Submitted by Mario BC (mario@bc.ufrpe.br) on 2016-09-23T12:47:02Z No. of bitstreams: 1 Rafaela Lima de Araujo.pdf: 5175233 bytes, checksum: a9acd861166189ddbd0b21c9b3ec9f11 (MD5) / Made available in DSpace on 2016-09-23T12:47:02Z (GMT). No. of bitstreams: 1 Rafaela Lima de Araujo.pdf: 5175233 bytes, checksum: a9acd861166189ddbd0b21c9b3ec9f11 (MD5) Previous issue date: 2014-01-20 / The Psidium guineense species, popularly known as strawberry guava, belongs to the Myrtaceae family, which is native to South America having a broad geographical distribution. In the Brazilian Northeast, is found on the coast, in the Forest Zone region. The fruit of the strawberry guava flavor is slightly reminiscent of the guava, although it is slightly more acidic and sharper scent. The culture of strawberry guava (P. guinenese) has no economic significance in the context of the national fruit production, not even existing commercial orchards. It is necessary, studies of genetic population level character, since there is little information on ecological and genetic aspects of the species. In order to provide information on the genetic resources of strawberry guava (P. guineense) in the State of Pernambuco, the objectives of this study were to evaluate the genetic diversity of four natural populations of P. guineense distributed in the Forest Zone of Pernambuco and characterize the genetic structure of these four populations from isozyme markers. Eighteen loci were used to estimate allele frequencies relating to 114 individuals in four populations: Itamaracá Arariba, Marieta and Palmares. Populations had generally high level of polymorphism and average of 1.5 alleles per locus. The heterozygosity observed was higher than heterozygosity expected, which ranged between 0.22 and 0.23 and the heterozygosity expected ranged between 0.34 and 0.40. The fixation index ranged from = -0.794 to = -0.549, presenting all negative, indicating the absence of inbreeding and heterozygosity excess. The estimated population in pairs all showed gene flow are generally high, ranging from Nm = 3.23 to Nm = 20.77 showing a high proportion of migrants among populations. / A espécie P. guineense, conhecida popularmente como araçá, pertence à família Mirtácea, que é originária da América do Sul apresentando uma ampla área de distribuição geográfica. Nos Estados do Nordeste Brasileiro, é encontrada no litoral, na região da Zona da Mata. O fruto do araçazeiro tem sabor que lembra um pouco o da goiaba, embora seja ligeiramente mais ácido e de perfume mais acentuado. A cultura do araçá (P. guinenese) não possui expressão econômica no contexto da fruticultura nacional, não existindo, inclusive, pomares comerciais. Faz-se necessário, estudos de caráter genético a nível populacional, uma vez que existem poucas informações sobre aspectos ecológicos e genéticos da espécie. Com o intuito de disponibilizar informações sobre os recursos genéticos do araçá (P. guineense) no Estado de Pernambuco, os objetivos do presente trabalho foram avaliar a diversidade genética de quatro populações naturais de P. guineense distribuídas na Zona da Mata do Estado de Pernambuco e caracterizar a estrutura genética dessas quatro popurações a partir de marcadores isoenzimáticos. Dezoito locos foram utilizados para estimar as frequências alélicas referentes aos 114 indivíduos, nas quatro populações: Itamaracá, Arariba, Marieta e Palmares. As populações apresentaram em geral, alto nível de polimorfismo com média de 1,5 alelos por loco. A heterozigosidade observada apresentou-se maior do que a esperada, onde variou entre 0,22 e 0,23 e variou entre 0,34 e 0,40 . O índice de fixação variou de = -0,549 a = -0,794, apresentando-se negativo para todos, indicando ausência de endogamia e excesso de heterozigosidade. O fluxo gênico estimado para as populações aos pares apresentou-se em geral elevado variando de Nm= 3,23 a Nm= 20,77 evidenciando alta proporção de migrantes entre populações.

The semi-arid region of the Brazilian Northwest has been the focused of several studies aiming the alternatives to amplify the possibilities utilization of sources for population. Subsistence agriculture and extensive herds constitute the systems of production basically. Nevertheless some native species from this region among them the Umbu trees (Spondias tuberosa Arr. Cam.) has been contributed as an alternative source income in the dry periods. The goal of this study was to evaluate the biotechnological potential of umbu fruits, aiming to promote alternative income which can contribute to improve the life of communities in dry regions. It was utilized the following parameters: the umbu yeast identification; workmanship wine production, physical chemistry and sensorial umbu wine characteristics and the isozyme pattern of yeast isolated from umbu. Fresh fruits of umbu were isolated from 54 morphotypes and identified 17 species with nine isolated from workmanship wine umbu essays. The results to evidence the fruits of umbu trees (Spondias tuberosa) as good substrate to grow yeasts with a predominance of Ascomycete species to ferment wine. All samples were able to produce wine in a workmanship scale with physical chemistry and sensorial characteristics acceptable. The Kloeckera japonica species and Kluyveromyces marxianus produced the more acceptable umbu wine. Analyzing the isozyme patterns it was detected the existence of a intra and inter specific polymorphism which demonstrate the potentiality of this technique to distinguish the studied yeast. The umbu wine in a production system context presents as an alternative income in productive chain. / A região semi-árida do Nordeste brasileiro tem sido foco de diversos estudos na busca de alternativas que ampliem as possibilidades de utilização de seus recursos pela população. Os sistemas de produção são constituídos basicamente pela agricultura de subsistência e pela pecuária extensiva. Contudo, algumas plantas nativas da região, entre elas o Umbuzeiro (Spondias tuberosa Arr. Cam.), têm contribuído como uma fonte de renda alternativa nos períodos de seca. O objetivo desse estudo foi avaliar o potencial biotecnológico do umbu visando fornecer alternativas que possam contribuir com a melhoria de vida das comunidades do semi-árido. Utilizaram-se como parâmetros identificação de leveduras isoladas de polpa de frutos de umbuzeiro, produção semi-artesanal do vinho de umbu, análise das características físico-químicas e sensoriais dos vinhos produzidos e o perfil isoenzimático das leveduras isoladas de umbu. A partir das polpas frescas e congeladas de umbu foram isolados 54 morfotipos e identificadas 17 espécies de leveduras das quais nove isolados foram utilizadas nos ensaios de produção do vinho artesanal de umbu. Os resultados deste estudo evidenciaram que os frutos de umbuzeiro (Spondias tuberosa) se apresentam como bons substratos para o desenvolvimento de leveduras, havendo predominância de espécies ascomicéticas fermentadoras. Foi verificado, também, que todas as amostras testadas mostraram-se capazes de produzir vinhos em escala artesanal de bancada com características físico-químicas e sensoriais aceitáveis. As espécies Kloeckera japonica e Kluyveromyces marxianus produziram os vinhos de umbu de maior aceitabilidade. Por meio do perfil isoenzimático, percebeu-se a existência de um polimorfismo intra e interespecífico, demonstrando a potencialidade da técnica para a detecção de variabilidade intra e interespecífica para a caracterização e distinção das leveduras estudadas. O potencial biotecnológico do umbu, no contexto de produção de vinho se apresenta como mais uma alternativa na cadeia produtiva dessa fruta.

A grande biodiversidade nas florestas tropicais, a elevada exploração de florestas e as poucas espécies estudadas do ponto de vista genético levaram a este estudo que é essencial para o manejo sustentável e a conservação genética de espécies, sendo importante no controle da redução da diversidade genética natural permitindo que as espécies se mantenham vivas e reprodutivas ao longo dos tempos. Entre as diversas espécies arbóreas brasileiras submetidas a práticas de manejo, tem-se a candeia (Eremanthus erythropappus). Os objetivos foram quantificar e comparar a variabilidade genética intrapopulacional e sistema de reprodução de candeia e antes e após o manejo, através da técnica de eletroforese de isoenzimas em uma população natural de candeia. Para as progênies as heterozigosidades observadas foram altas 0,357 e 0,423 e heterozigosidades esperadas 0,403 e 0,425. Para os adultos foram 0,299 e 0,399. A porcentagem dos locos polimórficos variou de 76 a 100% entre progênies e adultos. O número de alelos por loco variou de 2,3 nos adultos e 2,57 em progênies antes e após o manejo. Nas progênies não houve diferença significativa entre estes valores indicando que a população encontra-se em EHW, tal fato pode ser comprovado pelo índice de fixação significativamente igual a zero (0,112 e 0,005) antes e após manejo, respectivamente. Os resultados do sistema de reprodução foram: taxa de cruzamento alta na análise antes (0,963) e pós manejo (0,967) indicando que a espécie é de reprodução mista; ocorreram cruzamentos entre aparentados tanto na população antes do manejo como na pós manejo, com estimativas entre 3 e 5%; as estimativas da correlação de autofecundação ( s rˆ ) foi alta nas duas populações (0,188 e 0,179), sendo ambas significativas, sugerindo que os indivíduos de autofecundação encontram-se aleatoriamente distribuídos nas progênies não existindo a tendência de algumas progênies apresentarem mais indivíduos de autofecundação do que outros. A correlação de paternidade foi significativamente diferente de zero (0,414 e 0,368), sugerindo que uma parte das progênies de cruzamentos foi gerada pelo mesmo parental materno e paterno, indicando haver a presença de cruzamentos biparentais dentro das populações. As estimativas do número médio de indivíduos polinizadores efetivos por árvore, foram baixas, em torno de 2 a 3 polinizadores por árvore. O sistema misto tem implicações na estimativa do tamanho efetivo de variância (Ne(v)), os valores estimados para este parâmetro foram de 1,99 e 2,08 para população antes do manejo e posterior ao manejo, respectivamente. O valor estimado para o coeficiente de coancestria dentro das progênies antes do manejo (0,229) e posteriores ao manejo (0,222) foram superiores em 83,2% e 77,6% ao esperado em progênies de meios-irmãos (0,125). O tamanho efetivo de endogamia foi de 178,4, 51 e 49 indivíduos em adultos, progênies antes e após o manejo, respectivamente. Os resultados indicam forte estrutura genética espacial na população, com árvores próximas até 200 m apresentando algum grau de parentesco, com 95% de probabilidade. Estes resultados indicam que a espécie é passível de manejo e que este não afetou a diversidade nesta geração. / The great biodiversity in the tropical forests, high level of forest exploitation and the few arboreal species which have been studied from the genetic point of view led to this study that is essential for the sustained handling and the genetic conservation of the species. They are important in controlling the natural genetic diversity reduction thus allowing the species to be alive and reproduce, in time. Among the various Brazilian arboreal species submitted to the handling practice there is the candeia (Eremanthus erythropappus). The objectives have been of quantifying and comparing the intrapopulational genetic variability and the mating system of candeia and before and after the allozyme electrophoresis technique has been applied in a natural population of candeia two different times (before and after the handling). In the progenies, the observed heterozigosity was high (0,357 and 0,423) and expected heterozogosity (0,403 and 0,425).For the adults the results were (0,299 and 0,399). The percentage of the polymorphic loci varied from 76 % to 100% among progenies and adults. The number of alleles per loci varied from 2,3 in adults and 2,57 in progenies before and after the handling. In the progenies there was no significant difference between those values indicating that the population is in.HWE. That fact can be proven by the fixation index significantly equal to zero (0,112 and 0,005) before and after handling, respectively. The results of the reproduction system were: the crossing rate was high in the analysis that was done before (0,963) and in the one that was done after the handling ( 0,967). The estimates of the self-fertilization correlation were high in the two populations (0,188 and 0,179), and both were significant suggesting that the selffertilization individuals are distributed at random in the progenies and there is no tendency of some progenies to present more individuals of self fertilization than others. The paternity correlation was significantly different from zero (0,414 and 0,368), suggesting that a part of the progenies of crossings were bred by the same maternal and paternal begetter thus indicating that there was the presence of biparental crossings within the populations. The mixed system is involved in the average effective number variance (Ne(v), the estimated values for that parameter were of 1,99 and 2,08 for the population before the handling and after the handling, respectively. The estimated value of the coefficient of coancestry within the progenies before the handling ( 0,229) and after the handling (0,222) were superior in 83,2% and 77,6% than the expected in progenies of half-sib (0,125).The effective size of inbreeding was of 178,4, 51 and 49 individuals in adults, progenies before and after the handling, respectively. The results indicate a strong spatial autocorrelation analysis in the population, with trees next to each other as far as approximately 200 m presenting some degree of relationship, with 95% of probability. Those results indicate that the species is unresistant to handling and that did not affect the diversity in this generation and future works must be considered to assure the sustainability of the handling that is done from the genetic point of view.

Presence and range of phenotypic plasticity in the Agrostis stolonifera polyploid complex (Poaceae) was studied in the territory of the Czech Republic. Plants were cultivated under different experimental conditions. Stomatal size of different Agrostis stolonifera cytotypes was measured. Flow cytometry was applied for genome size estimation. Ploidy levels were determined for more than 150 samples of Agrostis stolonifera as well as several specimens of Agrostis canina, A. capillaris, A. gigantea, A. rupestris and A. vinealis. Absolute DNA content was estimated in all studied species. Isozyme analysis was used to test the possibility of the hybrid origin of pentaploid individuals of Agrostis stolonifera species.

Acetohydroxyacid synthase is a multisubunit enzyme that catalyses the first committed step in the biosynthesis of the branched chain amino acids viz., valine, leucine and isoleucine. In order to understand the structural basis for the observed allosteric feedback inhibition in AHAS, the regulatory subunit of AHAS isozymes I from E. coli was cloned, expressed, purified and the conditions were optimized for solution NMR spectroscopy. IlvN was found to exist as a dimer both in the presence and absence of the feedback inhibitor. Using high-resolution multidimensional, multinuclear NMR experiments, the structure of the dimeric valine-bound 22 kDa IlvN was determined. The ensemble of twenty low energy structures shows a backbone root mean square deviation of 0.73 ± 0.13 Å and a root mean square deviation of 1.16 ± 0.13 Å for all heavy atoms. Furthermore, greater than 98% of the backbone φ, ψ dihedral angles occupy the allowed and additionally allowed regions of the Ramachandran map. Each protomer exhibits a βαββαβα topology that is a characteristic feature of the ACT domain fold that is observed in regulatory domains of metabolic enzymes. In the free form, IlvN exists as a mixture of conformational states that are in intermediate exchange on the NMR timescale. Important structural properties of the unliganded state were probed by H-D exchange studies by NMR, alkylation studies by mass spectrometry and other biophysical methods. It was observed that the dynamic unliganded IlvN underwent a coil-to-helix transition upon binding the effector molecule and this inherent conformational flexibility was important for activation and valine-binding. A mechanism for allosteric regulation in the AHAS holoenzyme was proposed. Study of the structural and conformational properties of IlvN enabled a better understanding of the mechanism of regulation of branched chain amino acid biosynthesis. Solution structural studies of 32 kDa Protease-VPg (PVPg) from Sesbania mosaic virus (SeMV) Polyprotein processing is a commonly found mechanism in animal and plant viruses, by which more than one functional protein is produced from the same polypeptide chain. In Sesbania Mosaic Virus (SeMV), two polyproteins are expressed that are catalytically cleaved by a serine protease. The VPg protein that is expressed as a part of the polyprotein is an intrinsically disordered protein (by recombinant expression) that binds to various partners to perform several vital functions. The viral protease (Pro), though possessing the necessary catalytic residues and the substrate binding pocket is unable to catalyse the cleavage reactions without the VPg domain fused at the C-terminus. In order to determine the structural basis for the aforementioned activation of protease by VPg I undertook the structural studies of the 32 kDa PVPg domains of SeMV by solution NMR spectroscopy. NMR studies on this protein were a challenge due to the large size and spectral overlap. Using a combination of methods such as deuteration, TROSY-enhanced NMR experiments and selective ‘reverse-labelling’, the sequence specific assignments were completed for ~80% of the backbone and 13C nuclei. NMR studies on mutants such as the C-terminal deletion mutant, I/L/V to A mutants in VPg domain were conducted in order to identify the residues important for aliphatic-aromatic interactions observed in PVPg. Attempts were made to obtain NOE restraints between Pro and VPg domains through ILV labelled samples; however these proved unsuccessful. It was observed that ‘natively unfolded’ VPg possessed both secondary and tertiary structure in PVPg. However, 30 residues at the C-terminus were found to be flexible. Even though atomic-resolution structure could not be determined, the region of interaction between the domains was determined by comparing NMR spectra of Pro and PVPg. The conditions for reconstitution of the Protease-VPg complex by recombinantly expressed Pro and VPg proteins were standardised. These studies lay the foundation for future structural investigations into the Protease-VPg complex.

The proven ability to ferment Saccharomyces cerevisiae on a large scale presents an attractive target for producing chemicals and fuels from sustainable sources. Efficient and predominant carbon flux through to ethanol is a significant engineering issue in the development of this yeast as a multi-product cell chassis used in biorefineries. In order to evaluate diversion of carbon flux away from ethanol, combinatorial deletions were investigated in genes encoding the six isozymes of alcohol dehydrogenase (ADH), which catalyse the terminal step in ethanol production. The scarless, dominant and counter- selectable amdSYM gene deletion method was optimised for generation of a combinatorial ADH knockout library in an industrially relevant strain of S. cerevisiae. Current understanding of the individual ADH genes fails to fully evaluate genotype-by-genotype and genotype-by-environment interactions: rather, further research of such a complex biological process requires a multivariate mathematical modelling approach. Application of such an approach using the Design of Experiments (DoE) methodology is appraised here as essential for detailed empirical evaluation of complex systems. DoE provided empirical evidence that in S. cerevisiae: i) the ADH2 gene is not associated with producing ethanol under anaerobic culture conditions in combination with 25 g l-1 glucose substrate concentrations; ii) ADH4 is associated with increased ethanol production when the cell is confronted with a zinc-limited [1 μM] environment; and iii) ADH5 is linked with the production of ethanol, predominantly at pH 4.5. A successful metabolic engineering strategy is detailed which increases the product portfolio of S. cerevisiae, currently used for large-scale production of bioethanol. Heterologous expression of the cytochrome P450 fatty acid peroxygenase from Jeotgalicoccus sp., OleTJE, fused to the RhFRED reductase from Rhodococcus sp. NCIMB 978 converted free fatty acid precursors to C13, C15 and C17 alkenes (3.81 ng μl-1 total alkene concentration).