A HP1076 null mutant has been constructed to provide a better understanding of the biological significance of HP1076 in H. pylori . The DeltaHP1076 mutant displays impaired motility and resistance to the antibiotic drug metronidazole. Using a proteomic study, an overall of 40 differentially expressing proteins involved in metabolism and pH homeostasis for bacterial survival, adhesion for colonization, virulence factor to gastric epithelial cells and antigenic proteins have been identified. The virulence factor, Cag pathogenicity island protein (Cag 26) and urease UreA and UreB are confirmed to have enhanced and reduced expression in null mutants. These findings may provide new insight into the infection of H. pylori. / FliS is an export chaperone that binds to flagellin molecules in cytosol in order to prevent pre-mature polymerization. Disruption of FliS would result in formation of shorter flagella and impaired adhesion ability to epithelial cells. Previous yeast two-hybrid study has identified various FliS associated proteins in H. pylori, but with no known implications. Here, we have demonstrated the interaction of FliS and a hypothetical protein HP1076 by biochemical and biophysical methods. Moreover, HP1076 possesses anti-aggregation ability on insoluble FliS-mutants and chaperone activity. Thus, HP1076 is proposed to be a co-chaperone that promotes the folding and chaperone activity of FliS. FliS is demonstrated to have a broad range of substrate specificity that binds to flagellin and flagellar related proteins which may play a key role in flagellar export system different from other flagellated bacteria. / Helicobacter pylori is a pathogenic bacterium and adheres to the gastric mucosal cells. Chronic infection would lead to gastritis or peptic ulceration and is one of the leading causes of gastric cancer. Formation of functional flagella is essential for infection, that it aids in motility of bacteria and colonization on gastric epithelial cells. The process is complex and involves more than 50 proteins in assembly of structural proteins, regulatory proteins, an export apparatus, a motor and a sensory system. Cytosolic chaperones are required to bind to exported proteins in order to facilitate the export or prevent the aggregation of proteins in cytosol. Divergence is found in flagellar system H. pylori that may account for survival inside gastric environment. / The crystal structures of FliS, HP1076 fragment and FliS/HP1076 complex are determined at 2.7A, 1.8A and 2.7A resolution respectively to provide better understanding of their molecular interactions. FliS consists of four helices and HP1076 consists of helical rich bundle structure with three helices and three beta strands that share similar fold to that of a flagellin homologue, hook-associated protein and FliS, suggesting HP1076 is involved in flagellar system. The FliS/HP1076 complex reveals an extensive electrostatic and hydrophobic binding interface which is distinct from the flagellin binding pocket on FliS. HP1076 stabilizes two alpha helices of FliS and therefore the overall bundle structure. Our findings provide new insights into the flagellar export chaperones and other secretion chaperones in Type III secretion system. / Lam, Wai Ling. / Adviser: An Wing-Ngor. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 223-243). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-10T14:20:09Z No. of bitstreams: 1 Eliane Mayumi Inokuti.pdf: 262975 bytes, checksum: 46e8a0576767e569d0817560c99aae40 (MD5) / Made available in DSpace on 2017-03-10T14:20:09Z (GMT). No. of bitstreams: 1 Eliane Mayumi Inokuti.pdf: 262975 bytes, checksum: 46e8a0576767e569d0817560c99aae40 (MD5) Previous issue date: 2012-07-30 / The fungi Rhizoctonia spp. is an important soilborne plant pathogen. The objective of this study was to evaluate the effectiveness of baits to quantify the saprophytic activity of Rhizoctonia in soil and determine the relationship between saprophytic and pathogenic activities in order to fit an equation of pathogenic activity risk in soils for cowpea and common bean planting. In the evaluation of baits, soils from three locations were packed in trays and infested with an isolate of R. solani (50 mg colonized substrate kg-1 soil). After seven days, soil samples were transferred to gerboxes and sown six baits: beet, cowpea, maize and sorghum seeds, cowpea segment stalks and toothpick segments. After 48 h at 25 ° C, the baits were transferred to the Ko & Hora modified medium. The wood toothpick bait led to the detection of higher levels of saprophytic activity in all soils. The bait toothpick was evaluated against eight isolates and six inoculum densities of R. solani, demonstrating highly effective in all situations. In the analysis of the relationship between saprophytic and pathogenic activities, were used 12 soils collected in areas for cowpea and common bean planting. The saprophytic activity was evaluated using toothpick baits and the pathogenic activity was assessed by the distribution of soils in trays, planting of cowpea seeds and assessment of Rhizoctonia canker severity. There was a significant (P≤0.05) positive correlation (r = 0.7698) between the saprophytic (ATS) and pathogenic (ATP) activities. The regression equation ATP = 1 / (0.5822 to 0.0056 ATS) was estimated with high precision (R2 = 0.9930, P≤0.05), indicating that the risk of pathogenic activity of Rhizoctonia in soils for cowpea and common bean planting can be estimated from the analysis of saprophytic activity. / O fungo Rhizoctonia spp. é um importante fitopatógeno habitante do solo. O objetivo deste trabalho foi avaliar a eficácia de iscas para quantificação da atividade saprofítica de Rhizoctonia no solo e determinar a relação entre atividade saprofítica e atividade patogênica, visando ajustar uma equação de risco de atividade patogênica em áreas destinadas ao plantio de feijão-caupi e feijão-comum. Na avaliação das iscas, solos de três localidades foram acondicionados em bandejas e infestados com um isolado de R. solani (50 mg de substrato colonizado kg-1 solo). Após sete dias, amostras dos solos foram transferidas para caixas gerbox e semeadas seis iscas: sementes de beterraba, feijão-caupi, milho e sorgo, segmentos de talos de feijão-caupi e segmentos de palito de dente. Após 48 h a 25 ºC, as iscas foram transferidas para o meio de Ko & Hora modificado. A isca de palito de dente de madeira propiciou a detecção dos maiores níveis de atividade saprofítica em todos os solos. A isca de palito de dente foi avaliada em relação a oito isolados e seis densidades de inóculo de R. solani, demonstrando elevada eficácia em todas as situações. Na análise da relação entre as atividades saprofítica e patogênica, foram utilizados 12 solos coletados em áreas destinadas ao cultivo de feijão-caupi e feijão-comum. A atividade saprofítica foi avaliada com iscas de palito de dente e a atividade patogênica foi avaliada pela distribuição dos solos em bandejas, plantio de sementes de feijão-caupi e avaliação da severidade da rizoctoniose. Houve correlação positiva (r = 0,7698) significativa (P≤0,05) entre as atividades saprofítica (ATS) e patogênica (ATP). A equação de regressão ATP=1/(0,5822-0,0056 ATS) foi estimada com elevada precisão (R2 = 0,9930; P≤0,05), indicando que o risco de atividade patogênica de Rhizoctonia nos solos destinados ao cultivo de feijão-caupi e feijão-comum pode ser estimado a partir da análise da atividade saprofítica.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-20T14:31:46Z No. of bitstreams: 1 Luis Gustavo Chaves da Silva.pdf: 1253285 bytes, checksum: 0a1b6f21c6f4abf5783394812a777ab7 (MD5) / Made available in DSpace on 2017-03-20T14:31:46Z (GMT). No. of bitstreams: 1 Luis Gustavo Chaves da Silva.pdf: 1253285 bytes, checksum: 0a1b6f21c6f4abf5783394812a777ab7 (MD5) Previous issue date: 2011-02-28 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The anthracnose, fungal disease caused by Colletotrichum gloeosporioides, stands out as an important disease of cashew (Anacardium occidentale), which may lead to losses of over 40% in production. The cashew nut is the most important agricultural product of the state of Ceará, about 350 thousand hectares, generating more than 100 thousand direct/indirect jobs and exports over 150 million dollars in almonds. It is also important for the states of Piauí and Rio Grande do Norte. There are few and outdated research about disease and the needs basic information for programs improvement and integrated management of diseases. By presenting large morphological and genetic variability, it is difficult to generalize basic aspects of the species. The aim of this study was to characterize populations of C. gloeosporioides occurring on cashew trees in various regions of Brazil. Used 220 isolates obtained from cashew leaves, from 22 different areas of the states: Amazonas, Ceará, Maranhão, Paraíba, Pernambuco, Piauí, Rio Grande do North and São Paulo. Proceeded with pathogenic tests and molecular markers species-specific to all isolates. Then a representative from each collection area was selected to perform the other ratings, which are: morphological, molecular, epidemiological and physiological. The species-specific markers β-tubulin and ITS, noted the existence of only one group of 10 isolates of the same area, not being confirmed for C. gloeosporioides or C. acutatum, but morphological characteristics of C. gloeosporioides. Using seven primers ISSR markers and 100 representatives in 22 areas, it was possible identify the formation of four distinct genetic groups of C. gloeosporioides, where the unmarked isolated by ITS or β-Tubulin, not was contained in one or in groups. With the pathogenic test using 5 clones, it was possible to distinguish three groups and another 3 isolates distinct from other. Conclude that the species C. gloeosporioides is prevalent in cashew plantations in the areas studied. / A Antracnose, doença fúngica ocasionada por Colletotrichum gloeosporioides, destaca-se como doença importante do cajueiro (Anacardium occidentale), podendo levar a perdas de mais 40% na produção. A castanha de caju é o produto agrícola mais importante do estado do Ceará, com cerca de 350 mil hectares plantados, geração de mais de 100 mil empregos diretos e indiretos e exportações acima de 150 milhões de dólares em amêndoas. Também é importante para os estados do Piauí e Rio Grande do Norte. Há poucas e desatualizadas pesquisas sobre a doença, havendo a necessidade de informações básicas para os programas de melhoramento e manejo integrado da fitomoléstia. Por apresentar grande variabilidade morfológica e genética, torna-se difícil a generalização de aspectos básicos da espécie. O objetivo deste trabalho foi caracterizar populações de C. gloeosporioides que ocorrem em cajueiros de diversas regiões do Brasil. Foram usados 220 isolados, obtidos de lesões de folhas de cajueiro, provenientes de plantios de 22 áreas distintas dos estados do Amazonas, Ceará, Maranhão, Paraíba, Pernambuco, Piauí, Rio Grande do Norte e São Paulo. Procedeu-se com testes de patogenicidade e marcadores moleculares espécie específicos para todos isolados. Em seguida, um representante de cada área de coleta foi selecionado para realização das demais avaliações, sendo elas: morfológicas, moleculares, epidemiológicas e fisiológicas. Os marcadores espécie específicos ITS e β-Tubulina, constataram a existência de apenas um grupo de 10 isolados de uma mesma área, não sendo confirmados para C. gloeosporioides ou C. acutatum, porém com características morfológicas de C. gloeosporioides. Usando 7 primers ISSR e 100 marcadores nos representantes das 22 áreas, foi possível identificar a formação de 4 grupos genéticos distintos de C. gloeosporioides, onde o isolado não marcado por ITS ou β-Tubulina, não ficou contido em nem um dos grupos. Com o teste de patogenicidade utilizando 5 clones, foi possível diferenciar 3 grupos e mais outros 3 isolados distintos dos demais. Conclui-se que a espécie C. gloeosporioides é prevalente em plantios de cajueiro nas áreas estudas.

Orientador: Antonio Batista Filho / Banca: Carlos Gilberto Raetano / Banca: Edson Luiz Lopes Baldin / Banca: José Eduardo Marcondes de Almeida / Banca: Luis Garrigós Leite / Resumo: A cana-de-açúcar colhida sem queima é uma realidade em todo o Estado de São Paulo e os ataques da cigarrinha-da-raiz da cana estão cada vez mais freqüentes e intensos. O controle biológico desta praga com o fungo Metarhizium anisopliae também vem se desenvolvendo e adquirindo relevada importância. Com isso, este trabalho foi conduzido com os seguintes objetivos: avaliar a produção de conídios de diferentes isolados para o controle da cigarrinha-das-raízes, a partir de dois tipos de arroz; avaliar a eficiência dos isolados selecionados como mais produtivos em populações naturais de Mahanarva fimbriolata, na cultura da cana-de-açúcar colhida mecanicamente e; verificar o manejo da população de M. fimbriolata em áreas de cana-de-açúcar colhidas sem queima da palha, observando a influência da umidade sobre sua ocorrência. Foram avaliados 14 isolados, os quais foram produzidos em arroz tipo 1 e arroz parboilizado, em sacos de polipropileno, incubados em sala climatizada para desenvolvimento do fungo. Avaliou-se a concentração e viabilidade de cada isolado para os dois tipos de arroz. Os isolados que apresentaram maior produtividade foram aplicados em campo para avaliar a patogenicidade dos mesmos à cigarrinha-das-raízes, sendo eles: ESALQ 1037, IBCB 425, IBCB 353, IBCB 410, F 99 e IBCB 333. Foram pulverizados 2 kg/ha de arroz+fungo, contendo 1,0 x 1012 conídios/ha, além do tratamento com o inseticida tiametoxam 250 WG e a testemunha, sem aplicação. As avaliações foram realizadas aos 15, 30, 60, 90 e 120 dias após a aplicação (DAA), observando-se o número de ninfas e adultos de M. fimbriolata vivos, mortos, parasitados ou não, em cada parcela. A partir dos resultados, foi possível observar que os isolados IBCB 410 e F 99 causaram, respectivamente, mortalidades de 66,67 e 33,33% para ninfas, aos 15 DAA. Após 30 DAA, os isolados IBCB 425, IBCB 353 e IBCB 333 ...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The sugar cane is harvested without burning a reality throughout the state of Sao Paulo and the attacks of the leafhopper-root cane are increasingly frequent and intense. Biological control this pest with the Metarhizium anisopliae also has been developing and acquiring increasing importance. Therefore, this work was conducted to evaluate the production of conidial selected for the control of root speatlebug in two types of rice, evaluate the efficiency of the isolates selected as the most productive in natural populations of M. fimbriolata, the culture of cane sugar harvested mechanically, monitor the population of M. fimbriolata in areas of sugarcane harvested without burning the straw, and the influence of temperature and humidity on its occurrence. We evaluated 14 isolates, which were produced in rice type 1 and parboiled rice in polypropylene bags, incubated in a room for fungal growth and were evaluated the concentration and viability of each isolate for both types of rice. The isolates that had higher yields were applied in the field to assess the pathogenicity of the same root speatlebug, namely: ESALQ 1037, IBCB 425, IBCB 353, IBCB 410, F 99 and IBCB 333. Were sprayed 2 kg / ha of rice + fungus, containing 1,0 x 1012 conidia / ha, in addition to treatment with the insecticide tiametoxam 250 WG and the control. Evaluations were performed at 15, 30, 60, 90 and 120 days after the application, noting the number of nymphs and adults of M. fimbriolata alive in each plot, and the number of nymphs and adults dead, infected and non-parasitized. From the results, it was observed that isolates IBCB 410 and F 99 caused mortality to nymphs of 66.67 and 33.33%, respectively, at 15 days after application. After 30 days of spraying, the isolates IBCB 425, IBCB 353 and IBCB 333 had efficiencies... (Complete abstract click electronic access below) / Doutor

Xylella fastidiosa (Xf) é o agente causal de doenças em várias culturas economicamente importantes. Recentemente foi identificada como sendo o agente causal da clorose variegada dos citros (CVC), doença que representa um grande problema para os citricultores paulistas, pois vem causando perdas econômicas significativas. Trata-se de uma bactéria gram-negativa, fastidiosa e restrita ao xilema das plantas hospedeiras. Um fato observado na interação Xf - citros, é que plantas infectadas por Xf podem demonstrar sintomas da doença um longo tempo após a infecção. Isto faz supor que a expressão de fatores de patogenicidade e/ou virulência ocorre somente após a população de Xf na planta atingir altas densidades celulares. Como fatores de patogenicidade/virulência de algumas bactérias são regulados por sensores de quorum, seria interessante determinar se esse mecanismo de regulação gênica ocorre em Xf, e quais genes bacterianos são regulados por densidade celular. O objetivo deste trabalho foi verificar se genes de patogenicidade putativos de Xf são modulados pela densidade celular em meio de cultura. Para tal, Xf foi cultivada em PW líquido, células no início da fase exponencial (baixa densidade celular) e na fase estacionária (alta densidade celular) foram coletadas, e RNA total foi extraído. Fragmentos de 20 genes de Xf relacionados à patogenicidade putativos foram transferidos em arranjos ordenados para membranas de náilon, e hibridizados, sob condições de alta estringência, com sondas complexas de primeiras fitas de cDNA marcadas com fosfatase alcalina. A detecção foi feita por quimioluminescência. Sob as condições experimentais utilizadas, os genes fur (XF-2344), gumC (XF-2369), protease-serina (XF-1851) e rsmA (XF-0125) tiveram sua expressão significativamente suprimida sob condições de alta densidade celular (p < 0,05). Já a expressão de rpfF (XF-1115) foi induzida significativamente (p < 0,06) sob condições de alta densidade celular. Os genes gumD (XF-2367), gumJ (XF-2362) e rpfA (XF-0290) tiveram a expressão detectada somente sob condições de alta densidade celular, enquanto a expressão de rpfB (XF-0287) foi detectada somente sob condições de baixa densidade celular. A expressão de celulase (XF0818), mdoH (XF-1623), pluxR (XF-0972), protease-RE (XF-1823), rpoN (XF-1408), xpsK (XF-1523) e xpsL (XF-1524) não foi afetada significativamente pela densidade populacional em meio PW, enquanto a expressão dos genes luxR/UHPA (XF-2608), poligalacturonase (XF-2466), rpfC (XF-1114) e rsmB (XF-0928) não foi detectada em ambas as condições. Usando RT-PCR, os transcritos de todos os genes, exceto gumC, foram detectados sob condições de baixa e alta densidade celular, indicando que a expressão de alguns genes relacionados à patogenicidade putativos em meio de cultura é muito baixa. Os resultados obtidos sugerem que os EPS são sintetizados por Xf predominantemente em condições de alta densidade celular em meio PW. Adicionalmente, os dados sugerem que Xf sintetiza uma molécula sinal similar à de Xcc em meio PW sob condições de alta densidade celular, e que a disponibilidade de ferro pode ser importante para a regulação gênica em condições de alta densidade celular. É possível que o padrão de expressão gênica observado possa ser alterado em função do meio de cultivo ou in planta. / Xylella fastidiosa (Xf) is the causal agent of several economically important plant diseases. Recently, Xf has been identified as the causal agent of citrus variegated chlorosis (CVC), a disease that represents a major economic problem for citrus growers in the São Paulo State. Xf is a gram-negative, fastidious and xylem-limited bacterium. Based on the fact that plants may show disease symptoms a long time after infection in Xf-citrus interaction, we hypothesize that the expression of pathogenicity/virulence factors in Xf occurs after the bacterial population reach high cell densities. Since pathogenicity/virulence factors of some bacteria are quorum-sensing regulated, it would be interesting to determine whether this mechanism of genetic regulation occurs in Xf, and which genes are regulated by cell-density. In order to determine whether Xf putative pathogenesis-related genes are modulated by cell-density in culture media, Xf was grown in liquid PW and cells were sampled at early log (low cell density) and stationary phase (high cell density) and total RNA was extracted. Fragmentos of 20 putative pathogenicity-related genes from Xf were hybridized on ordered arrays nylon membranes to alkaline phosphatase-labeled first strand cDNA from low and high celldensity conditions as probes, at high stringency conditions. Detection was performed using chemiluminescence. Our results indicate that the following putative pathogenicity-related genes are significantly suppressed (p < 0.05) at high cell-density conditions: fur (XF-2344), gumC (XF-2369), serine-protease (XF-1851) and rsmA (XF-0125). The expression of rpfF (XF-1115) was significantly induced (p < 0.06) at high cell-density conditions. Expression of gumD (XF-2367), gumJ (XF-2362) and rpfA (XF-0290) was detected only at high cell-density conditions, whereas expression of rpfB (XF-0287) was detected only at low cell-density conditions. The expression of cellulase (XF0818), mdoH (XF-1623), pluxR (XF-0972), protease-RE (XF-1823), rpoN (XF-1408), xpsK (XF-1523) and xpsL (XF-1524) was not affected by the Xf population density in PW medium, whereas expression of luxR/UHPA (XF-2608), polygalacturonase (XF-2466), rpfC (XF-1114) and rsmB (XF-0928) was not detected under our experimental conditions. Using RT-PCR, transcripts for all genes, except gumC, were detected under low or high cell densities, indicating that the expression of some putative pathogenesis-related genes in culture medium is very low. Our results suggest that EPS may be synthesized by Xf mainly at high cell density conditions in PW medium. Additionally, our data suggest that Xf may synthesize a signal molecule similar to the Xcc in PW medium at high cell density conditions, and that iron availability may be important for gene regulation at high cell density conditions. It is possible that gene expression patterns observed under our experimental conditions may be altered with the culture medium used or when Xf grows in planta.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2015-02-26T13:54:49Z No. of bitstreams: 2 Tese - Cecília do Nascimento Peixoto - 2006.pdf: 6636718 bytes, checksum: b112892e216b4dc189c4e9cb3dd28f4c (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2015-02-26T13:55:31Z (GMT) No. of bitstreams: 2 Tese - Cecília do Nascimento Peixoto - 2006.pdf: 6636718 bytes, checksum: b112892e216b4dc189c4e9cb3dd28f4c (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-02-26T13:55:31Z (GMT). No. of bitstreams: 2 Tese - Cecília do Nascimento Peixoto - 2006.pdf: 6636718 bytes, checksum: b112892e216b4dc189c4e9cb3dd28f4c (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2006-03-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The morphological and cultural characteristics of G. graminis var. graminis isolates from rice and grasses were studied. The fungus forms two types of mycelia, dark macrohyphae that join laterally to form runner hyphae or rhizomorphs and hyaline or infectious microhyphae, as well as fan shaped hyphae characteristic of the pathogen. Pigmented and lobed hyphopodia on lower leaf sheaths were formed both under natural conditions and artificial inoculations of plants. The perithecia containing asci and ascospores were found on leaf sheaths lesions on field samples. The perithecia were produced on leaf sheaths of inoculated plants as well as on detached sterilized leaf sheaths and on culture medium, potato-dextrose-agar (PDA). Hyphae and hyphopodia were formed from germination tubes of ascospores, and the hyphae under moist stress conditions produced chlamydospores which were initially hyaline and later attained dark color. The culture of Ggg, was characterized by fluffy aerial mycelium, white in the initial stages of growth and later with age, the colony color changed from dirty-white or mouse gray to almost black. The marked diagnostic colony characteristic of whorled appearance was the curling back of marginal hyphae. The amount and time of formation of perithecia varied among the isolates tested. The virulence test conduced with 20 isolates of rice and grasses, showed differences in aggressiveness both on rice seedlings and adult plants. In general, isolates from rice were more aggressive on rice than isolates from grasses. The test with four levels of inoculum (0, 5, 1.0, 2.0, 4.0 g per plant of autoclaved sorghum grains) and two plant ages showed that 60-day old were more susceptible than 35-day old plants. The spontaneous infection of healthy plants was observed in the greenhouse indicating the role of ascospores in the dissemination of black sheath rot in rice. Furthermore, the pathogenicity of ascospores of Ggg on rice plants was confirmed by inoculations tests. Six fields of upland rice were surveyed in the advanced stages of maturation for the incidence of black sheath rot. The disease incidence on tillers, under natural conditions of infection, ranged from 68 to 100%. The pathogenicity of 20 isolates retrieved from rice and grasses were studied. All isolates were pathogenic to rice and grasses such as baranyard grass (Echinochloa crusgalli), fountain grass (Pennisetum setosum) signal grass (Brachiaria sp), crab grass (Digitaria horizontalis), plantain signal grass (Brachiaria plantaginea), indian goose grass (Eleusine indica) and southern sandbur (Cenchrus echinatus). Winter cereals such as wheat, oat, rye, barley and triticale as well as sorghum, corn, and millet exhibited different degrees of susceptibility to the isolate Ggg-a 01. Significant differences were observed in relation to characteristic symptoms on the culm, lesion height, number of tillers or dead plants, presence of characteristic mycelium, fan shaped hyphae, production of hyphopodia and perithecia. The formation of perithecia was not observed on leaf sheaths of inoculated plants of millet, sorghum, southern sandbur and maize. All inoculated wheat plants were killed indicating more susceptibility than other cereals. The resistance of 58 upland rice genotypes were tested in the greenhouse, utilizing rice isolate Ggg-a 01. Of the genotypes assessed, the lesion height of SCIA16 and SCIA08 was significantly shorter compared to the highly susceptible genotype CNAS10351. The progress and dissemination of black sheath rot in rice was studied during two years under field conditions in savanna sensu lato ‘cerrado’. The central line of each plot was inoculated with isolate of Ggg to establish the infection foci. The soil was infested with four levels of inoculum (5.0, 10.0, 20.0 and 40.0 g of autoclaved sorghum grains containing mycelium / 40 cm) and main tiller of plants (4, 8, 16 and 32, tillers per plot/ 40 cm) were inoculated with 2.0 cm-long detached leaf sheaths containing perithecia by insertion between the culm and leaf sheath of the tiller. There was no significant effect of inoculum level on the disease severity obtained by soil infestation with mycelium as well as the plants infected with perithecia. However, the total area under disease progress curve was significantly smaller for plant infection with perithecia than for soil infestation by mycelium, during 2002/2003. The evaluation of disease incidence for the analysis of gradients was based on infected tillers in 1.6 square meter area, five lines on either side of the inoculated 40 cm-long central line. The analysis according models of Gregory (1968) and Kiyosawa & Shiyomi (1972) showed the existence of gradients in the first year, both for levels of inoculum of soil infection by mycelium and plant infection with perithecia. In the second year (2004/2005), there was no well defined gradient for all the treatments. The disease progress was not affected by inoculum levels on soil or plant infections. Monomolecular model was found more adequate in tests conduced under greenhouse conditions while the models of Gompertz and monomolecular, better described the disease progress under field conditions. / Foram estudadas características morfológicas e culturais de isolados de Gaeumannomyces graminis var. graminis provenientes de arroz e capins. O fungo se estabelece formando dois tipos de hifas: macrohifas, escuras, superficiais que se juntam lateralmente e formam cordões ou rizomorfas e microhifas, hialinas ou infecciosas, que penetram no hospedeiro. Forma também hifas em leque sobre as bainhas, a partir de macrohifas, que caracterizam o patógeno. Houve a formação de hifopódios lobados e pigmentados em bainhas, tanto em condições naturais como em inoculações. Observou-se peritécios contendo ascas e ascósporos, característicos do fungo, nas bainhas sobre as lesões em amostras coletadas no campo. Através de inoculação artificial, foram produzidos peritécios em bainhas de plantas, em bainhas destacadas e esterilizadas e em meio de cultura de batata-dextrose-ágar (BDA). Foram formadas hifas e hifopódios a partir de tubos germinativos dos ascósporos e as hifas crescidas em condições de estresse hídrico produziram clamidósporos, inicialmente hialinos e, posteriormente, de coloração escura. O micélio de Ggg, geralmente de aspecto aéreo fofo, é branco no início do crescimento, com variação de cor com a idade, do branco cinza ao marrom oliváceo e quase preto. Uma característica marcante é a aparência espiralada das macrohifas escuras nas bordas da colônia. Entre os isolados testados houve variação na quantidade de peritécios bem como na época de formação. Os testes de virulência realizados com vinte isolados provenientes de arroz e capins apresentaram diferenças em agressividade, tanto em plântulas quanto em plantas de arroz. Em geral, os isolados provenientes de arroz foram mais agressivos em arroz que os isolados de capins. O teste com quatro níveis de inóculo (0,5, 1,0, 2,0, e 4,0 g de inóculo por planta, multiplicado em grãos de sorgo autoclavados) e duas idades de plantas mostrou que as plantas inoculadas aos 60 dias após o plantio foram mais suscetíveis do que aquelas inoculadas aos 35 dias, requerendo menor nível de inóculo para a infecção. A patogenicidade de ascósporos de Ggg em plantas de arroz foi comprovada, bem como o papel dos ascósporos na disseminação do mal-do-pé do arroz. A incidência de mal-do-pé em lavouras de arroz de terras altas nas condições naturais de infecção variou de 68 a 100% de perfilhos infectados, entre seis lavouras avaliadas em fase avançada de maturação. Foi estudada também a patogenicidade dos vinte isolados de Ggg obtidos, provenientes de arroz e capins. Todos os isolados foram patogênicos a arroz e aos capins: capim arroz (Echinochloa crusgalli), capim avião (Pennisetum setosum), capim braquiária (Bachiaria sp.), capim digitaria (Digitaria horizontalis), capim marmelada (Brachiaria plantaginea), capim pé-degalinha (Eleusine indica) e capim timbete (Cenchrus echinatus). Os cereais de inverno, trigo, aveia, centeio, cevada e triticale, bem como sorgo, milho, e milheto apresentaram diferentes graus de suscetibilidade ao isolado Ggg-a 01. As diferenças foram significativas quanto a sintomas típicos na base do colmo, altura de lesão escura na bainha, número de perfilhos ou plantas mortas, presença de micélio característico, hifas em leque e produção de hifopódios e peritécios. Não foram observados peritécios em milheto, sorgo, timbete e milho e a maior suscetibilidade foi apresentada pelo trigo, com a morte de todas as plantas inoculadas. Foi testada a resistência de 58 genótipos de arroz de terras altas, utilizando o isolado Ggg-a 01 proveniente de arroz, em casa-de-vegetação. Entre os genótipos avaliados, SCIA16 e SCIA08 apresentaram altura de lesão significativamente menor, sendo considerados resistentes em relação ao genótipo CNAS10351, altamente suscetível. O progresso e disseminação do maldo- pé do arroz foram estudados durante dois anos, em condições de campo em solo de cerrado. Utilizou-se delineamento experimental de blocos completos ao acaso e quatro repetições. Cada parcela foi constituída de dezenove linhas de sete e cinco metros, respectivamente no primeiro e segundo ano, com espaçamento de quarenta centímetros. Foi inoculada a linha central de cada parcela com isolado de Ggg para estabelecer os focos de disseminação da doença. O solo foi infestado com micélio em quatro níveis de inóculo (5,0, 10,0, 20,0 e 40,0 gramas de grãos de sorgo autoclavados e colonizados com micélio / 40 cm da linha) e perfilhos foram inoculados (4, 8, 16 e 32 perfilhos / 40 cm da linha) com pedaços de bainhas de arroz de dois centímetros de comprimento, contendo peritécios e micélio, inseridos entre o colmo e a bainha. Não houve efeito de níveis de inóculo na severidade da doença, tanto para micélio no solo quanto para peritécios na planta, nos dois anos de experimento. Entretanto, a área total sob a curva de progresso da doença na safra 2002/2003 foi significativamente menor nas plantas infectadas com peritécios, do que nas plantas infectadas através de infestação do solo com micélio. A avaliação de incidência da doença para análise do gradiente foi baseada nos perfilhos contados em 1,6 metros quadrados, compostos de cinco linhas de quarenta centímetros de cada lado da fonte de inóculo, na linha central. A análise de gradiente, conforme modelos de Gregory (1968) e Kiyosawa & Shiyomi (1972) mostrou existência de gradiente no primeiro ano, tanto para níveis de inóculo quanto para os focos provenientes dos dois tipos de inóculo. No segundo ano (2004/2005), não houve gradiente definido para os tratamentos testados. O progresso da doença não foi afetado pelos níveis, tanto na infecção do solo com micélio, quanto na planta com peritécios. Em teste de ajuste de modelo matemático para estudos epidemiológicos, o modelo monomolecular foi o mais apropriado para estudos de mal-do-pé do arroz nas condições de casa-de-vegetação e os modelos de Gompertz e monomolecular são os que melhor descrevem o progresso da doença, nas condições de campo.

Based on matching analysis between different EBV strains, we found two nucleotide variations in miR-BART21 and four nucleotide changes in miR-BART22. Interestingly, two nucleotide variations upstream of mature miR-BART22 likely favor its biogenesis by Drosha/DGCR8 processing and we experimentally confirmed this augmentation by in-vitro Drosha digestion, and thus may underline the high and consistent expression of miR-BART22 in NPC tumors. / Infection with the Epstein-Barr virus (EBV) is a strong predisposing factor in the development of nasopharyngeal carcinoma (NPC). Many viral gene products including EBNA1, LMP1 and LMP2 have been implicated in NPC tumorigenesis, although the de novo control of these viral oncoproteins remain largely unclear. / MicroRNAs (miRNAs) are a class of small, non-coding RNAs with a size around 18--24 nucleotides with significant roles in regulating gene expression by either transcriptional silencing or translational suppression. As gene regulators, recent miRNA studies have emphasized the contribution of aberrant miRNA expression in cancer development. The recent discovery of EBV encoded viral miRNAs (ebv-miRNAs) in lymphoid malignancies has prompted us to examine the NPC-associated EBV miRNAs. In this study, we have systematically examined the NPC associated EBV genome for viral-encoded miRNA expression. By constructing small cDNA libraries from a native EBV positive NPC cell line (C666-1) and a xenograft (X2117), we screened about 3000 clones and detected several small EBV fragments, within which two novel ebv-miRNAs in the BARTs region were identified. These two newly identified miRNAs, now named miR-BART21 and miR-BART22, were proven to be abundantly expressed in most NPC samples by both Northern blot and QRT-PCR analysis. / Taken together, this thesis shows that two newly identified EBV-encoded miRNAs are highly expressed in latent EBV infection in NPC. Frequent expression of miR-BART22 can be explained partially by a specific EBV strain that is associated with NPC in our locality. Our findings emphasize the role of miR-BART22 in modulating LMP-2A expression. Because LMP-2A is a potent immunogenic viral antigen that is recognized by the cytotoxic T cells (CTLs), down-modulation of LMP-2A expression by mir-BART22 may permit escape of EBV-infected cells from host immune surveillance. / We attempted to predict the potential viral and cellular targets of miR-BART21 and miR-BART22 by public available computer programs, miRanda and RNAhybrid. A number of potential cellular mRNA targets were suggested, although many failed to be validated by luciferase reporter assay. However, we found a putative miR-BART22 binding site in the LMP2A-3'UTR. Although the LMP-2A transcript is consistently detected in NPC, only 6 out of 26 (23%) primary NPC tumors show weak LMP-2A expression by immunohistochemistry (IHC). The expression levels of miR-BART22 and LMP-2A mRNA have also been determined in eleven of these tumors. Interestingly, the LMP-2A mRNA expression level did not directly correlate with protein expression, and relatively low expression levels of miR-BART22 miRNA were observed in all 3 LMP-2A positive-primary tumors. The suppressive effect of miR-BART22 on LMP-2A was also experimentally validated by a series of dual luciferase reporter assays using reporter constructs containing the putative or mutated recognition site at the LMP-2A 3'UTR. By co-transfection of different amounts of miR-BART22 with the LMP-2A-3'UTR expression vector in reporter assay, we confirmed that miR-BART22 suppressed the LMP-2A protein level in a dose-dependent manner. Furthermore, transfection of miR-BART22 into HEK293 cells that had been stably transfected with pcDNA3.1-LMP-2A, which contains a complete LMP-2A ORF and 3'UTR, readily suppressed levels of the LMP-2A protein. / Lung, Wai Ming Raymond. / Adviser: To Ka Fai. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 197-226). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Li, Sai Kam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 162-179). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese (摘要） --- p.ii / Acknowledgements --- p.iii / Table of Content --- p.iv / Abbreviations --- p.xi / List of Figures --- p.xiv / List of Tables --- p.xvii / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Hepatitis B Virus / Chapter 1.1.1 --- General information --- p.1 / Chapter 1.1.2 --- Classification --- p.2 / Chapter 1.1.3 --- Virus life cycle and genome --- p.3 / Chapter 1.1.4 --- Hepatitis B virus X protein (HBx) --- p.7 / Chapter 1.2 --- Enigmatic functions of HB --- p.x / Chapter 1.2.1 --- HBx as a transactivator --- p.10 / Chapter 1.2.2 --- HBx as a cell cycle regulator --- p.12 / Chapter 1.2.3 --- HBx as an apoptosis modulator --- p.13 / Chapter 1.3 --- Etiology of HBV-mediated hepatocarcinogenesis --- p.14 / Chapter 1.4 --- Clinical mutants of HBV --- p.16 / Chapter 1.5 --- Hypothesis and aims of the research --- p.16 / Chapter 1.6 --- Basis of Tet-On system --- p.18 / Chapter CHPATER 2 --- EXPERIMENT MATERIALS / Chapter 2.1 --- Cell culture / Chapter 2.1.1 --- Cell-lines --- p.21 / Chapter 2.1.2 --- Culture medium --- p.22 / Chapter 2.1.3 --- Culture medium supplements --- p.23 / Chapter 2.2 --- Reagents for subcloning / Chapter 2.2.1 --- Reagents for polymerase chain reaction (PCR) --- p.24 / Chapter 2.2.2 --- Reagents for restriction enzyme digestion --- p.24 / Chapter 2.2.3 --- Reagents for ligation --- p.25 / Chapter 2.2.4 --- Reagents for electrophoresis --- p.25 / Chapter 2.2.5 --- Reagents for E. coli DH5a preparation --- p.25 / Chapter 2.2.6 --- Materials for bacterial culture work --- p.27 / Chapter 2.3 --- Reagents for subcellular localization study / Chapter 2.3.1 --- Reagents for cell staining --- p.28 / Chapter 2.3.2 --- Reagents for mounting slides --- p.29 / Chapter 2.3.3 --- Materials for site-directed mutagenesis --- p.29 / Chapter 2.4 --- Reagents for cell cycle analysis and cellular proliferation / Chapter 2.4.1 --- Reagents for cell cycle analysis --- p.29 / Chapter 2.4.2 --- Reagents for cellular proliferation study --- p.30 / Chapter 2.5 --- Reagents for protein expression study / Chapter 2.5.1 --- Cell lysis buffer --- p.30 / Chapter 2.5.2 --- Reagents for SDS-PAGE --- p.30 / Chapter 2.5.3 --- Reagents for Western blot --- p.33 / Chapter 2.5.4 --- Antibodies --- p.34 / Chapter 2.6 --- Reagents for gene expression study / Chapter 2.6.1 --- Reagents for RNA extraction --- p.36 / Chapter 2.6.2 --- Reagents for first strand cDNA synthesis --- p.37 / Chapter 2.6.3 --- Reagents for real-time PCR --- p.37 / Chapter 2.7 --- Reagents for establishment of Tet-On inducible stable cell-lines / Chapter 2.7.1 --- Reagents for MTT assay --- p.38 / Chapter 2.7.2 --- Reagents for selection of stable clones --- p.38 / Chapter 2.8 --- Vectors used in the project / Chapter 2.8.1 --- Vectors for subcellular localization study --- p.39 / Chapter 2.8.2 --- Vectors for establishment of Tet-on inducible cell-lines --- p.39 / Chapter 2.9 --- Primers used in the project / Chapter 2.9.1 --- Primers used for subcloning --- p.42 / Chapter 2.9.2 --- Primers used for site-directed mutagenesis --- p.43 / Chapter 2.9.3 --- Primers used in real-time chain polymerase reaction --- p.43 / Chapter CHAPTER 3 --- RESEARCH METHODS / Chapter 3.1 --- Subcloning of HBx and mutant genes into a green fluorescence protein (GFP) expression vector / Chapter 3.1.1 --- Amplification of HBxWt，HBxΔC44 and HBxAN60 genes --- p.45 / Chapter 3.1.2 --- Purification of PCR products --- p.46 / Chapter 3.1.3 --- Restriction enzyme digestion --- p.47 / Chapter 3.1.4 --- Ligation of gene products with pEGFP-C 1 vector --- p.47 / Chapter 3.1.5 --- Preparation of chemically competent bacterial cells E. coli strain DH5α --- p.47 / Chapter 3.1.6 --- Transformation of the ligation product into competent cells --- p.48 / Chapter 3.1.7 --- PCR confirmation of successful ligation --- p.48 / Chapter 3.1.8 --- Small scale preparation of bacterial plasmid DNA --- p.49 / Chapter 3.1.9 --- DNA sequencing of the cloned plasmid DNA --- p.50 / Chapter 3.1.10 --- Large scale preparation of target recombinant plasmid DNA --- p.50 / Chapter 3.2 --- Subcellular localization pattern study / Chapter 3.2.1 --- Cell transfection --- p.51 / Chapter 3.2.2 --- Mitochondria and nucleus staining --- p.52 / Chapter 3.2.3 --- Epi-fluorescence microscopy --- p.53 / Chapter 3.2.4 --- Analysis of fluorescence images --- p.53 / Chapter 3.2.5 --- In vitro site-directed mutagenesis --- p.53 / Chapter 3.3 --- Cell cycle phase analysis with flow cytometry / Chapter 3.3.1 --- Cell transfection --- p.55 / Chapter 3.3.2 --- Cell staining --- p.55 / Chapter 3.3.3 --- Flow cytometry --- p.55 / Chapter 3.4 --- Cellular proliferation quantification by BrdU proliferation assay / Chapter 3.4.1 --- Cell transfection --- p.57 / Chapter 3.4.2 --- BrdU ELISA measurement --- p.57 / Chapter 3.5 --- Protein expression / Chapter 3.5.1 --- Cell lysate collection --- p.58 / Chapter 3.5.2 --- Quantification of protein samples --- p.59 / Chapter 3.5.3 --- SDS-PAGE --- p.59 / Chapter 3.5.4 --- Western blot --- p.60 / Chapter 3.5.5 --- Western blot luminal detection --- p.60 / Chapter 3.6 --- Gene expression / Chapter 3.6.1 --- Primer design --- p.61 / Chapter 3.6.2 --- Cell transfection --- p.61 / Chapter 3.6.3 --- RNA extraction --- p.61 / Chapter 3.6.4 --- Reverse transcription for first strand complementary DNA (cDNA) --- p.63 / Chapter 3.6.5 --- Quantitative real-time PCR --- p.63 / Chapter 3.7 --- Establishment of Tet-On inducible stable cell-lines / Chapter 3.7.1 --- Subcloning of HBx gene into pTRE2 vector --- p.64 / Chapter 3.7.2 --- Construction of WRL68/Tet-On stable cell-lines --- p.64 / Chapter 3.7.3 --- Construction of WRL68/Tet-On HBx and mutants expression cell-lines --- p.68 / Chapter 3.7.4 --- Characterization of Tet-On gene expression monoclones --- p.69 / Chapter 3.8 --- Statistical analyses --- p.70 / Chapter CHPATER 4 --- STUDY ON MITOCHONDRIA TARGETING / Chapter 4.1 --- Establishment of pEGFP-Cl-HBx and mutants constructs --- p.71 / Chapter 4.2 --- Transactivation C-terminus domain is essential for granular localization --- p.73 / Chapter 4.3 --- Wild-type HBx localizes in mitochondria --- p.76 / Chapter 4.4 --- C-terminal transactivation domain is sufficient for mitochondria targeting --- p.79 / Chapter 4.5 --- Mapping of the HBx region crucial for mitochondria targeting --- p.81 / Chapter 4.6 --- The 111-117 amino acids in HBx do not work as a signal peptide --- p.83 / Chapter 4.7 --- Site-directed mutagenesis identifies the key amino acid at 115 in HBx for mitochondrial targeting --- p.85 / Chapter CHAPTER 5 --- CELL PROLIFERATION AND REGULATION / Chapter 5.1 --- Alteration of S-phase distribution in cell cycle --- p.88 / Chapter 5.2 --- Analysis of DNA synthesis using BrdU proliferation ELISA --- p.92 / Chapter 5.3 --- Differential molecular regulation of cell cycle --- p.94 / Chapter 5.4 --- Regulation of the mRNA expression levels of cyclin-dependent kinases inhibitors p2raf/cipl and p27kipl --- p.98 / Chapter CHAPTER 6 --- TRANSACTIVATION AND RAS/RAF/MAPK PHOSPHORYLATION / Chapter 6.1 --- Determination of p53-dependency of p21、vaf/cipl expression --- p.101 / Chapter 6.2 --- Ras/Raf/MAPK pathway activation by HBx variants / Chapter 6.2.1 --- ERK1/2 phophorylation by HBx variants --- p.104 / Chapter 6.2.2 --- ERK inhibition blocks the regulation effect on p53Wt and p21waf/cipl --- p.107 / Chapter 6.3 --- Transactivation activity on oncogenes/ proto-oncogenes / Chapter 6.3.1 --- Effect on c-myc (NM´ؤ002467) mRNA expression --- p.109 / Chapter 6.3.2 --- Effect on RhoC (NM_017744) and Rabl4 (NM´ؤ016322) mRNA expression --- p.112 / Chapter CHAPTER 7 --- CONSTRUCTION OF TET-ON INDUCIBLE CELL-LINES / Chapter 7.1 --- Establishment of WRL/Tet-On monoclonal cell-lines Page / Chapter 7.1.1 --- Determination of geneticin selection dosage --- p.116 / Chapter 7.1.2 --- Selection of the best WRL/TOn clone using luciferase assay --- p.118 / Chapter 7.2 --- Establishment of inducible WRL/TOn/Gene monoclonal cell-lines / Chapter 7.2.1 --- Determination of hygromycin selection dosage --- p.120 / Chapter 7.2.2 --- Selection of positive WRL/TOn/Gene clones with viral genes --- p.122 / Chapter 7.3 --- Characterization of TOXDC1 cell-line / Chapter 7.3.1 --- Cell morphology --- p.125 / Chapter 7.3.2 --- Growth pattern of TOXDC1 --- p.126 / Chapter 7.3.3 --- HBxAC44 induced p21waf/cipl mRNA expression --- p.127 / Chapter 7.3.4 --- Doxycycline concentration dependent HBxAC44 expression in TOXDC1 --- p.129 / Chapter CHAPTER 8 --- DISCUSSION / Chapter 8.1 --- Selection of cell model / Chapter 8.1.1 --- Selection of cell models --- p.130 / Chapter 8.1.2 --- Selection of truncation mutant --- p.131 / Chapter 8.2 --- Differential sub-cellular localization of HBx and its variants / Chapter 8.2.1 --- Mechanisms of mitochondria targeting --- p.132 / Chapter 8.2.2 --- Mitochondria as site of HBx-induced apoptosis --- p.134 / Chapter 8.2.3 --- Stimulation of calcium release from mitochondria by wild-type HBx --- p.135 / Chapter 8.3 --- Cell cycle distribution profiling and its regulations / Chapter 8.3.1 --- Cell cycle pattern and cell proliferation --- p.136 / Chapter 8.3.2 --- Differential cell cycle molecular pathway activation --- p.138 / Chapter 8.4 --- Ras/Raf/MAPK mediated transactivation by HBxWt and its mutants / Chapter 8.4.1 --- p53-mediated p21waf/cipl expression --- p.142 / Chapter 8.4.2 --- ERK-mediated p21waf/cipl and wild-type p53 mRNA expression --- p.143 / Chapter 8.4.3 --- Regulation of oncogenes/ proto-oncogenes expression --- p.147 / Chapter 8.5 --- General discussions on differential effects of HBxWt and HBxAC44 --- p.149 / Chapter 8.6 --- Establishment of Tet-On/HBxAC44 cell-line TOXDC1 --- p.153 / Chapter 8.7 --- Conclusions --- p.154 / Chapter 8.8 --- Future Prospects / Chapter 8.8.1 --- From mitochondria targeting to calcium signaling --- p.157 / Chapter 8.8.2 --- Construction of a complete cell cycle regulation pathway --- p.158 / Chapter 8.8.3 --- Elucidation of the transcriptional transactivation regulation --- p.159 / Chapter 8.8.4 --- To make the best use of the Tet-on stable cell-line TOXDC1 --- p.159 / Chapter 8.8.5 --- Study with other carboxy-terminal truncation mutants --- p.160 / Chapter 8.8.6 --- In vivo study --- p.160 / REFERENCES --- p.162

O fungo Moniliophthora perniciosa é o agente causal da vassoura de bruxa no cacaueiro (Theobroma cacao L.). Três biótipos distintos (biótipos -C, -L e -S) são reconhecidos de acordo com a especificidade quanto ao hospedeiro. O presente estudo teve como objetivo a análise da diversidade genética com o uso de marcadores microssatélites de 134 isolados dos biótipos -C, -L e -S de M. perniciosa coletados principalmente na Amazônia Brasileira e áreas de cultivo. A diversidade genética foi associada com virulência e/ou agressividade dos isolados a acessos diferenciais (suscetíveis ou resistentes) de T. cacao. Os biótipos -S e -L apresentaram uma diversidade gênica e genotípica superior em comparação com o biótipo-C. A população do biótipo-C com maior diversidade genotípica foi a do Acre, seguida pelo Oeste do Amazonas, ambas correspondendo a áreas em que se encontra cacaueiro nativo no Brasil. A população com menor diversidade genotípica foi a da Bahia, que corresponde a uma área onde a presença de M. perniciosa foi registrada mais recentemente, no final da década de 1980. Dos 134 isolados, 83 correspondem a genótipos multilocos únicos, sendo encontrados apenas dois indivíduos com genótipos idênticos para o biótipo-S, e nenhum para o biótipo-L. No biótipo-C foram identificados 61 genótipos multilocos em 111 isolados coletados em áreas de ocorrência natural e cultivo de cacau. Os dados de similaridade genética corroboram que o biótipo-C e também o -S que são homotálicos evoluíram de um biótipo heterotálico, possivelmente o biótipo-L. As populações do biótipo-C do estado do Pará e Leste do Amazonas compartilham ancestrais comuns em Ji-Paraná (RO) e Assis Brasil (AC), enquanto que a região sob a influência do rio Amazonas possui outra ascendência, que seria em Atalaia do Norte (Alto Solimões). Os genótipos multilocos da Bahia exibiram origens análogas as da população do Baixo Amazonas, com ascendência em Ji-Paraná (RO) e Alenquer (PA). Na avaliação de agressividade de M. perniciosa, a concentração do inóculo se mostrou determinante para a manifestação dos sintomas, com o aumento de plântulas com sintomas à medida que aumenta a concentração de basidiósporos. Dentre as progênies avaliadas, \'PA 195 x CAB 214\' apresentou menor proporção de plântulas com sintomas. Na inoculação com isolados de M. perniciosa oriundos do Acre e Amazonas, a proporção de plântulas com sintomas foi superior quando inoculadas com o isolado de Tabatinga (AM). O genótipo de cacaueiro que apresentou uma reação diferenciada foi o \'CAB 214\', para o qual nenhuma plântula apresentou sintomas quando inoculada com o isolado de Marechal Thaumaturgo (AC). Com o uso de microssatélites os genótipos multilocos de Tabatinga (AM) e Marechal Thaumaturgo (AC) foram identificados em grupos distintos. Na análise de treze genes de patogenicidade induzidos pela limitada disponibilidade de N, o gene 88KD foi o que demonstrou o maior número de transcritos acumulados. Os isolados que apresentaram uma mesma tendência em seus genes mais expressos foram Tabatinga (AM) e Óbidos (PA) que apesar de possuírem origens geográficas distintas, foram identificados no mesmo grupo na análise com microssatélites / The fungus Moniliophthora perniciosa is the causal agent of witches\'broom in cacao (Theobroma cacao L.). Three different biotypes (C-; S-; and L-biotypes) are recognized according to host specificity. The present study aimed to analyze the genetic diversity using microsatellite markers for 134 isolates of the C-, L- and S- biotypes of M. perniciosa collected mainly in the Brazilian Amazon and areas of cultivation. Genetic diversity was associated with virulence and/or aggressiveness of isolates in differential accesses (susceptible or resistant) of T. cacao. The L- and S- biotypes showed a higher genetic and genotype diversity compared with C-biotype. Of the 134 isolates, 83 corresponded to unique multilocus genotypes, and found only two individuals with identical genotypes for the S-biotype, and none for the L-biotype. In the C-biotype were identified 61 multilocus genotypes in 111 isolates collected in areas of natural occurrence and cultivation of cocoa. The genetic similarity data corroborate that the C- and S- biotypes that are apparently homothallic evolved from a heterothallic biotype, possibly L-biotype. The populations of C-biotype of the state of Para and Amazonas East share common ancestors, in Ji-Paraná (RO) and Assis Brazil (AC), while the region under the influence of the Amazon River has another descent that would be in the Atalaia do Norte (Upper Solimões). The multilocus genotypes from Bahia showed a similar origin of the population of the Lower Amazon, with ancestry in Ji-Paraná (RO) and Alenquer (PA). In the evaluation of aggressiveness of M. perniciosa, the inoculum concentration proved to be decisive for the manifestation of symptoms, with the increase of seedlings with symptoms as increases the concentration of basidiospores. Among the progenies, \'PA 195 x CAB 214\' showed a lower proportion of seedlings with symptoms. In inoculation with M. perniciosa from Acre and Amazonas, the proportion of seedlings with symptoms was higher when inoculated with the isolate from Tabatinga (AM). The genotype of cocoa that had a differentiated reaction was the \'CAB 214\', for which no plants showed symptoms when inoculated with the isolate Marechal Thaumaturgo (AC). With the use of microsatellite, the multilocus genotypes of Tabatinga (AM) and Marechal Thaumaturgo (AC) were identified in different groups. In the analysis of thirteen genes of pathogenicity induced by the limited availability of N, 88KD gene showed the highest number of transcripts. The isolates that showed a similar trend in their more expressed genes were Tabatinga (AM) and Óbidos (PA) that despite having different geographical origins were identified in the same group in the analysis with microsatellite

Colletotrichum species are considered one of the most economically important plant pathogens. They cause many losses in tropical, subtropical and temperate regions affecting a wide range of plant species. In tropical and subtropical regions C. gloeosporioides and C. acutatum are associated with significant losses on pre and post-harvest anthracnoses. There are still many features to understand about Colletotrichum biology and its systematics. The accurate identification of species involved with each anthracnose is of high relevance to establish management strategies to control the disease. Although the great advances on Colletotrichum systematics, species complex such as C. gloeosporioides and C. acutatum are used in a broad sense in Brazil. These complexes were recently investigated and showed to be highly genetic and geographic variable. In this study multigene analysis were carried out based on ITS, GAPDH, CHS-1, TUB2 and CAL or HIS3 partial sequences for strains of C. gloesporioides and C. acutatum complexes collected from fruit crops in Brazil. Strains from different countries and exepitypes and others sequences available on GenBank from the species accepted on both complexes were added on dataset. Six strains from C. gloeosporiodes complex and five for C. acutatum were selected based on multigene phylogeny to investigate the pathogenicity through inoculations on detached fruit. The multigene phylogenies showed the occurrence of species in Brazil related to those complexes with a high genetic variability among them. The phylogeny of Brazilian strains belonging to the C. gloeosporioides complex showed that C. siamense represents the most genetically and host-specific variable clade. In contrast, C. asianum clade grouped only strains isolated from mango. The strains from this clade used on pathogenic test were not able to infect avocado and one of the strains caused symptoms only on mango. All strains from Brazil grouped in one subclade within the C. fructicola clade and seem to represent a genetically distinct group. C. theobromicola is first reported causing anthracnose on acerola fruit. Three new species (C. polyphialidicum, C. paranaense and C. pruni) belonging to the C. acutatum complex were recognized and their morphologic descriptions were provided. The pathogenic test for the strains in the C. acutatum complex showed their cross infection ability, but in some cases the larger lesions were produced on the original host. Most brazilian strains from C. acutatum complex grouped in one subclade within the C. nymphaeae clade and seem to be genetically distinct. / Fungos do gênero Colletotrichum são considerados um dos mais importantes economicamente na Fitopatologia. Espécies desse gênero são encontradas amplamente disseminadas e estão associadas a diversas espécies de plantas hospedeiras. Em regiões tropicais e subtropicais, espécies dos complexos C. gloeosporioides e C. acutatum são a principal causa das antracnoses em pré e póscolheita de frutos e consequentemente causam significantivas perdas. Ainda há muitos aspectos a serem compreendidos sobre o gênero Colletotrichum, como a biologia e a sistemática. A acurada identificação das espécies associadas a antracnoses é de suma importância para o estabelecimento de estratégias de controle. No entanto, apesar dos grandes avanços na sistemática desse gênero, complexos de espécies como aquelas citadas acima são tratados de modo genérico no Brasil. Estes complexos de espécies foram recentemente estudados e considerados geneticamente e geograficamente variáveis. Neste sentido, o presente trabalho teve como objetivo caracterizar isolados de Colletotrichum spp. associados a diferentes frutos e regiões do Brasil por meio de análise filogenética. Para análise multilocus, foram utilizadas sequências parciais dos genes ITS, GAPDH, CHS-1, TUB2 and CAL ou HIS3. Sequências de espécies-tipos disponíveis no GenBank e de isolados de diferentes países foram adicionadas ao conjunto de dados. Com base nos resultados obtidos por meio de filogenia multilocus, seis isolados do complexo C. gloeosporiodes e cinco do complexo C. acutatum foram escolhidos para testes de patogenicidade cruzada. A espécie C. siamense, pertencente ao complexo C. gloeosporioides, foi a mais variável geneticamente e quanto ao hospedeiro de origem. Diferentemente, apenas isolados obtidos de manga se agruparam no clado C. asianum. Isolados agrupados neste clado não infectaram abacate e um dos isolados (CPC 20969) causou sintomas apenas em manga. No clado C. fructicola, isolados coletados no Brasil se agruparam em um subclado e parecem representar um grupo geneticamente distinto. A espécie C. theobromicola é relatada pela primeira vez em acerola. Foram identificadas três novas espécies, C. polyphialidicum, C. paranaense e C. pruni, pertencentes ao complexo C. acutatum. Isolados brasileiros agrupados no clado C. nymphaeae parecem representar um grupo geneticamente distinto, todos se agruparam em um subclado. Isolados do complexo C. acutatum utilizados no teste de patogenicidade provocaram sintomas nos hospedeiros testados, porém, em algumas inoculações, as lesões foram maiores no hospedeiro de origem.