Um dos principais componentes envolvidos no processo de divisão celular bacteriana é FtsZ, uma proteína homóloga à tubulina eucariótica. FtsZ polimeriza no interior da célula formando um anel ao qual dá-se o nome de anel Z, responsável pelo recrutamento de diversas outras proteínas de divisão, formando o divisomo. Como meio de sobrevivência sob condições adversas, alguns procariotos, como B. subtilis, podem sofrer um tipo de diferenciação celular que forma um organismo em estado latente, conhecido como esporo. A primeira etapa da formação do esporo é a mudança da posição do anel Z para mais próximo a um dos pólos da célula, produzindo duas células com tamanhos diferentes. SpoIIE é uma proteína fosfatase integral de membrana, que se localiza especificamente no septo assimétrico de uma célula em processo de esporulação. Além de um papel na ativação do fator de transcrição de esporulação σF, SpoIIE se liga a FtsZ e a auxilia na formação do septo assimétrico. Para definirmos a região de FtsZ responsável pela interação com SpoIIE, neste trabalho foram realizados ensaios de duplo-híbrido utilizando vetores com domínios de ativação e de ligação ao DNA do fator de transcrição GAL4 de levedura fusionados a diferentes porções de FtsZ, bem como a SpoIIE. Esses experimentos não forneceram informações sobre interação entre essas proteínas, já que através deles não foi possível reproduzir o resultado positivo descrito na literatura. Como alternativa ao duplo-hibrido para identificarmos o sítio de interação entre as duas proteínas, criamos uma triagem genética capaz de identificar mutantes de FtsZ que não interagem com SpoIIE, fazendo uso de uma biblioteca de mutantes de FtsZ já disponível no laboratório. Foi padronizada uma técnica de microscopia em larga escala em placas de 96 poços, que permitiu a triagem de mais de mil de mutantes de FtsZ, em busca de um em que SpoIIE-GFP induzido não localizasse no anel Z em célula vegetativa. Porém todos os mutantes triados ainda localizavam SpoIIE-GFP. Paralelamente, foi realizada uma triagem de supressão, utilizando como ponto de partida um mutante de SpoIIE que perdeu capacidade de interagir com FtsZ e buscando mutações em FtsZ que reestabelecessem a interação com SpoIIE mutante. Foram triados cerca de 35000 mutantes nesse ensaio, dentre os quais dezoito apresentaram o fenótipo esperado para um supressor. No entanto, todos os candidatos selecionados tratavam-se de falsos-positivos. O motivo que leva esses candidatos a apresentarem o fenótipo esperado sem reestabelecer a interação entre as duas proteínas ainda é desconhecido. A fim de confirmar se não haveria outras proteínas do divisomo responsáveis por intermediar a interação entre FtsZ e SpoIIE, foram feitos experimentos de co-localização de FtsZ e SpoIIE na ausência de DivIB e FtsA. Em ambos os casos SpoIIE ainda localiza no divisomo, descartando a possibilidade de que DivIB e FtsA sejam mediadores da interação FtsZ-SpoIIE. Por fim, foram realizados experimentos de co-localização de SpoIIE com mutantes de FtsZ previamente identificados em outros experimentos em nosso laboratório. Nesse experimento foi identificado que a expressão de SpoIIE-GFP induzida por IPTG é capaz de reestabelecer a frequência de divisão no mutante FtsZ-R376T, que normalmente é deficiente na formação de divisomos. Esse resultado reforça a idéia de que essas proteínas interagem diretamente, e sugere que SpoIIE é capaz de reestabelecer a atividade de FtsZ em um mutante que apresente falhas na polimerização. / One of the major components involved in bacterial cell division is FtsZ, a protein homologous to the eukaryotic tubulin. FtsZ polymerizes inside the cell forming a ring to which is given the name Z ring, wich is responsible for the recruitment of several other proteins division, forming the divisome. As a means of survival under adverse conditions, some prokaryotes such as B. subtilis may undergo a type of cell differentiation that results in an organism in a latent state, known as a spore. The first stage of the spore formation is to change the Z ring position closer to the poles of the cell, producing two cells of different sizes. SpoIIE is an integral membrane phosphatase protein, which is specifically located in the septum of an asymmetric cell in sporulation process. In addition to a role in the activation of the sporulation transcription factor σF, SpoIIE binds to FtsZ and assists in the formation of the asymmetric septum. To define the FtsZ region responsible for interaction with SpoIIE, in this work we performed tests using two-hybrid vectors with activation and DNA binding domains of the yeast transcription factor GAL4 fused to different portions of FtsZ and SpoIIE. These experiments did not provide information on the interaction between these proteins, since through them it was not possible to reproduce the positive results reported in the literature. As an alternative to the two-hybrid to identify the site of interaction between the two proteins, we created a genetic screening that can identify FtsZ mutants that cannot interact with SpoIIE, using a library of FtsZ mutants already available in the laboratory. We standardized a large scale microscopy using 96-well plates, allowing the screening of over a thousand mutants of FtsZ in search of a induced SpoIIE-GFP which would no longer localize at the vegetative cell Z ring. However, all the screened mutants still localized SpoIIE-GFP. In parallel, we performed a screening of suppression, using as a starting point a SpoIIE mutant that lost the ability to interact with FtsZ and searching for mutations in FtsZ that would reestablish interaction with the SpoIIE mutant. We screened approximately 35,000 mutants in this essay, eighteen of which showed the phenotype expected for a suppressor. However, all selected candidates were false positives. The reason why such candidates do show the expected phenotype without reestablishment of the interaction between the two proteins is still unknown. In order to confirm whether there would be other divisiome proteins responsible for mediating the interaction between FtsZ and SpoIIE, co-localization experiments were made using FtsZ and SpoIIE in the absence of DivIB and FtsA. In both cases SpoIIE still located in divisome, ruling out the possibility that DivIB and FtsA are essencial mediators of the SpoIIE-FtsZ interaction. Finally, co-localization experiments were carried out with SpoIIE and FtsZ mutants previously identified in other experiments in our laboratory. In this experiment it was identified that the expression of IPTG-induced SpoIIE-GFP is able to restore the division frequency in the FtsZ-R376T mutant, which normally is deficient in the formation of divisomes. This result reinforces the idea that these proteins interact directly, and suggests that SpoIIE is able to restore the activity of FtsZ in a mutant that presents defect in polymerization.

Submitted by Leticia Schettini (leticia@ufrrj.br) on 2016-09-21T15:12:59Z No. of bitstreams: 1 2011 - Camila Pinheiro Nobre.pdf: 2714138 bytes, checksum: bc436d879ddfded703d4d5fea9f9943c (MD5) / Made available in DSpace on 2016-09-21T15:12:59Z (GMT). No. of bitstreams: 1 2011 - Camila Pinheiro Nobre.pdf: 2714138 bytes, checksum: bc436d879ddfded703d4d5fea9f9943c (MD5) Previous issue date: 2011-02-17 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior-CAPES / The aim of this study was to observe the germination, production of glomalin and monitor development of species of mycorrhizal fungi (AMF) of the germplasm bank of Embrapa in root organ culture (ROC) of basil and bryophytes in vitro, analyzing their interaction with the hosts and the influence of the culture medium enriched with humic acids on fungal growth and bryophyte Lunularia cruciata. For this, some AMF species were selected and had their glomerospores extracted and subjected to surface disinfection process, placed in water-agar medium and temperature-controlled chamber to germinate. A germination test was conducted for 15 days, and the results were analyzed by ANOVA and Tukey test applied to 5% probability. Species with germinated glomerospores (Gigaspora margarita, Glomus manihots, Scutellospora heterogama and Glomus proliferum) were placed in ROC of purple basil where they had their growth observed until 100 days after inoculation. Also as part of the characterization of AMF species it was quantified the level of glomalin in the samples of multiplication and the results were subjected to analysis of variance and Scott-Knott test at 5% probability. In the second chapter it was investigated the effect of mycorrhizal association in ROC of purple basil, and in the third chapter the influence of different concentrations of humic acid and association with growth of Lunularia cruciata (area and length). The results were submitted to ANOVA and Tukey test at 5% probability. Scutellospora heterogama was the species with higher germination rates of glomerosporos, followed by Gigaspora margarita. The species of Glomus sporulated after formation of symbiosis. The amount of glomalin produced by different AMF was distinct, especially in total glomalin fraction. Different AMF species did not show difference in efficiency to promote development of Ocimum basilicum transformed roots. The growth of basil transformed roots in the MSR was extended from 15 days after inoculation with mycorrhizal fungi. The usage of humic acids in the culture medium in concentrations of 20 and 80 mg CL-1 enhanced growth of bryophyte L. cruciata, and its association with mycorrhizal fungi, as well as promoted the highest number of spores of Gl. proliferum. The association L. cruciata and AMF was characterized as mutualistic, since both had advantages in growth and sporulation. Gigaspora margarita and Glomus proliferum increased growth of Lunularia cruciata. / O objetivo do trabalho foi observar a germina??o e produ??o de glomalina e acompanhar desenvolvimento de esp?cies de fungos micorr?zicos arbusculares (FMA) do banco de germoplasma da Embrapa em ra?zes geneticamente modificadas de manjeric?o e em bri?fitas in vitro. Ainda, avaliar sua intera??o com os hospedeiros e a influ?ncia de meio de cultura enriquecido com ?cidos h?micos no crescimento do fungo e da bri?fita Lunularia cruciata. Para isso algumas esp?cies de FMAs foram selecionadas e tiveram seus glomerosporos extra?dos e submetidos ao processo de desinfesta??o superficial, colocados em meio Agar?gua e c?mara com temperatura controlada para germinar. Realizou-se teste de germina??o por 15 dias e os resultados foram submetidos a an?lise de vari?ncia e aplicado teste de Tukey ? 5% de probabilidade. Esp?cies com glomerosporos germinados (Gigaspora margarita, Glomus manihots, Scutellospora heterogama e Glomus proliferum) foram colocadas em ra?zes modificadas de manjeric?o roxo onde tiveram seu crescimento observado at? 100 dias ap?s a inocula??o. Ainda como parte da caracteriza??o de esp?cies de FMAs foi realizado a quantifica??o dos teores de glomalina nas amostras de multiplica??o sendo os resultados submetidos a an?lise de vari?ncia e aplicado teste de Scott-Knott ? 5% de probabilidade. No segundo cap?tulo foi verificado o efeito da associa??o FMAs em ra?zes modificadas de manjeric?o roxo e no terceiro cap?tulo a influ?ncia da associa??o ?cido h?mico em diferentes concentra??es, bri?fita Lunularia cruciata (?rea e comprimento) e FMAs. Os resultados foram submetidos ? an?lise de vari?ncia e teste de Tukey a 5% de probabilidade. Scutellospora heterogama foi a esp?cie com maiores taxas de germina??o de glomerosporos, seguida da Gigaspora margarita. As esp?cies de Glomus esporularam logo ap?s a forma??o da simbiose. A quantidade de glomalina produzida pelos diferentes FMAs foi distinta, em especial na fra??o glomalina total. As diferentes esp?cies de FMAs n?o apresentaram distin??o na efici?ncia de promover o desenvolvimento das ra?zes transformadas de Ocimum basilicum. O crescimento de ra?zes transformadas de manjeric?o em meio MSR foi ampliado a partir dos 15 dias ap?s a inocula??o de fungos micorr?zicos. O uso de ?cidos h?micos no meio de cultura em concentra??es de 20 e 80 mg C.L-1 incrementou o crescimento da bri?fita Lunularia cruciata e sua associa??o com fungos micorr?zicos arbusculares, assim como promoveram a maior esporula??o de Gl. proliferum. A associa??o Lunularia cruciata e FMAs foi caracterizada como mutualista j? que ambos apresentaram benef?cios em crescimento e esporula??o. Gigaspora margarita e Glomus proliferum promoveram maior crescimento de Lunularia cruciata.

Saccharomyces cerevisiae is an excellent experimental model organism to study various biological processes owing to its versatile genetics, biochemistry, and standard laboratory conditions. S. cerevisiae shows distinct biological responses under nutritional starvation conditions. S. cerevisiae undergoes dimorphic transition from a unicellular yeast form to a multicellular pseudohyphae (Gimeno et al., 1992) under nitrogen starvation, but in the complete absence of a fermentable carbon source, it undergoes gametogenesis called sporulation (Mitchell, 1994). While the signal transduction cascades and regulatory controls under nutritional starvation conditions are studied to great extent, the role of S. cerevisiae core RNA polymerase II (pol II) is not much understood. S. cerevisiae core RNA pol II consists of 12 subunits (Woychik and Hampsey, 2002), which is organized into a ten-subunit core and the Rpb4/7 subcomplex (Edwards et al., 1991). Rpb4/7 subcomplex is known to play important roles in stress survival (Choder 2004; Sampath and Sadhale, 2005.). S. cerevisiae rpb4 null diploid strains show reduced sporulation levels but exhibits a predisposition to pseudohyphal morphology (Pillai et al., 2003). Overexpression of Rpb7 partially rescues some of these defects (Sharma et al., 1999; Sheffer et al., 2001). Rpb7 is a highly conserved protein but Rpb4 is the least conserved amongst all RNA pol II subunits at the sequence level. Rpb4 and Rpb7 also affect different cellular functions, which are not directly dependent on each other. (a) Relative levels of RNA pol II subunits Rpb4 and Rpb7 differentially affect starvation response in Saccharomyces cerevisiae S. cerevisiae rpb4 null diploid strains show reduced sporulation levels as compared to wild type but exhibits pseudohyphal predisposition. Overexpression of RPB7 partially rescues the sporulation defect but results in an exaggeration of the pseudohyphae phenotype. We generated S. cerevisiae strains expressing different levels of Rpb4 and Rpb7 proteins in the same strains and analyzed their effect on sporulation and pseudohyphal morphology. We observed that sporulation is dependent on Rpb4 because sporulation level gradually increases with an increase in the Rpb4 protein level in the strain. Rpb7 reduces sporulation level but enhances pseudohyphal exaggeration in a dose-dependent manner. Rpb4 is dominant over Rpb7 in both the starvation responses because strain expressing an equimolar ratio of Rpb4 and Rpb7 protein exhibits RPB4+ phenotypes. (b) Domainal organization of Saccharomyces cerevisiae Rpb7 orthologs reflects functional conservation Rpb7 orthologs are known in eukaryotes and archaebacteria. The primary structure of Rpb7 is conserved. We chose Rpb7 orthologs from Candida albicans, Schizosaccharomyces pombe and Homo sapiens sapiens to investigate whether Rpb7 orthologs are also functionally conserved. We observed that all the orthologs tested are functionally conserved because they can complement the absence of RPB7 in S. cerevisiae. However, we uncovered functional differences amongst Rpb7 orthologs with respect to its function in rpb4 null strain and ess1 ts strain. Furthermore, we made N and C-terminal chimeric RPB7 constructs from these orthologs with S. cerevisiae Rpb7. These chimeras also can replace ScRpb7 in S. cerevisiae. However, functional differences were observed with each chimera pair in rpb4 null strain and ess1 ts strain, showing that the N and C-terminal domains of Rpb7 protein can be genetically dissected. The genetic observation on the domainal organization of Rpb7 orthologs is strengthened by the crystal structure of Rpb7 (Armache et al., 2005), which shows that Rpb7 is structurally organized into an N terminal RNP domain and a C terminal OB fold domain. (c) The Rpb7 subunit of Candida albicans RNA polymerase II induces lectin-mediated flocculation in Saccharomyces cerevisiae The Rpb7 ortholog of C. albicans is a conserved functional ortholog of ScRpb7. We observed that CaRpb7 induces Ca2+-dependent flocculation and agar-invasive growth in S. cerevisiae. CaRpb7 overexpression induces very high transcript levels of FLO1 and FLO11. We believe that the observed flocculation and agar-invasive phenotypes are due to Flo1 and Flo11 respectively, because Flo1 and Flo11 contribute mainly to cell-cell adhesion while Flo11 contributes mainly to cell-substrate adhesion (Verstrepen and Klis, 2006; Lo et al., 1998; Guo et al., 2000). Pathway analysis revealed that CaRpb7-induced flocculation is dependent on Mss11 transcriptional activator. Two-hybrid analysis revealed that CaRpb7 does not physically interact with transcriptional repressors known to repress FLO gene transcription, however genetic analysis revealed that CaRpb7 is epistatic to the repressor Sfl1. Rpb7 orthologs possess conserved domains with potential RNA binding ability (Orlicky et al., 1999) and ScRpb7 is known to play in mRNA stability (Lotan et al., 2007). The possibility of CaRpb7 specifically affecting the stability of FLO gene transcripts is being pursued.

Faithful chromosome segregation is essential for all living organisms. Bacterial chromosome segregation utilizes highly conserved directional SpoIIIE/FtsK translocases to move large DNA molecules between spatially separated compartments. These translocases employ an accessory DNA-interacting domain (gamma) that dictates the direction of DNA transport by recognizing specific DNA sequences. To date it remains unclear how these translocases use DNA sequence information as a trigger to expend chemical energy (ATP turnover) and thereby power mechanical work (DNA movement). In this thesis, I undertook a mechanistic study of directional DNA movement by SpoIIIE from the Gram-positive model bacterium Bacillus subtilis. Specifically, I was interested in understanding the information transfer within the protein from sequence recognition, to ATP turnover, and ultimately to chromosome translocation. How do DNA sequences trigger directional chromosome movement?

Nitrogen (N) fertilization is known to influence the susceptibility of many plants to a variety of diseases. In the case of diseases caused by Botrytis cinerea, the role of N fertilization appears to be variable, with high levels either fostering or reducing severity depending on the studies. To test whether this variability could be due to possible differences in the host plants, inoculum pressure or in the behavior of different strains of the pathogen, studies were carried out to investigate the effect of different N fertilization regimes on the susceptibility of tomato and lettuce to six isolates of B. cinerea. Possible epidemiological effects of N fertilization through the sporulation of the pathogen and on the pathogenicity of resulting secondary inoculum were also investigated on tomato. Plants were grown in a soil-less drip-irrigation system. Differential N nutrition ranging from 0.5 to 30 mM NO3- was applied for the last four weeks prior to inoculation on the leaves (lettuce) or on leaf pruning wounds (tomato) and incubation of the plants in conditions conducive to disease development. On the tomato stems, disease onset was delayed and overall severity was lower for all isolates on plants with higher N inputs, regardless of inoculum concentration. However, the rate of stem lesion expansion was differentially affected depending on the strains, decreasing with increasing N fertilization levels for the more aggressive isolates, while increasing for the less aggressive isolates.In contrast with tomato, high N fertilization increased disease severity on lettuce for all isolates tested. On tomato plant tissue, sporulation of B. cinerea decreased significantly with increasing N fertilization up to 15-30 mM NO3- and the pathogenicity of the spores was significantly influenced by the nutritional status of their production substrate. It was highest for spores produced on plants with very low or very high N fertilization (0.5 or 30 mM NO3-) and lowest for those from plants with moderate levels of N fertilization. Plant fertilization also strongly affected the efficacy of two biocontrol agents (Trichoderma atroviride and Microdochium dimerum) to protect pruning wounds of tomato against B. cinerea. The highest levels of protection were obtained with high N fertilization and related to a delay in symptom development on the stems, sometimes associated with a slowdown in lesion expansion. Histological studies showed that the decrease in disease severity at high N fertilization was associated to structural alteration of Botrytis mycelial cells. In the presence of a biocontrol agent, the effect on the pathogen was further associated to vacuolisation, glycogen deposition and mycelial cell death. Hypotheses to explain these results are discussed in light of the possible physiological effects of nitrogen fertilization on nutrient availability for the pathogen in the host tissue and of possible production of defense metabolites by the plant. These results also open new possibilities for including the manipulation of N fertilization as a tool for the integrated protection of vegetable crops

LIMA, Ticiana de Brito. Cultivo da alga marinha vermelha Solieria filiformis (Kützing) P.W. Gabrielson: textura de géis aquosos e lácteos. 2012. 71 f. Dissertação (Mestrado em Bioquímica) - Universidade Federal do Ceará, Fortaleza-CE, 2012. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-07-13T13:40:06Z No. of bitstreams: 1 2012_dis_tblima.pdf: 3568499 bytes, checksum: d48f3a061d05e6c3a9b0df95c1bf6115 (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-07-14T22:56:09Z (GMT) No. of bitstreams: 1 2012_dis_tblima.pdf: 3568499 bytes, checksum: d48f3a061d05e6c3a9b0df95c1bf6115 (MD5) / Made available in DSpace on 2016-07-14T22:56:09Z (GMT). No. of bitstreams: 1 2012_dis_tblima.pdf: 3568499 bytes, checksum: d48f3a061d05e6c3a9b0df95c1bf6115 (MD5) Previous issue date: 2012 / The red seaweed Solieria filiformis, abundant in Ceará, biossintetiza, among other polysaccharides, phycoloide called carrageenan of great commercial importance due to its properties thickeners, stabilizers and gelling agents. In Brazil, exotic species of marine algae are being grown on a commercial scale in order to meet the demand of industrial phycoloide. The aim of this study was to analyze the texture of aqueous gels made with milk and carrageenan extracted from seaweed S. filiformis, cultivated in Ceará through sporulation natural technique, in order to establish a comparison between these gels and those prepared with commercial carrageenans (iota and kappa carrageenan). The algae were grown through sporulation natural, where monthly PVC structures (25 mm) in size 25 x 44 cm, each containing a single nylon cord (10 mm) of 23 m length were kept at depths of 1 and 2 m over a period of nine months. For extraction of carrageenan were collected at both depths seaweed samples S. filiformis backstage with higher and lower biomass, some for the rainy season (January and April) and dry (October and November). Initially, the algae were dried, crushed in electric grinder and subjected to aqueous extraction (1.5% m / v), stirring every 20 minutes in a water bath at 90 °C for 4 h. The homogenates were filtered through nylon cloth, the waste discarded. The filtrates were subjected to filtration on a vacuum funnel sintered plate, and carrageenans obtained were frozen, lyophilized and weighed. The highest yields were obtained from carrageenan seaweed collected from the scenes that have accumulated during the dry spores (35.6% - November 2M to 45.3% - October 1M), while the algae that accumulated during the rainy season had the lowest incomes (29 6% - April 1M to 33.8% - April 2M). In contrast, levels of total sugars ranged between 37.6 and 50.5% and the content of carrageenan sulfate ranged from 21.6 to 33.3% according to the levels already checked to carrageenans of the iota type. The texture of aqueous gels in the presence of CaCl2 and 0.1% milk prepared with carrageenan S. filiformis and commercial concentrations of 0.5 and 1.5%, were analyzed for firmness, adhesiveness, cohesiveness, springiness, gumminess and chewiness. All samples with carrageenan derived from S. filiformis backstage obtained values of firmness, gumminess and chewiness than those found for ι-carrageenan commercial. Backstage November had the highest values in the parameter of firmness for aqueous gels and compared to ι-carrageenan commercial dairy. Adhesiveness, elasticity and cohesiveness showed values close to those found for ι-carrageenan commercial. The results for firmness and chewiness of κ-carrageenan were superior when compared to commercial car ι-carrageenan and S. filiformis. The gels formulated with milk obtained results strongly higher than those formulated with a solution of calcium chloride 0.1% due to interactions between the proteins and the milk carrageenan. With the data obtained it can be concluded that the use of carrageenan S. filiformis derived by sporulation natural cultivation is capable of forming gels when compared to stronger ι-carrageenan commercial could generate savings through the use of lower concentrations of the carrageenan commercial, to obtain the desired firmness of a product. / A alga marinha vermelha Solieria filiformis, abundante no litoral cearense, biossintetiza, dentre outros polissacarídeos, um ficocolóide denominado carragenana de grande importância comercial devido às suas propriedades espessantes, estabilizantes e gelificantes. No Brasil, espécies exóticas de algas marinhas vêm sendo cultivadas em escala comercial com o objetivo de suprir a demanda industrial desse ficocolóide. O objetivo desse trabalho foi analisar a textura de géis aquosos e lácteos elaborados com ι-carragenana extraída da alga S. filiformis, cultivada no litoral cearense através da técnica de esporulação natural, com a finalidade de estabelecer um comparativo entre esses géis e aqueles elaborados com carragenanas comerciais (ι e κ carragenanas). As algas foram cultivadas através de esporulação natural, onde mensalmente estruturas de PVC (25 mm) de tamanho 25 x 44 cm, contendo cada uma única corda de nylon (10 mm) de 23 m de comprimento foram mantidas em profundidades de 1 e 2 m durante um período de nove meses. Para extração da ι-carragenana foram coletadas em ambas profundidades amostras da alga S. filiformis dos bastidores que apresentaram maior e menor biomassa, determinadas para a estação chuvosa (Janeiro- 26,2 Kg e Abril- 46,5 Kg) e seca (Outubro- 53,0 Kg e Novembro- 31,7 Kg). Inicialmente, as algas foram secas, trituradas em moinho elétrico e submetidas à extração aquosa (1,5% m⁄v), sob agitação a cada 20 minutos, em banho-maria a 90 ºC durante 4 h. Os homogenatos foram filtrados em tecido de nylon, os resíduos descartados. Os filtrados foram submetidos a uma filtração à vácuo em funil de placa sinterizada, e as carragenanas obtidas foram congeladas, liofilizadas e pesadas. Os maiores rendimentos de ι-carragenana obtidos foram das algas coletadas dos bastidores que acumularam esporos no período seco (35,6%- Novembro 2M a 45,3%- Outubro 1M), enquanto as algas que acumularam no período chuvoso apresentaram os menores rendimentos (29,6%- Abril 1M a 33,8%- Abril 2M). Já os teores de açúcares totais variaram entre 37,6 e 50,5% e os teores de sulfato das carragenanas variaram de 21,6 a 33,3%, de acordo com os teores já verificados para carragenanas do tipo ι. A textura dos géis aquosos na presença de CaCl2 0,1% e lácteos preparados com as ι- caragenanas de S. filiformis e caragenanas comerciais nas concentrações de 0,5 e 1,5%, foram analisados quanto a firmeza, adesividade, coesividade, elasticidade, gomosidade e mastigabilidade. Todas as amostras com ι-carragenanas de S. filiformis dos bastidores obtiveram valores de firmeza, gomosidade e mastigabilidade superiores aos encontrados para ι-carragenana comercial. Os bastidores de novembro das amostras com ι-carragenanas de S. filiformis obtiveram os maiores valores no parâmetro de firmeza para os géis aquosos e lácteos quando comparados aos géis comerciais. Adesividade, elasticidade e coesividade mostraram valores próximos aos encontrados para ι-carragenana comercial. Os resultados obtidos para firmeza e mastigabilidade da κ-carragenana foram superiores quando comparados a ι-carragenana de S. filiformis e ι-carragenana comercial. Os géis formulados com leite reconstituído obtiveram resultados de firmeza superiores aos formulados com solução de cloreto de cálcio 0,1%, devido às interações entre as proteínas do leite e a ι-carragenana. Com os dados obtidos pode-se concluir que a utilização da ι-carragenana de S. filiformis oriunda de cultivo por esporulação natural é capaz de formar géis mais firmes quando comparada a ι- carragenana comercial, podendo gerar economia através da utilização de concentrações de carragenana inferiores a comercial, para se obter a firmeza desejada para um produto.

Made available in DSpace on 2014-06-11T19:28:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-05-15Bitstream added on 2014-06-13T20:58:17Z : No. of bitstreams: 1 rosa_j_me_jabo.pdf: 154567 bytes, checksum: 0d2897a42007e37a9a5f314b75f0f0f0 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) / Objetivou-se o ajuste de metodologia e seleção de genótipos de guandu para resistência a Macrophomina phaseolina a partir de material obtido pela Embrapa Pecuária Sudeste, e verificar o desenvolvimento micelial e esporulação do fungo em meios de cultura. O trabalho foi conduzido em casa de vegetação na UNESP/Jaboticabal no período de agosto de 2004 a dezembro de 2005. Para o ajuste de metodologia e seleção de genótipos resistentes ao fungo as sementes foram submetidas a escarificação com lixa d'água e inoculação artificial através do método de exposição das mesmas ao patógeno por diferentes períodos, que variaram de O a 72 horas. Foram avaliadas porcentagem de plantas sobreviventes e massa fresca. Já para o crescimento micelial e esporulação do fungo foi utilizado o método de sobreposição de discos de diferentes hospedeiros no meio de cultura. A escarificação das sementes contribuiu para a penetração do fungo nas mesmas o período de 24h de exposição das sementes ao fungo são suficientes para detectar diferenças no grau de resistência dos genótipos. Os genótipos mais resistentes são g167-97, g124-95, g27-94, g40-95, g154-95, g127-97 e g9m-97, e os mais suscetíveis são g48-95, g123-95, g8-95, g168-99 e g1m-95. A sobreposição de discos foliares de guandu em meio BDA e folha de papel de filtro em meio sojinha proporcionam um incremento na esporulação de M. phaseolina. / This work had the objective of determining the best schedule for artificial inoculation and select pigeon pea genotypes resistant to Macrophomina phaseolina in material obtained by Embrapa Pecuária Sudeste, and verify the mycelial growth and sporulation of the fungi in middle of culture. The work were carried in greenhouse at the UNESP/Jaboticabal, from August 2004 to December 2005. For the methodology and selection adjustment of resistant genotypes to the fungi the seeds were submitted scarified with water sandpaper and artificial inoculation the seeds were the contact method to fungi for different periods, which varied from O to 72 hours. They were evaluated percentage of surviving plants and fresh mass. For the mycelial growth and sporulation of the fungi was used the superposition of disks method of different hosts in the middle of culture. The scarified of the seeds contributed for penetration of the fungi at the seeds; the period of 24h of contact of the seeds to the fungi enough to detect differences in the resistance degree ofthe genotypes. The genotypes g167-97, g124-95, 927-94, g40-95, g154-95, g127-97 and g9m-97 were found to be the most resistant and most susceptible were g48-95, g123-95, g8-95, g168-99 and g1m-95. The treatment with superposition of the leaf disks of pigeon pea in BDA and disks of filter paper in middle of soybean extract were the treatments that provided better sporulation levei in the conditions of that experiment were half.

Um dos principais componentes envolvidos no processo de divisão celular bacteriana é FtsZ, uma proteína homóloga à tubulina eucariótica. FtsZ polimeriza no interior da célula formando um anel ao qual dá-se o nome de anel Z, responsável pelo recrutamento de diversas outras proteínas de divisão, formando o divisomo. Como meio de sobrevivência sob condições adversas, alguns procariotos, como B. subtilis, podem sofrer um tipo de diferenciação celular que forma um organismo em estado latente, conhecido como esporo. A primeira etapa da formação do esporo é a mudança da posição do anel Z para mais próximo a um dos pólos da célula, produzindo duas células com tamanhos diferentes. SpoIIE é uma proteína fosfatase integral de membrana, que se localiza especificamente no septo assimétrico de uma célula em processo de esporulação. Além de um papel na ativação do fator de transcrição de esporulação σF, SpoIIE se liga a FtsZ e a auxilia na formação do septo assimétrico. Para definirmos a região de FtsZ responsável pela interação com SpoIIE, neste trabalho foram realizados ensaios de duplo-híbrido utilizando vetores com domínios de ativação e de ligação ao DNA do fator de transcrição GAL4 de levedura fusionados a diferentes porções de FtsZ, bem como a SpoIIE. Esses experimentos não forneceram informações sobre interação entre essas proteínas, já que através deles não foi possível reproduzir o resultado positivo descrito na literatura. Como alternativa ao duplo-hibrido para identificarmos o sítio de interação entre as duas proteínas, criamos uma triagem genética capaz de identificar mutantes de FtsZ que não interagem com SpoIIE, fazendo uso de uma biblioteca de mutantes de FtsZ já disponível no laboratório. Foi padronizada uma técnica de microscopia em larga escala em placas de 96 poços, que permitiu a triagem de mais de mil de mutantes de FtsZ, em busca de um em que SpoIIE-GFP induzido não localizasse no anel Z em célula vegetativa. Porém todos os mutantes triados ainda localizavam SpoIIE-GFP. Paralelamente, foi realizada uma triagem de supressão, utilizando como ponto de partida um mutante de SpoIIE que perdeu capacidade de interagir com FtsZ e buscando mutações em FtsZ que reestabelecessem a interação com SpoIIE mutante. Foram triados cerca de 35000 mutantes nesse ensaio, dentre os quais dezoito apresentaram o fenótipo esperado para um supressor. No entanto, todos os candidatos selecionados tratavam-se de falsos-positivos. O motivo que leva esses candidatos a apresentarem o fenótipo esperado sem reestabelecer a interação entre as duas proteínas ainda é desconhecido. A fim de confirmar se não haveria outras proteínas do divisomo responsáveis por intermediar a interação entre FtsZ e SpoIIE, foram feitos experimentos de co-localização de FtsZ e SpoIIE na ausência de DivIB e FtsA. Em ambos os casos SpoIIE ainda localiza no divisomo, descartando a possibilidade de que DivIB e FtsA sejam mediadores da interação FtsZ-SpoIIE. Por fim, foram realizados experimentos de co-localização de SpoIIE com mutantes de FtsZ previamente identificados em outros experimentos em nosso laboratório. Nesse experimento foi identificado que a expressão de SpoIIE-GFP induzida por IPTG é capaz de reestabelecer a frequência de divisão no mutante FtsZ-R376T, que normalmente é deficiente na formação de divisomos. Esse resultado reforça a idéia de que essas proteínas interagem diretamente, e sugere que SpoIIE é capaz de reestabelecer a atividade de FtsZ em um mutante que apresente falhas na polimerização. / One of the major components involved in bacterial cell division is FtsZ, a protein homologous to the eukaryotic tubulin. FtsZ polymerizes inside the cell forming a ring to which is given the name Z ring, wich is responsible for the recruitment of several other proteins division, forming the divisome. As a means of survival under adverse conditions, some prokaryotes such as B. subtilis may undergo a type of cell differentiation that results in an organism in a latent state, known as a spore. The first stage of the spore formation is to change the Z ring position closer to the poles of the cell, producing two cells of different sizes. SpoIIE is an integral membrane phosphatase protein, which is specifically located in the septum of an asymmetric cell in sporulation process. In addition to a role in the activation of the sporulation transcription factor σF, SpoIIE binds to FtsZ and assists in the formation of the asymmetric septum. To define the FtsZ region responsible for interaction with SpoIIE, in this work we performed tests using two-hybrid vectors with activation and DNA binding domains of the yeast transcription factor GAL4 fused to different portions of FtsZ and SpoIIE. These experiments did not provide information on the interaction between these proteins, since through them it was not possible to reproduce the positive results reported in the literature. As an alternative to the two-hybrid to identify the site of interaction between the two proteins, we created a genetic screening that can identify FtsZ mutants that cannot interact with SpoIIE, using a library of FtsZ mutants already available in the laboratory. We standardized a large scale microscopy using 96-well plates, allowing the screening of over a thousand mutants of FtsZ in search of a induced SpoIIE-GFP which would no longer localize at the vegetative cell Z ring. However, all the screened mutants still localized SpoIIE-GFP. In parallel, we performed a screening of suppression, using as a starting point a SpoIIE mutant that lost the ability to interact with FtsZ and searching for mutations in FtsZ that would reestablish interaction with the SpoIIE mutant. We screened approximately 35,000 mutants in this essay, eighteen of which showed the phenotype expected for a suppressor. However, all selected candidates were false positives. The reason why such candidates do show the expected phenotype without reestablishment of the interaction between the two proteins is still unknown. In order to confirm whether there would be other divisiome proteins responsible for mediating the interaction between FtsZ and SpoIIE, co-localization experiments were made using FtsZ and SpoIIE in the absence of DivIB and FtsA. In both cases SpoIIE still located in divisome, ruling out the possibility that DivIB and FtsA are essencial mediators of the SpoIIE-FtsZ interaction. Finally, co-localization experiments were carried out with SpoIIE and FtsZ mutants previously identified in other experiments in our laboratory. In this experiment it was identified that the expression of IPTG-induced SpoIIE-GFP is able to restore the division frequency in the FtsZ-R376T mutant, which normally is deficient in the formation of divisomes. This result reinforces the idea that these proteins interact directly, and suggests that SpoIIE is able to restore the activity of FtsZ in a mutant that presents defect in polymerization.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-23T12:15:14Z No. of bitstreams: 1 Priscilla Anunciada Alves Moreira.pdf: 1192520 bytes, checksum: f783ff4175972095251ac5f078d7fe19 (MD5) / Made available in DSpace on 2017-03-23T12:15:14Z (GMT). No. of bitstreams: 1 Priscilla Anunciada Alves Moreira.pdf: 1192520 bytes, checksum: f783ff4175972095251ac5f078d7fe19 (MD5) Previous issue date: 2008-08-06 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The Alternaria black spot is considered one of the most common and destructive diseases of brassica, and although it may be caused by several species, Alternaria brassicicola is the predominant pathogen in both conventional and organic brassica crops in State of Pernambuco, Brazil. To assess the pathogenic variability of 120 isolates of A. brassicicola, was analyzed the virulence to brassica species, physiology aspects of the pathogen and sensitivity to fungicides. The isolates obtained from conventional and organic crops were inoculated under greenhouse conditions in 40 days old plants of broccoli, cabbage, cauliflower and Chinese cabbage and disease severity was assessed 10 days after the inoculation. Under laboratory conditions the following parameters were evaluated for all isolates: rate of growth mycelial, sporulation, and sensitivity to fungicides iprodione and tebuconazole. Based on the variables analyzed, it was found variability among isolates of A. brassicicola, but there were no significant (P>0.05) correlation among the analyzed variables, regardless of the cultivation system and the original host of each isolate. The variables were useful in distinguishing individual groups of isolated with similarity in virulence to the host, physiological characteristics and sensitivity to fungicides. The cross inoculations and the development of symptoms in brassica highlighted the absence of host specificity. All isolates weresensitive to the fungicides iprodione and tebuconazole. Based on the components of variance analysis, there was no significant effect of the cultivation system on the variables, except for disease severity on Chinese cabbage (P = 0.0391). Most of the variance results from the difference among isolates of A. brassicicola within the cropping systems and the host of origin. In the context multivariate, no effect was found regarding the host of origin of the isolates (P = 0.4993), however, there was great variability (P <0.0001) among isolates within the cropping systems and the host of origin. / A alternariose é considerada uma das doenças mais comuns e destrutivas das brássicas, e embora possa ser causada por várias espécies, Alternaria brassicicola é o agente patogênico predominante nos plantios convencionais e orgânicos de brássicas no Estado de Pernambuco, Brasil. Para avaliar a variabilidade patogênica de 120 isolados de A. brassicicola, foi analisada a virulência a espécies de brássicas, aspectos relacionados à fisiologia do patógeno e sensibilidade a fungicidas. Os isolados obtidos de cultivos convencionais e orgânicos foram inoculados em plantas de brócolis, couve-chinesa, couve-comum e couve-flor com 40 dias de idade, em casa de vegetação, onde a severidade da doença foi avaliada 10 dias após a inoculação. Em condições de laboratório, os isolados foram avaliados quanto a taxa de crescimento micelial, esporulação e sensibilidade aos fungicidas iprodione e tebuconazole. Com base nas variáveis analisadas, foi constatada a existência de variabilidade entre os isolados de A. brassicicola, mas não foram observadas correlações significativas (P=0,05) entre as variáveis avaliadas, independentemente do sistema de cultivo e do hospedeiro de origem dos isolados. As variáveis individuais foram úteis na distinção de grupos de isolados do patógeno com similaridade na virulência às hospedeiras, características fisiológicas e sensibilidade aos fungicidas. As inoculações cruzadas e o desenvolvimento de sintomas nas brássicas evidenciou a inexistência de especificidade por hospedeiro entre os isolados do patógeno. Todos os isolados foram sensíveis aos fungicidas iprodione e tebuconazole. Pela análise dos componentes de variância, não houve efeito significativo do sistema de cultivo sobre as variáveis analisadas, com exceção para severidade em couve-chinesa (P=0,0391). A maior parte da variância foi resultante da diferença entre os isolados de A. brassicicola dentro dos sistemas de cultivo e dos hospedeiros de origem. No contexto multivariado, não se constatou efeito do hospedeiro de origem dos isolados (P=0,4993), porém, houve grande variabilidade (P<0,0001) entre os isolados dentro dos sistemas de cultivo e dos hospedeiros de origem.

Made available in DSpace on 2015-04-11T13:55:51Z (GMT). No. of bitstreams: 1 Francy Mary Sousa.pdf: 1356959 bytes, checksum: a7db48bf3163f386db3bca96f187f815 (MD5) Previous issue date: 2010-11-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Corynespora cassiicola has been related causing damages in several crops of economic importance in Amazonas State, principally Solanaceae and Cucurbitaceae families affecting production by significative form. This study aimed to characterize C. cassiicola isolates through colony morphology, sporulation, mycelial growth and spore dimension on culture media PDA, PSA, OEA and CA. Five mm-diameter disks taken from colonies grown on PDA medium for 15 days were transferred to Petri dishes containing each medium under room temperature and continuous light. The study was installed using randomized block design in a factorial 4 x 5 x 3 (culture media x isolates x hosts) with five replications. The effects of culture media varied to isolates of same host and among different hosts. Mycelial growth was observed in CA. The most spore production was observed in PDA and PSA media. A high variation of aspect and colonies coloration was observed among same host isolates and isolates of different hosts in all culture media. Variation trough spore dimension of C. cassiicola isolates was observed in all culture media tested. / O fungo Corynespora cassiicola já foi relatado causando danos em diversas culturas de interesse comercial no Estado do Amazonas, principalmente das famílias Solanaceae e Cucurbitaceae, afetando de forma significativa a produção. Este estudo teve como objetivo caracterizar isolados de C. cassiicola quanto a morfologia das colônias, esporulação, crescimento micelial e dimensão dos conídios nos meios de cultura BDA, BSA, AvA e CA. Discos de 5mm de diâmetro retirados de colônias crescidas em meio BDA por 15 dias, foram transferidos para o centro de placas de Petri contendo os meios de cultura e mantidas à temperatura ambiente sob luz contínua. O estudo seguiu esquema fatorial 4 x 5 x 3 (meios de cultura x isolados x hospedeiros) com cinco repetições. Os efeitos dos meios de cultura variaram para isolados do mesmo hospedeiro e entre hospedeiros diferentes. Maior crescimento micelial foi observado no meio CA. A maior produção de esporos foi observada nos meios BDA e BSA. Foi verificado uma ampla variação de aspecto e coloração das colônias entre os isolados do mesmo hospedeiro e entre isolados de hospedeiros diferentes em todos os meios de cultura. Também houve variação quanto às dimensões dos conídios dos isolados de C. cassiicola em todos os meios de cultura testados.