A cana-de-açúcar é uma gramínea com fotossíntese do tipo C4, com capacidade de acumular sacarose nos colmos em quantidades que excedem 50% de seu peso seco, característica única no reino vegetal (Moore, 1995). Sacarose e seu derivado mais importante, etanol, são dois produtos de grande importância mundial. Assim, o teor de sacarose em cana tem fundamental importância no aumento da produtividade dessas duas commodities. O melhoramento clássico parece ter alcançado seu limite, já que incrementos expressivos no teor de sacarose em novas variedades não têm sido observados e tudo aponta para a necessidade de estudos que levem a uma maior compreensão dos mecanismos moleculares associados à produção, transporte e acúmulo de sacarose em cana (Casu et al., 2005; Moore, 1995). Procurar seqüências promotoras de genes de interesse é importante para a obtenção de transgênicos, já que promotores constitutivos não apresentam resultados satisfatórios em cana (Lakshmanan et al., 2005). É também objetivo aqui hibridar mRNA de genótipos de cana-de-açúcar com segregação para acúmulo de sacarose, em uma plataforma customizada de oligos (Agilent), com aproximadamente 44k elementos, que compõe uma representatividade gênica não alcançada em esforços anteriores com microarranjos de cDNA. Genome walking foi a técnica utilizada na obtenção de regiões à montante do primeiro éxon, predito in silico, para três proteínas quinase de interesse, SASGMS11561, SASGMS16343 e SASGMS09047, que se mostraram moduladas em experimentos anteriores de hibridação com amostras segregantes para conteúdo de sacarose. Foi obtido sucesso nos três casos, tendo os fragmentos de DNA sido seqüenciados e oportunamente alinhados à montante dos correspondentes genes ortólogos em sorgo, bem como ao banco ainda em construção de contigs do genoma de cana-de-açúcar, obtidos por shotgun. A plataforma Agilent, com seus 43803 SAS únicos, mostrou-se uma ferramenta muito adequada para as hibridações de genótipos de mais alto Brix contra genótipos de mais baixo Brix. Um total de 569 genes diferencialmente expressos foram obtidos em pelo menos uma das três hibridações realizadas. Um grupo de genes, de diferentes categorias e perfis de modulação, foi validado por PCR em tempo real, obtendo uma taxa de aproximadamente 90%. Apesar do grande número de SAS diferencialmente expressos, por volta de 70% dos mesmos ainda se encontram não categorizados, seja por falta de similaridade de seqüência em bancos de dados de organismos próximos ou pela alta complexidade e esforço prático na cura desse processo de categorização manual. Assim, três fragmentos de seqüências promotoras para três proteínas quinase de interesse foram obtidos e seqüenciados, como parte dos esforços do grupo em formar um catálogo de promotores específicos para cana-de-açúcar. Um grupo de genes foi analisado dos resultados das hibridações por seus papéis relevantes nos processos que levam ao maior teor de sacarose em cana, devidamente corroborados por trabalhos do próprio grupo, bem como de outros / Sugarcane is a C4 plant with the unique characteristic of being capable of accumulating sucrose in its culms in quantities that exceed 50% of its dry weight (Moore, 1995). Sucrose and ethanol are highly valued products in the world of today. Sucrose content is a trait with fundamental importance in the on-going process of increasing productivity of these two sugarcane byproducts. Classic improvement of sugarcane seems to have reached its practical limits, given that it has become increasingly harder to obtain varieties with increased sugar content. This obstacle points towards the necessity of better comprehension of the molecular mechanisms associated to the production, transport and accumulation of sucrose in sugarcane (Casu et al., 2005; Moore, 1995). The search for promoter sequences of genes of interest is crucial for the production of transgenic lines, since the use of constitutive promoters in sugarcane has been highly problematic, leading to unsatisfactory results in most cases (Lakshmanan et al., 2005). Another objective was to hybridize sugarcane genotypes with contrasting sugar content in a customized Agilent oligo platform, containing approximately 44k elements, which signifies the best effort so far regarding gene representativeness. Genome walking was the chosen technique to obtain upstream regions of the first in silico predicted exon of three proteins kinases of interest, SASGMS11561, SASGMS16343 and SASGMS09047, all of them selected from previous hybridization experiments with contrasting sucrose content samples. Success was achieved in all three cases, and the obtained fragments were sequenced and aligned to their respective syntenic region on the sorghum genome as well as on contigs from an increasingly larger bank of genomic sugarcane sequences, from our group, which has been acquired using the shotgun sequencing method. The Agilent platform, with its 43803 unique sugarcane assembled sequences (SAS), has proven valuable as a powerful high scale tool for the hybridization of genotypes with contrasting Brix values (high versus low Brix). A total of 569 differentially expressed genes were obtained from at least one of the three experiments accomplished. A group of genes from different categories and modulation profiles was depicted and validated through real time PCR, with an approximate validation rate of 90%. Although the number of differentially expressed genes is high, around 70% of them is still uncategorized, mostly because of their unique identity and therefore lack of reference organisms to compare with and the high complexity and laboriousness of categorizing them manually. In summary, three promoter fragments from three different protein kinases of interest were obtained and sequenced, as a part of a greater effort to create a sugarcane promoter sequence catalogue. From the hybridization assays, a group of genes were analyzed, due to their putative importance in the processes that lead to a higher sucrose content in sugarcane, also corroborated by previous studies from our group as well as from others.

Made available in DSpace on 2014-08-20T13:32:46Z (GMT). No. of bitstreams: 1 dissertacao_railson_schreinert_dos_santos.pdf: 1331296 bytes, checksum: 2121f264ff3310f6f460595517aafdab (MD5) Previous issue date: 2012-07-27 / Rice (Oryza sativa L.) is one of the most important cereals in the world being cultivated in lowland ecosystems, which have a common characteristic: lack of drainage conditions. The lack of oxygen, due to submergence of seedlings, and iron toxicity are two of the major abiotic stresses which rice plants are subjected. Recently it was realized the importance of some ethylene responsive transcription factors (ERFs) in plant response to different stresses, especially to oxygen depletion. Considering it, two studies were made in order to evaluate the expression of ERFs under different stresses. In a first study seven genes were selected, based on previous results obtained from Genevestigator analysis, and had their transcriptional expression evaluated trough quantitative PCR (qPCR) in rice seedlings under oxygen depletion and iron overload. In the second study thirteen ERFs, which had no information available in the literature, also had their transcriptional levels evaluated in seedlings under conditions of hypoxia and anoxia through qPCR in a cultivar susceptible to flooding (Bonança) and a tolerant one (Nipponbare). As general conclusions it was once more emphasized the importance of ERF genes on plant response to environmental stresses. The specific function of each of these genes is yet to be studied. In silico analysis suggests the methylation of promoters as a possible way to regulate them. More studies about the function of these ERFs and about the role of methylation in plant stress response should be done. / O arroz (Oryza sativa L.) é um dos cereais mais importantes no mundo, sendo muito cultivado em ecossistemas de várzea, os quais apresentam uma característica comum: condições de deficiência de drenagem. A falta de oxigênio, devido à submergência das plântulas, e a toxidez por excesso de ferro são dois dos maiores estresses abióticos dentre os quais as plantas de arroz são submetidas. Recentemente percebeu-se a importância de alguns fatores de transcrição responsivos ao etileno (ERFs) na resposta vegetal à diferentes estresses, em especial à deficiência de oxigênio. Considerando-se o exposto efetuaram-se basicamente dois estudos visando avaliar a expressão de ERFs em diferentes estresses. Em um primeiro estudo sete genes foram selecionados a partir de resultados anteriores obtidos de análise no Genevestigator e estes tiveram sua expressão transcricional avaliada por PCR quantitativa (qPCR) em plântulas de arroz sob deficiência de oxigênio e excesso de ferro. No segundo estudo treze ERFs, os quais não apresentavam informações disponíveis na literatura, também tiveram seus níveis de expressão transcricional avaliados em plântulas sob condições de estresse por hipoxia e anoxia, também através de qPCR em uma cv. sensível à inundação (Bonança) e uma tolerante (Nipponbare). Como conclusões gerais se ressalta, novamente, a importância dos ERFs na resposta vegetal frente a estresses ambientais. A função específica de cada um destes genes ainda necessita ser estudada. Uma análise in silico nos promotores destes genes indica a metilação como possível forma de regulação transcricional. Mais estudos sobre a função destes ERFs e sobre o papel da metilação na resposta de arroz a estresses deverão ser feitas.

O presente estudo buscou avaliar os efeitos dos Mananoligossacarídeos (MOS) como aditivo alimentar no desempenho de primíparas suínas em final de gestação e em lactação, bem como no desempenho e na saúde dos leitões até os 65 dias de idade. O experimento foi realizado no Laboratório de Pesquisa em Suínos (VNP-FMVZ-USP) Pirassununga SP. O delineamento experimental foi inteiramente casualizado em arranjo fatorial de tratamentos, sendo um fator o fornecimento dos MOS para fêmeas e o segundo para os leitões. Foram utilizadas 17 primíparas prenhes, tratadas a partir de 81 &plusmn;1,36 dias de gestação com 0,2% de MOS na dieta. Aos 82 &plusmn; 1,36 dias de gestação as fêmeas foram submetidas à coleta de sangue e à vacinação contra rinite atrófica progressiva, sendo pesadas quinzenalmente até a transferência para a maternidade, aos 109 &plusmn; 1,36 dias de gestação. Na segunda quinzena as fêmeas tratadas apresentaram uma vantagem numérica no ganho de peso médio diário (P=0,063). Durante o parto foram colhidas amostras de sangue e colostro para titulação de anticorpos contra o antígeno vacinal, sendo consideradas positivas 66,67% das fêmeas MOS e 42,86% das fêmeas controle. Os leitões aleitados por fêmeas tratadas tiveram um maior ganho numérico de peso na primeira semana (p=0,0614), significativo na segunda semana (p=0,047). Na terceira semana foi introduzido o segundo fator através do oferecimento de alimento sólido (0,4% de MOS). No período total, do nascimento aos 21 dias, foi observada uma vantagem numérica (p=0,0989) no ganho de peso a favor dos leitões de fêmeas MOS. As fêmeas, contudo, não diferiram quanto à variação de peso ou consumo durante a fase de lactação. Aos 23 &plusmn; 1,91 dias de idade dos leitões foi realizado o desmame abrupto com a transferência dos leitões para unidade de creche, onde foram alojados em gaiolas para 4 animais. O peso ao desmame foi maior nos leitões aleitados por fêmeas tratadas (p<0,0001), bem como em todas as pesagens semanais até a quinta semana pós-desmame. O consumo na primeira semana sofreu influência da suplementação de MOS nas fêmeas e do maior peso à desmama se mostrando superior nesses animais (P=0,049) influenciando diretamente o ganho de peso, superior na segunda semana (p=0,002). O MOS para os leitões aumentou o consumo de alimento (p=0,0784), e a conversão alimentar na segunda semana (p=0,0103). Aos 14 dias pós-desmame foram colhidas amostras de sangue para hemograma e realizada a aplicação oral de 108 UFC de Salmonella Typhimurium. Foram observadas diariamente a consistência das fezes por 28 dias e aferidas as temperaturas retais por 9 dias. Leitões tratados com MOS e os leitões oriundos de fêmeas MOS tenderam a apresentar o pico de hipertermia mais cedo que os demais (p=0,0629 e p=0,0976, respectivamente) e os leitões alimentados com MOS tenderam a ter uma temperatura de pico mais baixa (p=0,0989). Na última semana os leitões de fêmeas MOS apresentaram um maior consumo (p=0,0007) e uma incidência de Salmonella numéricamente inferior em linfonodos mesentéricos e nas fezes colhidas aos 36 dias pós-desafio. Pode-se concluir que a suplementação de MOS para primíparas prenhes e lactentes pode melhorar o desempenho e a saúde entérica de seus leitões nas fases de aleitamento e creche. / The present study evaluated the effects of mananoligossacarides (MOS) as a feed additive on the performance of primiparous sows in late gestation and lactation, as well as on performance and health status of their progeny up to 65 days of age. The experiment was conducted in the Laboratory of Swine Research (FMVZ/USP) Pirassununga SP. For this purpose a completely random factorial design was used, factors which corresponded to (1) feeding sows with MOS and (2) feeding piglets with MOS, characterizing 4 treatments: MM Feeding MOS to both sows and piglets, MC Feeding MOS only to the sows, CM Feeding MOS only to piglets, CC Control diets for both sows and piglets. A total of seventeen pregnant gilts were used, and divided into 2 groups: MOS (n=9) and Control (n=8). MOS gilts had 0,1% MOS added to their diets from 81 &plusmn; 1,36 days of gestation onward. On day 82 &plusmn; 1,36 blood was collected and vaccination against Progressive Atrophic Rhinitis was conducted. Animals were weighted biweekly until 109 &plusmn; 1,36 days of gestation, when transference to the farrowing unit was conducted. Between the first and second weighing, MOS gilts show a numerical advantage in daily weight gain (p=0,063). At farrowing, blood and colostrum samples were collected for determination antibodies titles against vaccine antigen, being considered positive 66,67% and 42,86% of MOS and Control sows, respectively. Piglets nursed by MOS sows had a numerical advantage in weight gain in the first week (p=0,0614), with statistical significance in the second week (p=0,047). On the third week, the second factor was introduced by the offering of solid feed (0,4% MOS). During the suckling period, from birth to 21 days of age, a numerical advantage in weight gain was observed for MOS sows piglets (p=0,0989). The sows themselves did not differ in weight change or feed consumption. At 23 &plusmn; 1,91 days of age, weaning was conducted, as piglets were transferred to nursing facilities and allocated in pens of 4 animals. Weight of MOS sows piglets was higher at weaning (p<0,001) and until the 5th week port-weaning. Feed consumption was affected by MOS supplementation of sows and by the higher weaning weight (p=0,0049), directly influencing weight gain, which was superior in the 2nd week. Feeding MOS to piglets enhanced feed consumption (p=0,0784) and feed conversion (p=0,0103) on the 2nd week. At 14 days of age, blood samples were collected for hemogram analysis and an oral dose of 108 CFU of Salmonella Typhimurium administered. Fecal consistency and rectal temperature were evaluated for 28 and 9 days, respectively. Piglets treated with MOS and MOS sows\' piglets tended to show a peek of hyperthermia earlier (p=0,0629 e p=0,0976, respectively). Piglets fed with MOS tended to show a lower temperature peek (p=0.0989). In the last week, MOS sows\' piglets show a higher feed consumption (p=0.0007) and a numerically inferior Salmonella incidence in mesenteric lymph nodes and feces 36 days after the bacterial challenge. In conclusion, supplementing primiparous sows with MOS during gestation and lactation can enhance their body weight gain and the performance and intestinal health of the offspring during the suckling and nursing period.

Philosophiae Doctor - PhD / Promoter detection, especially in prokaryotes, has always been an uphill task and may remain so, because of the many varieties of sigma factors employed by various organisms in transcription. The situation is made more complex by the fact, that any seemingly unimportant sequence segment may be turned into a promoter sequence by an activator or repressor (if the actual promoter sequence is made unavailable). Nevertheless, a computational approach to promoter detection has to be performed due to number of reasons. The obvious that comes to mind is the long and tedious process involved in elucidating promoters in the &lsquo;wet&rsquo; laboratories not to mention the financial aspect of such endeavors. Promoter detection/prediction of an organism with few characterized promoters (M.tuberculosis) as envisaged at the beginning of this work was never going to be easy. Even for the few known Mycobacterial promoters, most of the respective sigma factors associated with their transcription were not known. If the information (promoter-sigma) were available, the research would have been focused on categorizing the promoters according to sigma factors and training the methods on the respective categories. That is assuming that, there would be enough training data for the respective categories. Most promoter detection/prediction studies have been carried out on E.coli because of the availability of a number of experimentally characterized promoters (+- 310). Even then, no researcher to date has extended the research to the entire E.coli genome. / South Africa

BCG é uma das vacinas mais usadas no mundo. Avanços na manipulação genética têm permitido o seu uso como carreador de antígenos heterólogos, porém o aprimoramento dos sistemas de expressão se faz necessário, sendo o promotor um importante elemento, uma vez que regula o nível de produção do antígeno, induzindo uma resposta imunológica adequada. Avaliamos a atividade de diferentes promotores de micobactérias, como o PAg, PAN, PBlaF*, Phsp60 e um promotor ainda não caracterizado do micobacteriófago L5, usando o gene gfp como repórter da expressão, todos clonados no vetor extracromossomal, pLA71. Foi possível avaliar as cepas de M. smegmatis e BCG fluorescentes para quase todas as construções e alguns plasmídeos pLA71-p mostraram características diferentes dependentes da micobactéria transformada. Numa escala de força de expressão, os diferentes promotores se apresentaram como fraco (pLA71-PAN-gfp), médio (pLA71-PBlaf*-gfp) e forte (pLA71-Phsp60-gfp). Os rBCG foram usados para infecção de macrófagos e a atividade dos promotores não foi afetada após a internalização. Para ensaio de localização, camundongos foram inoculados com BCG e foi possível confirmar a presença de colônias (recombinantes ou não) nos pulmões após 1 e 3 dias de inoculação, por plaqueamento em meio sólido e por microscopia confocal. / BCG is one of the most widely used vaccines in the world. Advances in genetic manipulation have allowed their use as a carrier for heterologous antigens, however the improvement of systems of expression is necessary, the promoter being an important element, since it regulates the expression level of the antigen, inducing an adequate immune response. We evaluated the activity of different promoters of mycobacteria, such as PAg, PAN, PBlaF* and Phsp60, and the not yet characterized promoter of the micobacteriophage L5, using GFP as a reporter gene expression activity, all cloned in the extrachromosomal vector, pLA71. It was possible to evaluate promoters in the M. smegmatis and BCG strains, fluorescent for almost all constructions and some pLA71-p plasmids showed different characteristics dependent on the transformed mycobacterium. The different promoters showed expression levels as weak (pLA71-PAN-gfp), medium (pLA71-PBlaf*-gfp) and strong (pLA71-Phsp60-gfp). The rBCG were used for infection of macrophages and the activity of the promoters wasnt affected after internalization. For BCG location test, mice were inoculated and it was possible to confirm the presence of colonies (recombinant or not) in the lungs after 1 and 3 days after inoculation by plating on solid medium and by confocal microscopy.

Indiana University-Purdue University Indianapolis (IUPUI) / Cyclin Dependent Kinase regulation is often manipulated by cancer cells to promote unlimited proliferation. P27 is an important regulator of Cyclin E/CDK 2, which has been found in low amounts in many types of malignant cancers. Lovastatin has been shown to cause cell cycle arrest in the G1 phase of the cell cycle by increasing the P27 protein. There has been some question, however, if lovastatin regulates P27 at the transcriptional or translational level. Although it has been claimed that P27 expression regulation is due to an IRES located in its 5’UTR, other studies suggested that P27 expression is regulated at the level of transcription. To further investigate the regulation mechanism of P27 expression, the 5’-UTR of P27 and its deletion mutants were examined using a luciferase reporter gene in HeLa cells following exposure to lovastatin. It was found that lovastatin stimulated a significant 1.4 fold increase in its promoter activity of the full length 5’UTR (575). Deletion of 35 nucleotides from the 5’ end of the UTR eliminated the lovastatin-induced increase in promoter activity. Further mapping analyses of the first 35 bases showed that two regions, M1 (575-559) and M3 (543-527), were less sensitive to lovastatin than the other mutated constructs. Since M1 and M3 still showed some activity, a construct was created with deletions in both the M1 and M3 regions. This showed no increase in luciferase activity when exposed to lovastatin. Looking at RNA levels, there was a 1.5 fold increase in RNA when the full length 5’UTR was inserted into HeLa cells and exposed to 81 µM of lovastatin. In contrast, there was no increase in RNA when M1/M3 (575-559; 543-527) was inserted into HeLa cells and exposed to 81 µM of lovastatin. In addition, there was a 1.6 fold increase in endogenous P27 RNA levels after HeLa cells were exposed to 81 µM of lovastatin. In all of these experiments, there seems to be two promoters that work cooperatively: M1 (575-559) and M3 (543-527).

<p> Promoted and supported catalysts were studied with an electron probe microanalyzer. Investigations we re made on promoted fused iron ammonia synthesis catalysts regardirlg the general morphology of the catalysts, the distribution of promoters, and the reduction and poisoning processes . The effect of the promoters on the reduction process , the effect of the impurity silica on the promoter distributions and the nature of the poisoning are discussed. Supported catalysts were prepared by impregnating porous y-alumina spheres with solutions of chromium and/or copper compounds. Concentration profiles for chromium and/or copper , i.e., concentration as a function of distance from the center, were determined on sectioned spheres. Different distributions of these elements on the alumina support were obtained by varying the chemical used, the concentration and the amount of the solution impregnated. Explanations are given for the physical and chemical processes involved during the impregnation . The amount of chromium or copper impregnated is compared with the amount present in each particle. </p> / Thesis / Doctor of Philosophy (PhD)

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