what should be elucidated in the future.

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2016-10-05T10:38:29Z No. of bitstreams: 2 Dissertação - Enio Saraiva Soares - 2016.pdf: 2392510 bytes, checksum: 37b84e9c9bc76eaf406e92f41d3828bb (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-10-05T10:58:52Z (GMT) No. of bitstreams: 2 Dissertação - Enio Saraiva Soares - 2016.pdf: 2392510 bytes, checksum: 37b84e9c9bc76eaf406e92f41d3828bb (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-10-05T10:58:52Z (GMT). No. of bitstreams: 2 Dissertação - Enio Saraiva Soares - 2016.pdf: 2392510 bytes, checksum: 37b84e9c9bc76eaf406e92f41d3828bb (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-04-29 / Currently there are different approaches to synthesize and discover new compounds, but the pursuit of these products on biodiversity is still advantageous. In bioprospecting microorganisms, which often are seeking their properties that can be exploited in biotechnology products. This is the case of chitinases, enzymes that degrade chitin. Chitinases (EC 3.2.1.29) are glycosyl hydrolases type enzymes that specifically cleave β-1,4 bonds between N-acetylglucosamines units of chitin with sizes ranging from 20 kDa to 90 kDa. The main producers of chitinase are the bodies that have chitin in their cell wall or exoskeleton, such as insects, crustaceans, fungi, algae, among others. This study aimed to select and identify producing bacteria chitinase in soil samples from different coastal regions of southern Brazil. Seventeen soil samples, collected close to fishing for shellfish waste disposal sites, were prepared and seeded in four minimum culture medium containing colloidal chitin as the sole source of carbon and energy, incubated and the colonies were isolated and purified. After yielded a total of thirteen isolates that were submitted to enzymatic index test, stressed that four isolates. The four isolated genomic DNA was extracted, amplified and purified, and sequenced region encoding 16S rRNA of these organisms. Bacteria were then pooled and identified by construction of a phylogenetic tree. The results showed the presence of the species Paenibacillus illinoisensis and Paenibacillus chitinolyticus and two members of the genus Bacillus. Future studies may indicate the possibility of its use as a source of genes for biotechnological applications such as the production of new biopesticides. / Existem atualmente diferentes abordagens para se sintetizar e descobrir novos compostos, mas a busca desses produtos na biodiversidade ainda é vantajosa. Na bioprospecção de microrganismos, o que muitas vezes se busca são as suas propriedades que possam ser aproveitadas em produtos biotecnológicos. Esse é o caso das quitinases, enzimas capazes de degradar a quitina. As quitinases (EC 3.2.1.29) são enzimas do tipo glicosilhidrolases, com tamanhos que variam de 20 kDa até 90 kDa, que clivam especificamente as ligações β-1,4 entre unidades de N-acetilglicosaminas da quitina. Os principais produtores de quitinases são os organismos que possuem quitina no seu exoesqueleto ou parede celular, como insetos, crustáceos, fungos, algas, bactérias entre outros. O presente estudo teve como objetivo selecionar e identificar bactérias produtoras de quitinases em amostras de solos de diferentes locais litorâneos da região Sul do Brasil. Dezessete amostras de solo, coletadas próximo a locais de descarte de resíduos de crustáceos por pescadores, foram preparadas e semeadas em meio de cultura mínimo contendo quitina coloidal como única fonte de carbono e energia, incubadas e as colônias foram isoladas e purificadas. Ao fim obteve-se um total de treze isolados de bactérias, que foram submetidas ao teste de índice enzimático, que destacou desses quatro isolados. O DNA genômico de quatro isolados foi extraído, amplificado e purificado, sendo sequenciada a região codificadora do gene 16S rRNA destes microrganismos. As bactérias foram então agrupadas e identificadas pela construção de uma árvore filogenética. Os resultados mostraram a presença das espécies Paenibacillus illinoisensis e Paenibacillus chitinolyticus além de dois membros do gênero Bacillus. Estudos futuros poderão indicar a possibilidade de seu uso como fonte de genes para aplicação biotecnológica, como a produção de novos bioinseticidas.

anglický Phlebotomine sand flies (Diptera: Psychodidae) are the only proven vectors of Leishmania parasites (Kinetoplastida: Trypanosomatidae). In Nematoceran Diptera, including sand flies, adults produce a type 1 peritrophic matrix (PM) which is secreted in response to the distension of the midgut caused by blood meal. The PM is an acellular envelope composed of chitin fibres and proteins, which protects the midgut epithelium against abrasion and pathogens and improves digestion. In hematophagous insects, the PM also plays a central role in heme detoxification. Female sand flies acquire Leishmania with a bloodmeal and the parasites undergo complicated development in their gut finished by the colonization of the stomodeal valve. The PM is one of the most important barriers in Leishmania development and its role in the vector competence of the S. schwetzi is the main topic of this master thesis. The PM's kinetic and morfology in the S. schwetzi is compared with other three sand fly species which differ in susceptibility to L. donovani. The key role of the PM in S. schwetzi vector competence is finally proved by disrupting the PM using the exogenous chitinase from Beauveria bassiana. Under these artificial conditions, the Leishmania parasites (L. donovani and L. major) are able to exit the PM,...

Tall fescue (Festuca arundinacea Schreb.) has great utility as a low maintenance turfgrass in the northern and transition zone regions of the United States. However, it is difficult to successfully maintain tall fescue of high quality over consecutive summers because of its susceptibility to the fungal pathogen Rhizoctonia solani, which causes the disease brown patch. Not only is brown patch aesthetically unpleasing in a stand of tall fescue but it can also thin out the turf and allow for the encroachment of undesirable weedy species. Cultivar selection, cultural practices, mixing turf species and timing of pesticide applications all can impact the epidemiology of brown patch in tall fescue. Research was conducted in tall fescue to quantify chitinase activity in different cultivars, elucidate the impact of mowing height and nitrogen fertility on brown patch and bermudagrass (Cynodon dactylon L.) encroachment, to evaluate seeding mixtures of tall fescue with hybrid bluegrass (Poa pratensis x Poa arachnifera) on diseases and weeds as well as measuring the impact of the herbicide bispyribac-sodium on brown patch. Chitinase activity was greater in the tall fescue cultivar that was less susceptible to brown patch. In the mowing-fertility studies, cutting tall fescue at 10 cm generally reduced brown patch and bermudagrass encroachment compared to 6 cm. Mixing hybrid bluegrass with tall fescue reduced disease and weed species infestations compared to tall fescue alone. Applying bispyribac-sodium earlier in April resulted in less brown patch and better weed control compared to application in May. Based on this research brown patch severity and subsequent weed species infestations can be reduced by selecting a tall fescue cultivar with a high basal level of chitinase, mowing it at 10 cm and mixing it with a hybrid bluegrass cultivar. / Ph. D.

Ching, Ka Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves [69-80]). / Abstract also in Chinese. / Abstract (in English) --- p.ii / Abstract (in Chinese) --- p.v / Acknowledgement --- p.viii / Table of Contents --- p.ix / List of Tables --- p.xii / List of Figures --- p.xiii / Glossary of Terms and Abbreviations --- p.xv / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Introduction of Atopic Eczema (AE) --- p.1 / Chapter 1.1.1 --- Definition and classification of AE --- p.1 / Chapter 1.1.2 --- Epidemiology --- p.3 / Chapter 1.1.2.1 --- The hygiene hypothesis --- p.5 / Chapter 1.2 --- Pathogenesis and Etiology --- p.6 / Chapter 1.2.1 --- Biphasic type-1/type-2 T-helper lymphocyte (Thl/Th2) immunological responses --- p.6 / Chapter 1.2.2 --- Nature and involvements of immunoglobin E (IgE) --- p.8 / Chapter 1.2.3 --- Microbial colonization --- p.9 / Chapter 1.2.4 --- Cytokines involvement --- p.10 / Chapter 1.2.5 --- Pruritus inducing neurotrophic factors --- p.11 / Chapter 1.2.6 --- "Food allergens, aeroallergens" --- p.12 / Chapter 1.2.7 --- Dysregulation of innate immune system --- p.13 / Chapter 1.2.7.1 --- Dysregulation of antimicrobial peptides --- p.14 / Chapter 1.2.7.2 --- Skin barrier impairment --- p.14 / Chapter 1.2.8 --- Genetic predisposition --- p.15 / Chapter 1.3 --- Assessments of Atopic Eczema (AE) --- p.17 / Chapter 1.3.1 --- AE severity assessment --- p.17 / Chapter 1.3.1.1 --- Scoring of atopic dermatitis (SCORAD) system --- p.17 / Chapter 1.3.1.2 --- Nottingham eczema severity score (NESS) --- p.20 / Chapter 1.3.2 --- Dermatological parameter - skin hydration (SH) and transepidermal water loss (TEWL) --- p.22 / Chapter 1.4 --- Chitinase (CHIA) --- p.22 / Chapter 1.4.1 --- Chitin and CHIA --- p.22 / Chapter 1.4.2 --- Association of acid mammalian chitinase (AMCase) with asthma --- p.23 / Chapter 1.4.3 --- Hygiene hypothesis implies: AMCase and allergy relationship --- p.24 / Chapter Chapter 2: --- Hypothesis and Objectives --- p.25 / Chapter 2.1 --- Hypothesis - based on CHIA involvements in canine AE --- p.25 / Chapter 2.2 --- Hypothesis --- p.25 / Chapter 2.3 --- Objective 226}0ؤ based on AMCase single nucleotide polymorphism (SNPs) in asthma susceptibility --- p.25 / Chapter 2.4 --- Objectives --- p.27 / Chapter Chapter 3: --- Methodology --- p.28 / Chapter 3.1 --- Recruitment of cases and controls --- p.28 / Chapter 3.2 --- Assessment of clinical parameters --- p.29 / Chapter 3.2.1 --- Scoring of atopic dermatitis (SCORAD) system --- p.29 / Chapter 3.2.2 --- Nottingham eczema severity score (NESS) --- p.29 / Chapter 3.2.3 --- Dermatologic parameters --- p.29 / Chapter 3.2.3.1 --- Cutaneous bacterial colonization --- p.29 / Chapter 3.2.3.2 --- Skin hydration (SH) and transepidermal water loss (TEWL) --- p.30 / Chapter 3.3 --- Peripheral blood collection and genomic deoxyribonucleic acid (DNA) extraction --- p.30 / Chapter 3.4 --- Acid mammalian chitinase (AMCase) polymorphism genotyping --- p.31 / Chapter 3.4.1 --- Polymerase chain reactions (PCR) amplification of AMCase gene --- p.31 / Chapter 3.4.1.1 --- List of PCR reagents --- p.32 / Chapter 3.4.1.2 --- Electrophoresis reagents --- p.33 / Chapter 3.4.2 --- Restriction fragment length polymorphism (RFLP) analysis of AMCase and confirmation with direct sequencing --- p.33 / Chapter 3.5 --- Statistical analysis --- p.34 / Chapter Chapter 4: --- Results and Data Analysis --- p.36 / Chapter 4.1 --- Results --- p.36 / Chapter 4.1.1 --- Demographic data of cases and controls --- p.36 / Chapter 4.1.2 --- PCR amplification and RFLP analysis of AMCase gene --- p.37 / Chapter 4.1.3 --- PCR cycle sequencing of the PCR fragments --- p.40 / Chapter 4.2 --- Data analysis --- p.41 / Chapter 4.2.1 --- Data overview --- p.41 / Chapter 4.2.2 --- Genotypes distribution of AMCase polymorphisms --- p.43 / Chapter 4.2.2.1 --- Allele frequency comparison of AMCase single nucleotide polymorphism (SNPs) by chi-square --- p.43 / Chapter 4.2.2.2 --- Allele frequency comparison of AMCase SNPs by logistic regression analysis --- p.44 / Chapter 4.2.3 --- Haplotype frequency estimation via maximum likelihood algorithm --- p.45 / Chapter 4.2.4 --- Association of AMCase polymorphism with Atopic Eczema (AE) clinical parameters --- p.47 / Chapter 4.2.4.1 --- Peripheral blood eosinophil counts --- p.48 / Chapter 4.2.4.2 --- Serum immunoglobin E (IgE) level --- p.49 / Chapter 4.2.4.3 --- Dermatologic factors --- p.49 / Chapter 4.2.4.3.1 --- Cutaneous Staphylococcus aureus colonization --- p.49 / Chapter 4.2.4.3.2 --- Skin hydration (SH) and transepidermal water loss (TEWL) --- p.50 / Chapter Chapter 5: --- Discussion --- p.52 / Chapter 5.1 --- Data overview --- p.52 / Chapter 5.2 --- AMCase rs3806448 polymorphism was significantly different among AE cases and controls --- p.53 / Chapter 5.2.1 --- Allele frequency comparison of AMCase SNPs polymorphisms by chi-square --- p.53 / Chapter 5.2.2 --- Allele frequency comparison of AMCase SNPs polymorphisms by logistic regression analysis --- p.54 / Chapter 5.2.3 --- The possible genetic modification by rs3806448 homozygous recessive genotype --- p.55 / Chapter 5.3 --- "Significant difference of haplotype frequency, 2212 among case-control comparison" --- p.56 / Chapter 5.4 --- Strong associations between AMCase SNPs polymorphisms and clinical parameters of AE --- p.57 / Chapter 5.4.1 --- Peripheral blood eosinophil counts --- p.57 / Chapter 5.4.2 --- Dermatologic factors --- p.58 / Chapter 5.4.2.1 --- Cutaneous Staphylococcus aureus colonization --- p.58 / Chapter 5.4.2.2 --- Skin hydration (SH) and transepidermal water loss (TEWL) --- p.59 / Chapter 5.5 --- Limitation of the present study --- p.59 / Chapter Chapter 6: --- Conclusion and Future Prospect --- p.62 / Chapter 6.1 --- Conclusion --- p.62 / Chapter 6.2 --- Future prospect --- p.62 / Chapter Chapter 7: --- Appendices --- p.64 / Chapter Chapter 8: --- References --- p.69

Ho, Sai Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 144-164). / Abstracts in English and Chinese. / TABLE OF CONTENTS --- p.ii / ABSTRACT --- p.xii / 撮要 --- p.xv / LIST OF ABBREIVIATIONS --- p.xvi / LIST OF TABLES --- p.xvii / LIST OF FIGURES --- p.xix / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Overview of lectins --- p.1 / Chapter 1.1.1 --- History of lectins --- p.1 / Chapter 1.1.2 --- Definitions of lectins --- p.2 / Chapter 1.1.3 --- Classification and nomenclature of lectins based on structure --- p.2 / Chapter 1.1.4 --- Classification and nomenclature of lectins based on carbohydrate-bindingspecificity --- p.4 / Chapter 1.1.5 --- Structure of plant lectins --- p.4 / Chapter 1.1.6 --- Biological function of plant lectins --- p.5 / Chapter 1.1.6.1 --- Anti-viral activity of plant lectiins --- p.5 / Chapter 1.1.6.2 --- Lectins as plant defense proteins --- p.6 / Chapter 1.1.6.3 --- Insecticidal activity of plant lectins --- p.7 / Chapter 1.1.6.4 --- Anti-fungal activity of plant lectins --- p.7 / Chapter 1.1.6.5 --- Mitogenic activity of plant lectins --- p.7 / Chapter 1.1.6.6 --- Anti-tumor and anti-proliferative activity of plant lectins --- p.9 / Chapter 1.1.7 --- Background of legume lectins --- p.11 / Chapter 1.1.7.1 --- Structure of legume lectins --- p.11 / Chapter 1.1.7.2 --- Functions and activities of legume lectins --- p.12 / Chapter 1.2 --- Overview of serine protease inhibitors in plants --- p.14 / Chapter 1.2.1 --- Classification of serine protease inhibitor --- p.15 / Chapter 1.2.2 --- The main functions of plant serine protease inhibitors --- p.17 / Chapter 1.2.3 --- Commercial application of serine protease inhibirtors --- p.19 / Chapter 1.2.3.1 --- Medical application --- p.19 / Chapter 1.2.3.2 --- Transgenic application in agriculture --- p.22 / Chapter 1.3 --- Overview of Pathogenesis-related proteins in plants --- p.25 / Chapter 1.3.1 --- Overview of PR-5 family Thaumatin-like proteins (TLPs) --- p.27 / Chapter 1.3.1.1 --- Structural similarities among TLPs --- p.28 / Chapter 1.3.1.2 --- Antifungal activity of TLP --- p.31 / Chapter 1.3.2 --- Overview of Chinase-like proteins (CLPs) --- p.33 / Chapter 1.3.2.1 --- Classification of chitinase --- p.34 / Chapter 1.3.2.1.1 --- On the basis of amino acid sequence of glycosyl hydrolase --- p.34 / Chapter 1.3.2.1.2 --- On the basis of amino acid sequence of plant chitinase --- p.35 / Chapter 1.3.2.2 --- Antifungal activity of CLP --- p.36 / Chapter 1.3.3 --- Anti-freeze property of PR proteins --- p.38 / Chapter 1.3.4 --- Application of PR proteins in agriculture --- p.40 / Chapter 1.4 --- Rationale of the present study --- p.42 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.43 / Chapter 2.2 --- Preparation of crude extract --- p.44 / Chapter 2.2.1 --- Hokkaido large black soybean --- p.44 / Chapter 2.2.2 --- Emperor banana --- p.45 / Chapter 2.3 --- Purification --- p.45 / Chapter 2.4 --- Chromatography --- p.46 / Chapter 2.4.1 --- DEAE-cellulose chromatography --- p.46 / Chapter 2.4.2 --- Affi-gel Blue gel --- p.47 / Chapter 2.4.3 --- SP-Sepharse --- p.48 / Chapter 2.4.4 --- Mono Q HR 5/5 and Mono S HR 5/5 --- p.49 / Chapter 2.4.5 --- Superdex 75 and superdex 200 --- p.50 / Chapter 2.5 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.50 / Chapter 2.6 --- Protein concentration determination --- p.54 / Chapter 2.7 --- Preparation of rabbit reticulocyte lysate --- p.54 / Chapter 2.8 --- Determination of N-terminal amino acid sequence --- p.56 / Chapter 2.9 --- Assay of inhibition of hemagglutinating activity by different carbohydrates --- p.56 / Chapter 2.10 --- Thermal stability determination assays --- p.57 / Chapter 2.10.1 --- Stability at various temperatures --- p.57 / Chapter 2.10.2 --- Stability at 100°C --- p.57 / Chapter 2.11 --- Assay of pH dependence of hemagglutinating activity --- p.58 / Chapter 2.12 --- Assay of ion dependence of hemagglutinating activity --- p.58 / Chapter 2.13 --- Assay of antifungal activity --- p.58 / Chapter 2.14 --- Assay of trypsin inhibitory activity --- p.60 / Chapter 2.15 --- Assay of antibacterial activity --- p.61 / Chapter 2.16 --- Assay for cytotoxic activity on cancer cell lines --- p.61 / Chapter 2.17 --- Assay for HIV-1 reverse transcriptase (RT) inhibitory activity --- p.62 / Chapter 2.18 --- Assay of mitogenic activity --- p.63 / Chapter Chapter 3 --- Purification and Characterization of Defense-Related Proteins from their Respective Sources / Chapter 3.1 --- Purification and Characterization of a Lectin from the Seeds of Hokkaido large black soybean / Chapter 3.1.1 --- Introduction --- p.65 / Chapter 3.1.2 --- Results --- p.66 / Chapter 3.1.3 --- Purification --- p.68 / Chapter 3.1.3.1 --- Affinity chromatography on Affi-gel Blue gel --- p.69 / Chapter 3.1.3.2 --- Anion-exchange chromatography on DEAE-cellulose --- p.70 / Chapter 3.1.3.3 --- Anion-exchange chromatography on Mono Q column --- p.71 / Chapter 3.1.3.4 --- Gel filtration on Superdex 200 column --- p.72 / Chapter 3.1.3.5 --- Hemagglutinating activity at each purification step --- p.73 / Chapter 3.1.4 --- Characterization of Lectin --- p.74 / Chapter 3.1.4.1 --- Molecular mass determination --- p.74 / Chapter 3.1.4.2 --- N-terminal amino acid sequencing --- p.76 / Chapter 3.1.4.3 --- Assay of inhibition of hemagglutinating activity by different carbohydrates --- p.77 / Chapter 3.1.4.4 --- Thermal stability --- p.78 / Chapter 3.1.4.5 --- Assay of pH dependence of hemagglutinating activity --- p.80 / Chapter 3.1.4.6 --- Assay of ion dependence of hemagglutinating activity --- p.81 / Chapter 3.1.4.7 --- Assay for HIV-1 reverse transcriptase (RT) inhibitory activity --- p.82 / Chapter 3.1.4.8 --- Assay of mitogenic activity --- p.83 / Chapter 3.1.4.9 --- Assay of antibacterial activity --- p.84 / Chapter 3.1.5 --- Discussion --- p.86 / Chapter 3.2 --- Purification and Characterization of a Trypsin inhibitor from the Seeds of Hokkaido large black soybean / Chapter 3.2.1 --- Introduction --- p.93 / Chapter 3.2.2 --- Results --- p.94 / Chapter 3.2.3 --- Purification --- p.95 / Chapter 3.2.3.1 --- Anion-exchange chromatography on Mono Q column --- p.96 / Chapter 3.2.3.2 --- Gel filtration on Superdex 75 column --- p.98 / Chapter 3.2.3.3 --- Trypsin inhibitory activity at each purification step --- p.99 / Chapter 3.2.4 --- Characterization of trypsin inhibitory --- p.100 / Chapter 3.2.4.1 --- Molecular mass determination --- p.100 / Chapter 3.2.4.2 --- N-terminal amino acid sequencing --- p.102 / Chapter 3.2.4.3 --- Assay for HIV-1 reverse transcriptase (RT) inhibitory activity --- p.103 / Chapter 3.2.4.4 --- Antiproliferative effect on MCF-7 and Hep G2 cells --- p.104 / Chapter 3.2.4.5 --- pH and thermal stability --- p.105 / Chapter 3.2.5 --- Discussion --- p.106 / Chapter 3.3 --- Purification and Characterization of a Thaumatin-like protein and Chitinase-like protein from Emperor Banana / Chapter 3.3.1 --- Introduction --- p.108 / Chapter 3.3.2 --- Results --- p.109 / Chapter 3.3.3 --- Purification --- p.111 / Chapter 3.3.3.1 --- Affinity chromatography on Affi-gel Blue gel --- p.112 / Chapter 3.3.3.2 --- Cation exchange chromatography on Mono S column --- p.113 / Chapter 3.3.3.3 --- Gel filtration on Superdex 75 column --- p.114 / Chapter 3.3.3.3.1 --- Fraction MS 2 --- p.114 / Chapter 3.3.3.3.2 --- Fraction MS 4 --- p.115 / Chapter 3.3.3.3.3 --- Fraction MS 5 --- p.118 / Chapter 3.3.4 --- Characterization of the thaumatin-like protein --- p.121 / Chapter 3.3.4.1 --- N-terminal amino acid sequence determination --- p.121 / Chapter 3.3.4.2 --- Assay for antifungal activity --- p.122 / Chapter 3.3.4.3 --- Thermal stability --- p.124 / Chapter 3.3.4.4 --- pH stability --- p.125 / Chapter 3.3.4.5 --- Resistance to trypsin digestion --- p.125 / Chapter 3.3.4.6 --- Anti-HIV-1 reverse transcriptase activity --- p.126 / Chapter 3.3.4.7 --- Discussion --- p.127 / Chapter 3.3.5 --- Characterization of the two chitinase-like protein --- p.131 / Chapter 3.3.5.1 --- N-terminal amino acid sequence determination --- p.131 / Chapter 3.3.5.1.1 --- Emperor banana MS2 CLP --- p.131 / Chapter 3.3.5.1.2 --- Emperor banana MS4 CLP --- p.132 / Chapter 3.3.5.2 --- Assay for antifungal activity --- p.133 / Chapter 3.3.5.3 --- Discussion --- p.136 / Chapter Chapter 4 --- general discussion --- p.138 / References --- p.144

Thesis (MScAgric) -- University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Fungi are an extremely diverse group of organisms and, by acting as pathogens, they can colonise various other organisms, including humans, plants and animals. The effect of this is usually detrimental, not only to agricultural crops and livestock, but also to human well-being. The extensive farming of crops and livestock requires persistent control of fungal populations, commonly through the use of chemical fungicides. However, the exclusive use of fungicides is no longer a sustainable practice, as a result of serious problems, such as increasing fungicide resistance in pathogen strains, the high costs of fungicides, as well as concern about the environment. The search by producers and scientists for alternative control measures is an ongoing process. The fungal cell wall consists of polysaccharides that not only playa role in protection of the fungi, but also in relaying signals for the invasion and infection of susceptible hosts. Chitin, a polysaccharide composed of N-acteylglucosamine (GleNAc) residues linked by P-1,4 glucosidic linkages, is one of the major components of the fungal cell wall, where it plays an important role in the apical growth of the vegetative hyphae. Chitinases (EC 3.2.1.14) are abundant proteins produced by a variety of microorganisms and plants and are necessary for the hydrolysis of the chitin polymer. During the invasion of many plant species by a pathogen, the production of a specific group of proteins, designated pathogenesis-related (PR) proteins that include chitinases, is induced as part of their defence response. Due to the facts that pathogenic fungi contain chitin in their cell walls and that plant chitinases are induced upon pathogen attack, chitinases have been confirmed as an integral and crucial part of the plant's natural defence response. Chitinases have increasingly been targeted to upregulate plants' endogenous disease resistance mechanisms through transgenic overexpression in a variety of hosts. Several species of fungi, including various Trichoderma spp., are potent biocontrol agents of plant pathogenic fungi and insects. The antagonistic activities of these biological control agents towards phytopathogens are based on the secretion of extracellular hydrolytic enzymes, such as cell wall-degrading chitinase enzymes. However, biological control is not restricted to naturally occurring biocontrol agents. Through the process of genetic transformation, other fungal or yeast species can be enhanced to produce their own chitinases or other antimicrobial substances more effectively in order to yield potent biocontrol agents. Various types of chitinases have been applied in the production of fungal resistant plants and some research has been done on the application of chitinases, from a variety of microorganisms, as biological control agents. In contrast, very little is known about the antifungal activity of the Saccharomyces cerevisiae chitinase enzyme, encoded by the CTS1-2 gene. The CTS1-2 gene was utilised in this study as a candidate for overexpression in both yeast and plant expression systems to analyse the ability of the encoding chitinase to inhibit fungal growth. The first objective of this study involved the high level expression and optimisation of the secretion of the CTS1-2 gene in S. cerevisiae to render recombinant yeast with enhanced antifungal abilities and with possible applications as a biocontrol agent to control plant pathogenic fungi. It was hypothesised that high-level expression and efficient secretion would be prerequisites in a biocontrol yeast strain. To this end, two strong promoters and terminators were included in the study and the secretion of the chitinase gene was evaluated by testing three different secretion signals. The secretion signals included: the native CTS1-2 secretion signal, the S. cerevisiae mating pheromone a-factor (MFa1) secretion signal, as well as the Trichoderma reesei f3-xylanase 2 (XYN2) secretion signal. The phosphoglycerate kinase 1 (PGK1) and alcohol dehydrogenase 2 (ADH2) promoters and terminators were employed to achieve high-level expression. The results obtained from the analysis of the recombinant yeasts showed that the PGK1 promoter-terminator constructs yielded high level CTS1-2-expressing and chitinase-producing strains of S. cerevisiae PRY488. The ability of the different secretion signals to efficiently secrete the overexpressed chitinase was analysed and it was found that the non-native secretion signals delivered significantly more protein to the extracellular environment. It was thus evident that the performance of the MFa1 and XYN2 secretion signals was superior to that of the native secretion signal. The antifungal activities of the recombinant chitinases produced by these constructs were tested in in vitro assays against Botrytis cinerea. The enzymes led to a significant reduction in hyphal development, caused by extreme structural damage to the hyphal tips, the hyphal cell walls as well as the ability of the fungus to form reproductive and survival structures, thereby confirming the antifungal abilities of this enzyme. The ADH2 promoter-terminator constructs yielded CTS1-2 transcripts, but no chitinase activity could be detected with any of these strains. The reasons for this still remain unclear. The second objective of this study was to assess the potential of the yeast chitinase gene to upregulate defence against fungal infection in planta. In order to elucidate this, the CTS1-2 gene was constitutively overexpressed in tobacco plants, targeting the chitinase both to the intra- and the extracellular environment. The results obtained showed that the transgenic tobacco lines regenerated in this study stably integrated the transgene, exhibiting transgene expression as well as the production of a biologically active yeast chitinase enzyme. The F, progeny were rigorously tested for resistance to B. cinerea, and both in vitro and in planta assays confirmed that the yeast chitinase increased the plant's tolerance to fungal infection; some of the lines showed disease resistance of 65 and 70%. The plants expressing an extracellularly targeted chitinase gene are still under evaluation. Interesting results are expected relating to the effect of the chitinase on the plant surface with regards to disease resistance to fungal pathogens. In conclusion, the combined set of results from both the yeast and plant overexpression studies has confirmed the strong antifungal effect of yeast chitinases. The yeast CTS1-2 chitinase could be instrumental in the development of a new generation of yeast strains with improved antifungal capabilities. This enzyme could also play an important role in genetic transformation technologies aimed at enhanced disease resistance. / AFRIKAANSE OPSOMMING: Swamme omsluit 'n uiterste diverse groep organismes wat mense, plante en diere deur patogeniese aksie kan koloniseer. Die uitkoms hiervan op landbougewasse, die veebedryf en menslike gesondheid is gewoonlik skadelik. Uitgebreide gewas- en veeboerderye benodig voortdurende beheer van fungiese populasies, tipies deur van chemiese swamdoders gebruik te maak. Die uitsluitlike gebruik van swamdoders is egter nie meer 'n lewensvatbare praktyk nie, hoofsaaklik as gevolg van probleme soos die opbou van weerstand van patogeniese rasse teen swamdoders, die hoë kostes van die middels, asook besorgheid oor die omgewing. Die soektog na alternatiewe beheermaatreëls deur produsente en wetenskaplikes bly 'n aaneenlopende proses. Die swamselwand bestaan uit polisakkariede wat nie net In rol in die beskerming van die swam speel nie, maar ook betrokke is in die oordrag van aanvals- en infeksieverwante seine in 'n vatbare gasheer. Chitien, 'n polisakkaried bestaande uit N-asetielglukosamien (GlcNAc) residu's gekoppel deur 13-1,4glukosidiese bindings, is een van die hoofkomponente van die swamselwand, waar dit 'n belangrike rol in die apikale groei van vegetatiewe hifes speel. Chitinases (EC 3.2.1.14) is proteïene wat oorvloedig deur 'n verskeidenheid van mikroërganismes en plante geproduseer word, waar hulle vir die hidrolise van die chitien polimeer noodsaaklik is. Tydens die infeksie van verskeie plantspesies deur In patogeen, word die produksie van 'n spesifieke groep proteïene, die sogenaamde patogeen-verwante (PR) proteïene wat chitinases insluit, as deel van die plant se verdedigingsreaksie geïnduseer. Die feit dat patogeniese swamselwande chitien bevat en dat plantchitinases tydens infeksie geïnduseer word, het daartoe gelei dat dit bevestig is dat chitinases In integrale en kritiese deel van die plant se natuurlike verdedigingsreaksie uitmaak. Chitinases word toenemend geteiken in pogings om die plant se intrinsieke siekteweerstandsmeganismes te verbeter deur transgeniese ooruitdrukking daarvan in 'n verskeidenheid van gashere. Verskeie swamspesies, insluitend verskillende Trichodenna-spesies, is kragtige bio-antagoniste van plantpatogeniese swamme. Die antagonistiese aksies van hierdie biologiese beheeragente teenoor fitopatogene is gebaseer op die uitskeiding van ekstrasellulêre hidrolitiese ensieme, soos die selwandverterende chitinase ensieme. Nietemin is biologiese beheer nie net tot bio-antagoniste wat natuurlik voorkom beperk nie. Deur die proses van genetiese transformasie kan ander swam- of gisspesies verbeter word om hul eie chitinases of ander antimikrobiese substanse meer effektief te produseer, wat aanleiding sal gee tot kragtige bio-antagoniste. Verskeie tipes chitinases is al in die produksie van swambestande plante ingespan en uitgebreide navorsing is gedoen op die toepassing van 'n reeks chitinases, afkomstig van 'n verskeidenheid van mikroërganismes, as biologiese beheeragente. In teenstelling is baie min bekend oor die antifungiese aktiwiteite van die Saccharomyces cerevisiae chitinase ensiem, wat deur die CTS1-2 geen ge-enkodeer word. Die CTS1-2-geen is in hierdie studie gebruik vir ooruitdrukking in beide gis- en plantuitdrukkingsisteme om die chitinase se vermoë om swamgroei te inhibeer, te ondersoek. Die eerste oorkoepelende oogmerk van hierdie studie het hoë-vlak uitdrukking en optimalisering van sekresie van die CTS1-2-geen in S. cerevisiae behels, met die toekomstige doelwit om 'n rekombinante gis met verbeterde antifungiese eienskappe en met moontlike toepassings as 'n bio-antagonis teen plantpatogeniese swamme te ontwikkel. Die hipotese was dat hoë-vlak uitdrukking en voldoende sekresie voorvereistes vir 'n bio-antagonisras is. Omdié rede is twee sterk promotors en termineerders by hierdie studie ingesluit en is die sekresie van die chitinase-geen geëvalueer deur drie verskillende sekresieseine te toets. Die sekresieseine sluit in: die wilde-tipe CTS1-2 sekresiesein, die S. cerevisiae paringsferomoon a-faktor (MFa1) sekresiesein, en die Trichoderma reesei p-xilanase (XYN2) sekresiesein. Die fosfogliseraat kinase 1 (PGK1) en alkohol dehidrogenase 2 (ADH2) promotors en termineerders is gebruik om hoë-vlak uitdrukking te dryf. Die resultate wat vanaf die analises van die rekombinante giste verkry is, het getoon dat die PGK1 promotor-termineerder konstrukte hoë-vlak CTS1-2-uitdrukkende en chitinase-produserende S. cerevisiae PRY488 rasse opgelewer het. Die vermoë van die verskillende sekresieseine om die ooruitgedrukte chitinase voldoende uit te skei, is geanaliseer, en daar is gevind dat die heteroloë sekresieseine aansienlik meer proteïene na die ekstrasellulêre omgewing geloods het. Dit was dus duidelik dat die MFa1 en XYN2 sekresieseine beter as die wilde-tipe sekresiesein presteer het. Die antifungiese aktiwiteit van die rekombinante chitinases wat deur hierdie konstrukte geproduseer is, is ook in in vitrotoetse teen Botryits cinerea getoets. Die teenwoordigheid van die ensieme het gelei tot 'n aansienlike afname in hife-ontwikkeling, veroorsaak deur ekstreme strukturele skade aan die hifepunte, die hifeselwande, asook die vermoë van die swam om voortplanting- en oorlewingstrukture te vorm. Die ADH2 promotor-termineerderkonstrukte het CTS1-2 transkripte vertoon, maar geen chitinase-aktiwiteite kon in hierdie konstrukte waargeneem word nie. Die redes hiervoor is tot op hede onbekend. Die tweede oogmerk van hierdie studie was om die potensiaal van die gischitinase om swaminfeksie in planta teë te werk, te ondersoek. Die CTS1-2-geen is konstitutief ooruitgedruk in tabakplante, waarin die chitinase na beide die intra- en ekstrasellulêre omgewing geteiken is. Resultate het getoon dat die geregenereerde transgeniese tabaklyne die transgeen stabiel geïntegreer het, transgeenuitdrukking vertoon en dat 'n biologies aktiewe chitinase-ensiem geproduseer is. 'n F1-generasie is aan strawwe toetse onderwerp om weerstand teen B. cinerea te ondersoek. Beide die in vitro en in planta toetse het bevestig dat die gischitinase die plant se verdraagsaamheid teenoor swaminfeksie verhoog het; sommige lyne het siekteweerstand van tussen 65 en 70% getoon. Die plante wat 'n ekstrasellulêre chitinase produseer, word steeds geëvalueer. Interessante resultate word verwag aangaande die effek van die chitinase op die plant se oppervlak met betrekking tot siekteweerstand teen swampatogene. Ten slotte, die gekombineerde stel resultate wat vanaf beide die gis- en plantuitdrukkingstudies verkry is, het die sterk antifungiese effek van gischitinases bevestig. Die gis CTS1-2 kan instrumenteel wees in die ontwikkeling van 'n nuwe generasie gisrasse met verbeterde antifungiese eienskappe. Die ensiem kan ook 'n belangrike rol in genetiese transformasietegnologieë, wat op verbeterde siekteweerstand gemik is, speel.