O maracujá-amarelo (Passiflora edulis f. flavicarpa) é uma frutífera de importância econômica no Brasil, sendo apreciado para a produção de suco concentrado e para o consumo in natura, além de ser usado pela indústria farmacêutica na extração da passiflorina. O presente trabalho visou à exploração da biblioteca genômica inserida em BACs (Ped-B-Flav) por meio da técnica de BAC-end sequencing, visando prover os primeiros insights sobre a composição e organização genômica da espécie, além de gerar novos candidatos a marcadores moleculares. Ao todo foram realizadas 9.979 reações de sequenciamento com eficiência média de 89 %, resultando em 8.821 BES de alta qualidade, com tamanho variando entre 100 pb e 1.255 pb, tendo em média 596 pb, totalizando cerca de 5,7 Mpb de informação genômica. Foram identificados, ao todo, 610 potenciais novos marcadores microssatélites. Os motivos de tetranucleotídeos foram os mais abundantes, ou seja, 28,9 % do total, sendo as repetições AATT aquelas observadas com maior frequência, com 131 ocorrências. Foram identificados e classificados 4.394 (19,69 %) elementos repetitivos. Dentre estes elementos, os grupos dos retrotransposons gypsy e copia-like foram os mais abundantes, correspondendo a 10,08 % e 7,93 % das ocorrências, respectivamente. Além disso, foram encontradas 767 (8,7 %) sequências com alta identidade a regiões codificadoras de proteínas. Estas sequências foram classificadas e anotadas de acordo com o vocabulário controlado GeneOntology. Análises de mapeamento genômico comparativo revelaram três regiões microssintênicas com o genoma de Populus trichocarpa, uma com o genoma de Vitis vinifera e uma com o genoma de Arabdopisis thaliana, além de evidenciarem uma série de regiões rearranjadas em relação aos genomas de referência. O presente estudo mostrou que os BES de Passiflora edulis são uma excelente fonte de informações sobre o genoma da espécie, principalmente no que tange à diversidade gênica, identificação de elementos transponíveis e ao potencial para o desenvolvimento de novos marcadores genéticos. Igualmente, foi possível empregar essas sequências na identificação de regiões microssintênicas entre o genoma do maracujá-amarelo e de outras espécies vegetais próximas. / Yellow passion fruit (Passiflora edulis f. flavicarpa) is of considerable economic importance to Brazil. It is used to produce juice concentrate and also marketed for consumption as a fresh fruit. In the pharmaceutical industry, it is used to produce passiflora extract. The aim of this study was to explore the BAC (Bacterial Artificial Chromosome) genomic library (Ped-B-Flav) using BAC-end sequencing (BES) to provide some initial insights into the composition and organization of the species genome, and to generate new candidates for molecular markers. Altogether, 9,979 sequencing reactions were performed, with an average efficiency of 89 %, resulting in 8,821 high-quality BES, of average length ranging from 100 bp to 1255 bp, and consisting of an average 596 bp, totaling some 5.7 Mb of genomic information. In all, we identified 610 potential new microsatellite markers. Tetranucleotide motifs (28.9%) were the most abundant and AATT was the most frequently observed motif, with 131 occurrences. We identified and classified 4,394 (19.69 %) repetitive elements. Retrotransposon gypsy (10.8%) and copia-like (7.93%) elements were the most abundant. Furthermore, we found 767 (8.7 %) sequences very similar to those of protein coding regions. These sequences were classified and annotated according to gene ontology controlled vocabulary. Comparative genomic mapping revealed three regions showing microsynteny with the genome of Populus trichocarpa, one with Vitis vinifera genome and one with the Arabdopisis thaliana genome. In addition it revealed a series of rearranged regions in comparison to the reference genomes. This study showed that Passiflora edulis BES form an excellent source of information on the genome of the species, especially in regard to genetic diversity, identification of transposable elements and potential for the development of new genetic markers. It was also possible, using these sequences, to identify regions showing microsynteny with other plant species.

A majority of commercial rose varieties bloom repeatedly throughout the year, as compared to most rose species, other woody ornamentals, and fruit crops that bloom once a year. This recurrent flowering feature of the commercial roses resulted from a flowering mutation named everblooming (evb). The mutation is recessive to once blooming and is found in the rose species Rosa chinensis. Although several molecular maps have been developed for rose, little is known about the evb gene, except for its classic genetics. The purpose of this study was to develop a large-insert bacterial artificial chromosome (BAC) library as a starting tool for molecular cloning and analysis of the evb gene by map-based cloning. To construct the large-insert BAC library, nuclear megabase-size DNA was isolated from the recurrent blooming diploid species, Rosa chinensis cv. Old Blush. The DNA was then partially digested with BamHI and separated on agarose gels by multi-phase pulsed-field gel electrophoresis. Size selected fragments estimated between 100 kb and 150 kb in size were cloned into the pECBAC1 BAC vector and the clones having rose DNA inserts were arrayed in 80 384-well microplates individually, with each clone being barcoded. The library contains 30,720 clones, has an average insert size of 108 kb and covers roughly 5.9x genome equivalents, with a >99% probability of isolating a single-copy clone from the library. The library is now available to be screened with the genes cloned from other species that control vernalization and floral development and will be used in mapbased cloning of the evb gene using a Rosa wichuraiana (‘Basye’s Thornless’) x ‘Old Blush’ backcross population.

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No. of bitstreams: 1 Moretto_gm_me_bot.pdf: 1154041 bytes, checksum: e226bf5e802a12577fe08f1fc48c0b49 (MD5) Previous issue date: 2017-02-07 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A barreira hematoencefálica é o principal obstáculo para que drogas possam chegar até o cérebro e realizar o tratamento de neuropataologias. Além disso, as doenças ligadas ao Sistema Nervoso Central (SNC) são a segunda categoria de doenças potencialmente fatais, ficando atrás apenas de problemas ligados ao sistema cardiovascular. Devido à baixa eficácia que os tratamentos comuns apresentam, o uso de nanomateriais para o tratamento e diagnóstico deste tipo de patologia tem ganhado grande interesse da área acadêmica. Este estudo buscou elucidar parâmetros relacionados à nanopartículas magnéticas (NPMs) no cérebro, assim como os efeitos do manitol – fármaco conhecido por sua ação diurética e grande uso no tratamento de anormalidades na pressão intracraniana - em parâmetros como o tempo de meia-vida das NPMs (T1/2), acúmulo em diferentes órgãos, intensidade máxima de sinal, entre outros. Foram utilizados três grupos de animais que receberam NPMs, sendo que em dois deles a administração das NPMs ocorreu 15 minutos e 30 minutos após a administração do manitol. Os resultados apresentaram diferentes intensidades máximas para cada grupo, assim como um diferente acúmulo de NPMs no baço e no sangue para animais que receberam manitol, indicando alterações causadas por este fármaco em sistemas biológicos. O sistema BAC se mostrou eficaz na detecção de NPMs no cérebro, assim como na posterior análise da biodistribuição destas em organimos vivos. Além disso, os resultados de biodistribuição e tempo de circulação encontrados permitirão estabelecer novas aplicações deste protocolo para implementação de nanopartículas magnéticas como agentes de contraste e carreadores de fármacos no SNC. Este estudo buscou aumentar a possibilidade da aplicação do sistema BAC na caracterização e avaliação de parâmetros farmacocinéticos de NPMs em sistemas biológicos. / The blood-brain barrier is the main obstacle for drugs to reach the brain and treat neuropathologies. In addition, diseases linked to the Central Nervous System (CNS) are the second category of potentially fatal diseases, behind only problems related to the cardiovascular system. Due to the low effectiveness that common treatments present, the use of nanomaterials for the treatment and diagnosis of this type of pathology has gained great interest in the academic area. This study sought to elucidate parameters related to magnetic nanoparticles (NPMs) in the brain, as well as the effects of mannitol - drug known for its diuretic action and great use in the treatment of abnormalities in intracranial pressure - in parameters such as the half-life time of NPMs (T1 / 2), accumulation in different organs, maximum signal intensity, among others. Three groups of animals that received NPMs were used, and in two of them the administration of the NPMs occurred 15 minutes and 30 minutes after the administration of mannitol. The results presented different maximum intensities for each group, as well as a different accumulation of NPMs in the spleen and blood for animals that received mannitol, indicating changes caused by this drug in biological systems. The BAC system proved to be effective in the detection of NPMs in the brain, as well as in the subsequent analysis of the biodistribution of these into living organism. In addition, the results of biodistribution and time of circulation found will allow to establish new applications of this protocol for the implementation of magnetic nanoparticles as contrast agents and carriers of drugs in the CNS. This study aimed to increase the possibility of applying the BAC system in the characterization and evaluation of pharmacokinetic parameters of NPMs in biological systems.

O maracujá-amarelo (Passiflora edulis f. flavicarpa) é uma frutífera de importância econômica no Brasil, sendo apreciado para a produção de suco concentrado e para o consumo in natura, além de ser usado pela indústria farmacêutica na extração da passiflorina. O presente trabalho visou à exploração da biblioteca genômica inserida em BACs (Ped-B-Flav) por meio da técnica de BAC-end sequencing, visando prover os primeiros insights sobre a composição e organização genômica da espécie, além de gerar novos candidatos a marcadores moleculares. Ao todo foram realizadas 9.979 reações de sequenciamento com eficiência média de 89 %, resultando em 8.821 BES de alta qualidade, com tamanho variando entre 100 pb e 1.255 pb, tendo em média 596 pb, totalizando cerca de 5,7 Mpb de informação genômica. Foram identificados, ao todo, 610 potenciais novos marcadores microssatélites. Os motivos de tetranucleotídeos foram os mais abundantes, ou seja, 28,9 % do total, sendo as repetições AATT aquelas observadas com maior frequência, com 131 ocorrências. Foram identificados e classificados 4.394 (19,69 %) elementos repetitivos. Dentre estes elementos, os grupos dos retrotransposons gypsy e copia-like foram os mais abundantes, correspondendo a 10,08 % e 7,93 % das ocorrências, respectivamente. Além disso, foram encontradas 767 (8,7 %) sequências com alta identidade a regiões codificadoras de proteínas. Estas sequências foram classificadas e anotadas de acordo com o vocabulário controlado GeneOntology. Análises de mapeamento genômico comparativo revelaram três regiões microssintênicas com o genoma de Populus trichocarpa, uma com o genoma de Vitis vinifera e uma com o genoma de Arabdopisis thaliana, além de evidenciarem uma série de regiões rearranjadas em relação aos genomas de referência. O presente estudo mostrou que os BES de Passiflora edulis são uma excelente fonte de informações sobre o genoma da espécie, principalmente no que tange à diversidade gênica, identificação de elementos transponíveis e ao potencial para o desenvolvimento de novos marcadores genéticos. Igualmente, foi possível empregar essas sequências na identificação de regiões microssintênicas entre o genoma do maracujá-amarelo e de outras espécies vegetais próximas. / Yellow passion fruit (Passiflora edulis f. flavicarpa) is of considerable economic importance to Brazil. It is used to produce juice concentrate and also marketed for consumption as a fresh fruit. In the pharmaceutical industry, it is used to produce passiflora extract. The aim of this study was to explore the BAC (Bacterial Artificial Chromosome) genomic library (Ped-B-Flav) using BAC-end sequencing (BES) to provide some initial insights into the composition and organization of the species genome, and to generate new candidates for molecular markers. Altogether, 9,979 sequencing reactions were performed, with an average efficiency of 89 %, resulting in 8,821 high-quality BES, of average length ranging from 100 bp to 1255 bp, and consisting of an average 596 bp, totaling some 5.7 Mb of genomic information. In all, we identified 610 potential new microsatellite markers. Tetranucleotide motifs (28.9%) were the most abundant and AATT was the most frequently observed motif, with 131 occurrences. We identified and classified 4,394 (19.69 %) repetitive elements. Retrotransposon gypsy (10.8%) and copia-like (7.93%) elements were the most abundant. Furthermore, we found 767 (8.7 %) sequences very similar to those of protein coding regions. These sequences were classified and annotated according to gene ontology controlled vocabulary. Comparative genomic mapping revealed three regions showing microsynteny with the genome of Populus trichocarpa, one with Vitis vinifera genome and one with the Arabdopisis thaliana genome. In addition it revealed a series of rearranged regions in comparison to the reference genomes. This study showed that Passiflora edulis BES form an excellent source of information on the genome of the species, especially in regard to genetic diversity, identification of transposable elements and potential for the development of new genetic markers. It was also possible, using these sequences, to identify regions showing microsynteny with other plant species.

Listeria monocytogenes is one of the toughest challenges the food industry is facing today. While quaternary ammonium compounds (QAC) are typically applied at very high concentrations to kill L. monocytogenes, there are some factors that can cause its reduced efficacy in the food processing environment. Sublethal concentrations of stagnated QAC have been found both in processing plants and in waste waters released to the environment. Therefore, the purpose of this study was to investigate the ability of two L. monocytogenes strains, Bug600 (serotype 1/2a) and ScottA (serotype 4b) to persist in lethal and sublethal concentrations of first generation QAC (benzalkonium chloride, BAC) and second generation QAC (Ster-BAC), and if these strains would regrow when conditions had improved. Using BAC or Ster-BAC concentrations that mimic those found occurring in some environments, we determined L. monocytogenes survival, persistence, and recovery in high/low nutrient conditions, and also isolated two distinct morphotypes.

Biogenic Acid Corrosion (BAC) is the biodeterioration of concrete caused by biological and chemical activities of bacteria that grow in an acidic environment. Such environments are typical in sewer systems, which collect wastewater from households, industries, and urban and storm water runoff, and convey this to wastewater treatment plants. Since these sewer systems transport large volumes of sewage and are invariably buried under the ground, concrete pipes are customarily used. Concrete is widely known as a robust, flexible, and durable material in many aggressive environments, yet it can suffer from severe sewer corrosion. Numerous techniques have been employed to eliminate or control the problem, including applying chemical or biological agents that decrease acid production, and surface treatment techniques that inhibit chemical attack and acid penetrability into the concrete. However, these techniques are expensive and variably effective, and some may lead to a loss of structural integrity and performance during the long service life of more than 50 years, due to a lack of long-term performance testing before they are introduced into the market. The most reliable and affordable approach remains to modify concrete by incorporating readily available binders with high chemical and physic mechanical potential in resisting corrosion. Numerous studies have approached this technique using Portland Cement (PC) based systems with Supplementary Cementitious Materials (SCMs). However, other binder systems such as Calcium Aluminate Cement (CAC) and Calcium Sulpho-Aluminate cement (CSA) have been less studied, despite showing higher potential in suppressing biogenic corrosion. Furthermore, most biogenic prediction models cannot effectively predict the performance of these binders in a sewer environment, especially when they are incorporated with different aggregates. For instance, the Life Factor Method (LFM), which is a common and often preferred method in sewer concrete design, is not formulated to enable the prediction of the corrosion rate of concrete comprising CAC, or specific PCbased systems with SCMs such as slag, fly ash, and silica fume. The LFM model consists of two parts; the acid environment generation part, and the acid resistance part, also sometimes called the ‘material factor', which defines the potential of concrete to resist corrosion due to acid. However, the current material factor in the model involves only an alkalinity factor (total calcium oxide content in concrete) as a significant resistance provider, while not considering other chemical compositions that significantly contribute to providing concrete with acid resistance. A previous amendment of the model at the University of Cape Town (UCT) resulted in a 'material factor' approach that was highly empirical and complex, and therefore less practical and comprehensive in application. The model also could not handle modern binder systems in conjunction with aggregates, and the criteria for its refinement were somewhat self-contradictory. Therefore, this study aimed to fundamentally re-think and improve the LFM model to cover a broader range of binder systems and aggregate types with their performance in different sewer conditions. Firstly, the deterioration mechanisms of concretes with different binder systems and different types of aggregates were studied in different live sewer environments. Secondly, the mechanism of deterioration of the binder systems was further studied using a reactive transport modelling approach to understand the critical phases that govern the deterioration. Using the information from the first two goals, the LFM model was then improved and advanced to cover a wider range of binder systems, aggregate types, and sewer environments. The study characterised the behaviour of three sewer sites in the Cape Town Metro (i.e., Langa Pump station (LPS) manhole, Northern Area Sewer manhole 19 (NAS M19) and manhole 54 (NAS M54)), monitoring techniques (i.e., visual observations, mass and thickness change, and concrete surface pH), The study also evaluated the influence of different concrete mixes using concrete microstructural analysis (Scanning Electron Microscopy (SEM), Quantitative Evaluation of Minerals (QEMSCAN), and X-Ray Diffraction (XRD) analysis), and reactive transport modelling (i.e., HYTEC modelling tool). 2 The concrete mixes were grouped into two batches: LH concrete and UCT concrete mixes. The LH concrete mixes consisted of four binder systems, i.e., a blend of 80% Sulphate Resisting Portland Cement and 20% Fly Ash (SRPC+FA), a similar blend with 11% iron-based additive (SRPC+FA+HC), a blend of 80% SRPC and 20% ground Limestone (SRPC+LS), and CSA. These mixes were cast with calcite and siliceous aggregates by Lafarge Holcim (LH) in Lyon, France and delivered to the UCT laboratory for sewer exposure. Before exposure, it was observed that the mixes exhibited significant compaction voids, but their condition was such as to permit sewer exposure with the expectation of gathering useful information on these mixes. The UCT concrete mixes were prepared at UCT with local dolomite and siliceous aggregates, but using the same LH binders, i.e., SRPC+FA, SRPC+FA+HC, with some additional local binders, i.e. Portland cement blended with limestone (CEM II A-L), a blend of 50% CAC and 50% SRPC, CSA, and CAC. The LH concrete mixes were exposed to all three sites, while the UCT concrete mixes were exposed only to the LPS and NAS M19 after observing that NAS M54 was minimally aggressive. In terms of reactive transport modelling, only UCT concrete mixes were studied. Regarding sewer characterisation, the LPS manhole has the most aggressive environment, followed by NAS M19 and NAS M54. The aggressivity of the LPS was due to its high H2S gas concentration and sewer hydraulic actions as it receives wastewater from a pump station. NAS M19 is located in the midsection of the sewer line and collects a mixture of domestic and industrial wastewater. It experiences occasional flooding, mainly during the winter season. NAS M54 is an upstream manhole with maximum gas concentrations below 10 ppm. Therefore, concrete mixes exposed at the LPS exhibited more severe corrosion than at NAS M19, while at NAS M54, only minor signs of corrosion were observed after two years. Also, it was observed that the aggressivity of the sewers varied with the seasons, with higher gas production during hot periods. BAC monitoring and concrete microstructural analyses indicated that the Portland-based concretes (SRPC-based and CEM II A-L) experienced more severe deterioration compared to alumina-based concretes (CAC-based and CSA). CAC performed the best, followed by a blend of CAC+ SRPC concrete and then CSA, due to the formation of gibbsite and the high neutralisation potential provided by alumina bearing phases over calcium oxide-bearing phases such as calcium silicate hydrates and portlandite. In terms of Portland-based concretes, it was observed that blending SRPC concrete with fly ash improved the resistance potential over CEM II A-L, iron-based additives had little influence, and ground limestone in conjunction with calcite aggregates provided more acid-soluble material to neutralise the acid. In terms of aggregate performance, siliceous aggregates do not react with acid, and as a result, they eventually detach from the exposed surface. Dolomite and calcite aggregates dissolve in acid, and with magnesium carbonate, the rate of dolomite dissolution was slightly higher than calcite. The corrosion rate of the concrete depended on the relationship between the rate of deterioration of the cement matrix and the rate of deterioration of the aggregate matrix. Thus, concrete with similar cement and aggregate deterioration rates has a uniform corrosion front and a slower rate of corrosion, and vice versa. The LFM model was modified with the information from the BAC monitoring and concrete microstructural analysis. The revised model has two key parameters: the sewer environment factor and the material resistance factor; the latter includes the acid neutralisation factor of the binder system and the aggregate reactivity factor. The sewer environmental factor evaluates the rate of acid generated on the exposed concrete surface while considering various factors associated with H2S gas adsorption and oxidation. The material resistance factor, on the other hand, evaluates the quantity of acid to be neutralised by a specific volume of exposed concrete while considering the influence of binder and aggregate. Ultimately, a ratio of sewer environmental and material resistance factors can assist in predicting and providing the corrosion rate of any concrete mix with any binder and aggregate types when subjected to a sewer environment. The corrosion rates predicted by this model correlated well with field-measured corrosion rates both in this study and in previous sewer studies at UCT. Therefore, this study provides engineers with a relatively simple tool for predicting the corrosion rate of sewer concrete, with recommendations for selecting the most durable sewer concrete mix designs.

Xenopus leavis was a favorite model organism during the 20th. century, but nowadays it has been replaced by diploid Xenopus tropicalis, which has not only shorter generation time, but also smaller genom. One of the disadvantages of Xenopus tropicalis is the lack of full physical and linkage map. According to JGI genome database (assembly 4.1) there are unmapped regions on short arm of the chromosome 2 and 7 . Several BAC clones ( with a single or dual-end sequence) has been found to be located within this region, according to a recent assembly 7.1. However , it isn't clear whether 100bp length of BAC ends is enough to place entire BAC clone into the genom of Xenopus tropicalis. In order to prove correct inclusion of these BAC clones into JGI database, several BAC clones, which are supposed to be located on short arm of chromosome 2, were picked. Using fluorescence in situ hybridisation, the signal of these BAC clones was localised on the short arm of chromosome 1 instead of chromosome 2 and in most cases they had opposite orientation. It means that the 100bp lenght of BAC ends propably isn't sufficient to place entire BAC clone on chromosome. New working protocol of BAC DNA isolation and labeling was established.

Tese de Mestrado Integrado. Engenharia Civil - Especialização em Estruturas. Faculdade de Engenharia. Universidade do Porto. 2011

No description available.

VP16 initiates the HSV replication cycle by activating immediate early (IE) gene expression. It recruits the RNA pol II through an acidic C-terminal domain. The defective VP16 encoded by the V422 mutant of HSV-1 possesses a truncated C-terminal domain. Therefore, V422 replication is suppressed in most cell-lines, except U2OS osteosarcoma cells. The permissive phenotype of U2OS cells stems from a failure to express one or more inhibitory factors that are produced in restrictive cells. The initial project was designed to identify these host inhibitory factors in restrictive cells of V422, using siRNA silencing technology. To facilitate the siRNA screen, a GFP reporter gene has been inserted into the thymindine kinase (TK) gene of the V422 genome and the wild-type KOS genome. This thesis provides information about characterizing the kinetics of GFP expression from recombinant viruses at both protein and mRNA levels, during different infection times in HeLa and Vero cells. / Virology