A conversão da proteína prion celular normal (PrPc), cuja função ainda esta sob investigação, para a forma infecciosa (PrPsc) é a causa de algumas doenças neurodegenerativas em humanos e animais. Vários estudos têm sido realizados e mostram que PrPc pode participar de processos normais como memória, estresse oxidativo, neuritogênese e outros. Portanto, a elucidação dos processos de regulação de sua expressão é importante tanto para definir uma estratégia para controlar a infecção quanto para entender melhor a função fisiológica de PrPc. Este trabalho tem por objetivo avaliar a expressão do gene de PrPc, a partir da regulação da atividade de seu promotor frente a drogas que foram eleitas de acordo com a composição dos elementos de resposta a fatores de transcrição nele contidos. Para isto o promotor foi clonado em um vetor contendo o gene \"reporter\" de luciferase, células C6 e PC-12 foram transfectadas e clones com expressão estável de luciferase foram selecionados. Os resultados dos tratamentos dos clones celulares mostram que éster de forbol (TPA) e AMPc induzem a atividade do promotor de 1,5 a 3 vezes, ácido retinóico (RA) diminui esta atividade em cerca de 50% enquanto que NGF e Dexametasona não têm efeito. A dependência da conformação da cromatina na regulação deste gene também foi testada utilizando-se Tricostatina A (TSA), esta droga foi capaz de aumentar de 10 a 4.000 vezes a atividade do promotor, o que foi seguida tanto pela indução de expressão do RNAm quanto da proteína PrPc. Este efeito parece não ser generalizado a todos os promotores uma vez que esta droga não alterou expressão de GAPDH e de β-actina. Quando TPA e AMPc foram associados à TSA uma potencialização do efeito indutor destas drogas foi observada e a associação de RA e TSA mostrou que RA reduz a indução gerada por TSA. Estes novos dados indicam que a regulação de PrPc é extremamente dependente da conformação da cromatina. / Conversion of the cellular normal prion protein (PrPc), whose physiological function is still under investigation, to an infectious form called prion is the cause of some neurodegenerative diseases. Therefore, the elucidation of PrPc gene regulation is important both to define a strategy to control the infection and to better understand PrPc function. We cloned the rat PrPc gene promoter region into a luciferase reporter vector, transfected C6 and PC-12 cells and isolated clones with stable luciferase expression. The phorbol ester TPA and cAMP induced promoter activity by 1.5 to 3 times, retinoic acid decreased it by 50% while NGF and dexamethasone had no effect. We also tested the dependence of chromatin conformation for PrPc promoter activity using Trichostatin A (TSA), which was able to highly increase not only promoter activity but also PrPc rnRNA and protein leveIs. Moreover, the TSA effect seems to be restricted since any alteration was observed regarding GAPDH (Glyiceraldehyde 3-phosphate desydrogenase) and β-actin expression. When cAMP, TPA or retinoic acid were associated with TSA a potentiation of their primary effects was observed. These new data indicate that PrPc gene regulation is highly dependent on disruption of chromatin fiber assembly what permits assess of trascription factors.

O presente trabalho de pesquisa se refere às atividades e às relações desenvolvidas em torno da agroindústria brasileira Korin Agropecuária Ltda. Essa empresa possui uma origem peculiar uma vez que foi fundada com o propósito de colocar em prática os princípios, conceitos e métodos originados da Agricultura Natural, sistema agrícola preconizado por Mokiti Okada (Japão, 1882-1955). Okada enfatizou a necessidade de um perfeito equilíbrio entre as atividades humanas e as forças da natureza, para se alcançar bons resultados na produção, privilegiando a segurança dos alimentos, a interação e o respeito à natureza e a saúde e o bem estar socioeconômico dos agentes participantes deste sistema. Segundo Okada, a Agricultura Natural é um dos pilares de sustentação de uma civilização e sociedade ideais, onde a saúde, a prosperidade e a paz são predominantes. Assim, enquanto método produtivo enquadra-se perfeitamente na ideia de uma agricultura sustentável, pelo não uso de adubos químicos solúveis, agrotóxicos, antibióticos, melhoradores de desempenho e outros insumos industriais, veiculando uma abordagem socioambiental da atividade agrícola, com enfoque na qualidade diferenciada dos alimentos. Especificamente sobre a produção avícola de corte e postura convencional discutimos os problemas decorrentes do uso excessivo de antibióticos e promotores de crescimento, apresentando a evolução técnico-produtiva deste sistema de integração vertical alternativo no qual estas substâncias não são utilizadas e o bem estar animal é considerado. Prioritariamente abordamos, neste trabalho, a sustentabilidade sob o ponto de vista da multifuncionalidade da agricultura, tratando também do desenvolvimento da empresa sob a ótica dos Sistemas Agroalimentares Localizados - SIAL. Considerando essas abordagens, realizamos pesquisa qualitativa junto a 28 produtores predominantemente familiares integrados à empresa, aplicando questionário semiestruturado para explorar e analisar suas atitudes, percepções e experiências em relação aos conceitos e métodos da empresa, ao meio ambiente, segurança alimentar e suas condições socioeconômicas. No que diz respeito à sede da empresa, apresentamos um estudo, que contou com nossa colaboração, sobre a sustentabilidade de seu sistema de produção através de método que utiliza indicadores socioambientais, desenvolvido pela Embrapa Meio Ambiente. Trata-se do APOIA - Novo Rural, voltado para análise de unidades produtivas rurais. A análise integrada de sustentabilidade avalia a gestão ambiental do estabelecimento rural, tendo como base o contexto local e as práticas de manejo adotadas. A agroindústria em questão obteve um excelente índice integrado de sustentabilidade (0,87), justificado pelo seu histórico de mais de 20 anos de práticas relacionadas à Agricultura Natural. Peculiarmente, este complexo em torno da Agricultura Natural está inserido na confluência das divisas de duas unidades de conservação do estado de São Paulo, as Áreas de Proteção Ambiental - APA Corumbataí e a APA Piracicaba, o que promove sinergias com vistas a alcançar modelos ambientalmente adequados de produção agroalimentar. Os dados obtidos neste estudo junto aos avicultores integrados nos permitiram verificar que a prática da Agricultura Natural tem contribuído sinergicamente para que as dimensões da multifuncionalidade da agricultura sejam reconhecidas no território em questão, dinamizando e consolidando um sistema agroalimentar localizado sob uma perspectiva de sustentabilidade na produção agropecuária. / This research study refers to the activities and relationships developed around a Brazilian agrifood company named Korin Agropecuária Ltda. With a peculiar origin, this company was founded with the purpose of putting into practice the principles, concepts and methods originated from the Nature Farming system. NF is an agricultural approach created and advocated by Mokiti Okada (Japan, 1882-1955). Okada emphasized the necessity of a perfect balance between human activities and the nature forces in order to achieve good results in the production, favouring food safety, nature interaction and respect, health, and the social-economic well-being of all players of this system. According to Okada, Nature Farming is one of the pillars of true civilization and an ideal society where health, prosperity and peace are predominant. Thus, while a productive method, perfectly fits in the idea of sustainable agriculture since its methodological aspects as the non-use of chemical fertilizers, pesticides, antibiotics, growth promoters and other synthetic inputs, leading to a social-environmental approach to agriculture, with a focus on differentiated quality food. Regarding chicken meat and eggs regular production, we discuss the concerns about the overuse of antibiotics and growth promoters, showing the technical evolution of this particularly verticalized system where these substances are not used and the animal welfare is highly take into consideration. As a priority, we address in this work, sustainability from the point of view of the multifunctionality of agriculture and also addressing the company\"s development from the perspective of Localized Agrifood Systems. Considering these approaches, we conducted a qualitative research on twenty eight integrated farmers who are linked to the company, applying semi-structured questionnaire. The objective was to explore and describe their attitudes, perceptions and experiences in relation to the company concepts and methods towards the environment, food safety, and their socio-economic conditions. With regard to corporate headquarters, located in a farm on a rural area, we present a study on the sustainability of the production system through a social-environmental indicator developed by Embrapa, named APOIA - NovoRural. This indicator was designed to analyse rural productive units with focus on the environmental management based on local context. The company have achieved an excellent sustainability index (0.87), due to its history for more than twenty years of Nature Farming\'s handling. Peculiarly this Nature Farming complex is inserted at the confluence of two conservation units of the state of São Paulo, called Environmental Protected Areas of Corumbataí and of Piracicaba, which reinforces the need to search for environmentally suitable models of agrifood production. The data obtained from this study with the integrated farmers have allowed us to verify that the dimensions of multifunctionality of agriculture are being recognized within this particular territory, streamlining and consolidating this agrifood localized system under a sustainability perspective in agriculture.

A metilação do DNA é uma alteração epigenética que atua na regulação da expressão gênica. A deficiência de vitaminas (cobalamina, B6 e folato) pode interferir na taxa de metilação. O efeito da deficiência destas vitaminas foi determinado em estudos com cultura de células e em animais. No entanto, são raros os estudos realizados com seres humanos. Os objetivos deste trabalho foram: avaliar a taxa de metilação do DNA de leucócitos na região promotora dos genes Interferon gama (IFNγ), Serpin B5 e Stratifin; verificar se existe associação entre as concentrações das vitaminas e dos metabólitos com a taxa de metilação do DNA dos 3 genes; e analisar quais são os fatores de predição para a taxa de metilação do DNA (variável dependente) considerando como variáveis independentes os valores séricos das vitaminas e metabólitos, em mulheres com idades gestacionais de 16, 28 e 36 semanas. Cento e oitenta e três mulheres foram convidadas a participar desse estudo, porém apenas 96 completaram o estudo prospectivo. Foram determinadas as concentrações séricas da cobalamina (Cbl), folato, vitamina B6, S-adenosilmetionina (SAM), S-adenosilhomocisteína (SAH), ácido metilmalônico (MMA), homocisteína total (tHcy) e folato eritrocitário. A taxa de metilação nos 3 genes foi determinada por qMSP (Quantitative Methylation Specific - Polimerase Chain Reaction). Várias mulheres estavam fazendo uso de suplementação com ácido fólico e/ou polivitamínicos. Diante deste fato foram formados 4 subgrupos: Grupo 1 (constituído por mulheres que não usaram suplementação), Grupo 2 (mulheres que usaram suplementação em todas as idades gestacionais estudadas - 16, 28 e 36 semanas), Grupo 3 (mulheres que usaram suplementação no início da gestação até a 16ª semana) e Grupo 4 (mulheres que usaram suplementação na 16ª e 28ª semana gestacional). Não houve diferença entre as taxas de metilação do DNA dos genes IFNγ, Serpin B5 e Stratifin durante o período gestacional estudado. As taxas de metilação no gene IFNγ do grupo 1 foram maiores, quando comparadas as taxas dos demais grupos. Em modelos de regressão linear múltipla, considerando o período gestacional como um todo, apenas a vitamina B6 e a tHcy foram diretamente associadas aos valores da taxa de metilação do gene IFNγ. No entanto, a vitamina B6 foi inversamente associada, enquanto que tHcy esteve diretamente associada aos valores da taxa de metilação do gene Stratifin. A taxa de metilação não sofre alterações durante a gestação; a vitamina B6 e a tHcy foram os fatores de predição para as taxas de metilação do DNA na região promotora dos genes IFNγ e Stratifin. / DNA methylation is an epigenetic modification that regulates gene expression. Cobalamin, vitamin B6 and folate deficiencies can impair the DNA methylation index. Studies involving cultured cells and animals have evaluated the effect of vitamin deficiencies in DNA methylation. However, few studies were conducted in humans. The goals of this study were: to evaluate the leukocyte DNA methylation index in the promoter region of interferon gamma (IFNγ), Serpin B5 and Stratifin genes; to assess the association between vitamins and metabolites concentrations and DNA methylation index in three genes; and to examine the predictive factors for DNA methylation index using as independent variables: serum folate, serum cobalamin, serum vitamin B6, erythrocyte folate, methylmalonic acid (MMA), total homocysteine (tHcy) in three gestational ages (16th, 28th and 36,th weeks). A hundred eighty three women were included, but only 96 completed the prospective study. The serum concentrations of cobalamin, folate, vitamin B6, S-adenosylmethionine (SAM), Sadenosylhomocysteine (SAH), MMA, tHcy were determined. The erythrocyte folate concentration was also evaluated. The DNA methylation index was determined in three genes by qMSP (Quantitative Methylation Specific - Polymerase Chain Reaction). Several women were taking folic acid supplementation and/or multivitamins. Four groups were formed according to supplementation use: Group 1 (women who take no supplementation), Group 2 (women who took supplements at 16th, 28th and 36th weeks of pregnancy), Group 3 (women who took supplements in early pregnancy and up to 16 weeks) and Group 4 (women who took supplements in the 16th and 28th week of pregnancy). There was no difference between the DNA methylation index in the IFNγ, Serpin B5 and Stratifin genes during the gestational periods studied. The DNA methylation index in the IFNγ gene in group 1 was higher than those indexes from other groups. In multiple linear regression models considering the gestational periods as a whole, only vitamin B6 and tHcy were directly associated to DNA methylation index in IFNγ gene. However, vitamin B6 was inversely associated, whereas tHcy was directly associated with values of DNA methylation in Stratifin. The DNA methylation index does not change during pregnancy, vitamin B6 and tHcy were the predictors of DNA methylation in the promoter region of IFNγ and Stratifin genes.

Chromatin remodeler proteins exert an important function in promoting dynamic modifications in the chromatin architecture, rendering the transcriptional machinery available to the condensed genomic DNA. Due to this central role in regulating gene transcription, deregulation of these molecular machines may lead to severe perturbations in the normal cell functions. Loss-of-function mutations in the CHD7 gene, a member of the chromodomain helicase DNA-binding (CHD) family, are the major cause of the CHARGE syndrome in humans. The disease is characterized by a variety of congenital anomalies, including malformations of the craniofacial structures, peripheral nervous system, ears, eyes and heart. In this context, several studies have already shown the importance of CHD7 for proper function of the neural stem cells (NSCs). Interestingly, we found that CHD7 mRNA levels are upregulated in gliomas, when compared to normal brain tissue, therefore, we hypothesized that CHD7 might have a role in the pathogenesis of these tumors. To investigate the possible oncogenic role of CHD7 in glioblastoma (GBM), we adopted gain- and loss-of-function approaches in adherent GBM cell lines. Using CRISPR_Cas9 genome editing, we found that CHD7 deletion suppresses anchorage-independent growth and reduces spheroid invasion in human LN-229 cells. Moreover, deletion of CHD7 delayed tumor growth and improved overall survival in an orthotopic xenograft glioma mouse model. Conversely, ectopic overexpression of CHD7 in LN-428 and A172 cells was found to increase cell motility and invasiveness in vitro and LN-428 tumor growth in vivo. RNAseq analysis showed that alterations of CHD7 expression levels promote changes in several molecular pathways and modulate critical genes associated with cell adhesion and locomotion. However, the mechanisms underlying the effects of CHD7 overexpression in glioma tissue are still not understood. Here, we also generated recombinant plasmid with functional CHD7 promoter activity reported by luciferase assay. This powerful tool should enable future studies to determine the direct targeting relationship between different signal transduction pathways and CHD7 geneexpression. In summary, our findings indicate that GBM cells expressing a high level of CHD7 may exist and contribute to tumor infiltration and recurrence. Further studies should warrant important clinical-translational implications of our findings for GBM treatment. / As proteínas remodeladoras de cromatina exercem importante papel, promovendo modificações dinâmicas na arquitetura da cromatina e dando acesso à maquinaria transcricional ao DNA genômico condensado. Devido à esta função central na regulação da transcrição gênica, a desregulação dessas máquinas moleculares pode levar a perturbações graves na função normal das células. Assim, por exemplo, mutações do tipo perda de função no gene CHD7, um membro da família \"chromodomain helicase DNA-binding\" (CHD), são a principal causa da síndrome de CHARGE em humanos. A doença é caracterizada por uma variedade de anomalias congênitas, incluindo malformações das estruturas craniofaciais, sistema nervoso periférico, orelhas, olhos e coração. Neste contexto, vários estudos já mostraram a importância da proteína CHD7 para o funcionamento normal de células-tronco neurais (NSCs). Curiosamente, descobrimos que os níveis de mRNA de CHD7 estão mais fortemente expressos em gliomas, quando comparados ao tecido cerebral normal, portanto, nós hipotetizamos que CHD7 poderia ter um papel na patogênese desses tumores. Para investigar o possível papel oncogênico de CHD7 em glioblastoma (GBM), utilizamos enfoques de ganho e perda de função em linhagens celulares aderentes de GBM. Utilizando a técnica de CRISPR_Cas9 para edição do genoma, demonstramos que a deleção do gene CHD7 suprime o crescimento independente de ancoragem e reduz a invasão de esferóides em células LN-229 humanas de GBM. Além disso, a deleção de CHD7 reduziu o crescimento do tumor e melhorou a sobrevida em modelo de injeção ortotópica xenográfica em camundongo. Por outro lado, verificou-se que a super-expressão ectópica de CHD7 nas células LN-428 e A172 aumenta não só a motilidade celular e a capacidade de invasão in vitro, mas, também, o crescimento do tumor de LN-428 in vivo. A análise de RNA-seq mostrou que o nocauteamento da sequência codificadora de CHD7 e sua super-expressão promovem alterações em diversas vias moleculares, modulando genes críticosassociados à adesão e locomoção celular. No entanto, os mecanismos subjacentes aos efeitos da super-expressão de CHD7 em tecidos de glioma ainda não são compreendidos. Neste trabalho, geramos um plasmídeo recombinante contendo um fragmento da região promotora de CHD7, o qual se mostrou funcional em ensaios de luciferase. Esta ferramenta permitirá que estudos futuros possam identificar a relação direta entre as diferentes vias de transdução de sinal e a expressão do gene CHD7. Em resumo, nossos achados indicam que células de GBM expressando um alto nível de CHD7 podem existir e contribuir para a infiltração e recorrência do tumor. Estudos posteriores deverão avaliar as possíveis implicações dos resultados apresentados neste trabalho para a translação clínica no tratamento de pacientes com GBM.

O remifentanil é um opióide potente comparável ao fentanil, mas, possui rápida eliminação plasmática e por isso necessariamente deve ser adiministrado por infusão contínua. A administração transdérmica pode liberar fármacos de forma prolongada, contínua e controlada. Características que facilitariam a administração do remifentanil, contornariam sua limitação farmacocinética e ampliariam os casos em que este fármacos seria útil. Lipossomas deformáveis são formados por fosfolipídios e por tensoativo que normalmente é um surfactante de cadeia simples que aumenta a flexibilidade da bicamada lipídica da membrana e torna os lipossomas deformáveis. Os lipossomas deformáveis são capazes de disponibilizar, de forma expressiva, várias substâncias transdermicamente incluindo macromoléculas e podem ser um eficiente sistema de liberação controlada de remifentanil. O presente estudo desenvolve diferentes dispersões de lipossomas deformáveis de remifentanil e caracteriza essas dispersões quanto a tamanho de diâmetro dos lipossomas, eficiência de encapsulação dos lipossomas, elasticidade da membrana, permeação e retenção cutânea in vitro. As dispersões de lipossomas deformáveis preparadas foram compostas por remifentanil, fosfatidilcolina de soja, tensoativo tween 80 ou tween 20 e etanol a 10% ou a 20% e foram preparadas por dois métodos: sonicação e hidratação de filme e sonicação. O tamanho e a distribuição de tamanho foram obtidos por espalhamento dinâmico de luz, a eficiência de encapsulação por diálise, a elasticidade por filtração, seguido de determinação do tamanho. Células de difusão de Franz e pele de orelha de porco foram usadas para estudar a permeação e a retenção cutânea in vitro. Os lipossomas deformáveis preparados apresentaram pequeno diâmetro (40 a 71 nm) quando comparado a outros na literatura e seus tamanhos foram dependentes da formulação. Todos os lipossomas deformáveis preparados apresentaram boa elasticidade e permeação dependente da eficiência de encapsulação e do tamanho de diâmetro. A solução hidroetanólica com remifentanil e a mistura física dos excipientes com remifentanil não apresentaram permeação e retenção. A eficiência de encapsulação dos lipossomas deformáveis é coerente com outros resultados na literatura e foi dependente da formulação e do método de preparação. A avaliação da permeação de dispersões de lipossomas deformáveis com remifentanil não encapsulado foi feita para se obter informações sobre os mecanismos de ação pelos quais estes lipossomas agem. Verificou-se que o transporte de substâncias encapsuladas nos lipossomas foi o mecanismo de maior importância / Remifentanil is a potent opioid comparable to fentanyl. Remifentanil has rapid systemic elimination and, therefore, it necessarily has to be administered by continuous parenteral infusion. Transdermal delivery can provide extended, continuous and controlled delivery of drug. Characteristics that could make the remifentanil administration easier, could resolve its phamacocinetcs limitation and could enlarge the cases that this drug would be useful. Deformable liposomes consist of phospholipid and surfactant that is often a single chain surfactant that increases the membrane lipid bilayer flexibility and makes the liposome deformable. Deformable liposomes are able to expressively improve skin delivery of many drugs, including macromolecules and could be an efficient controlled delivery system of remifentanil. The aim of this study has been to develop different deformable liposomes dispersions of remifentanil and to characterize those dispersions by liposome diameter size, liposome entrapment efficiency, membrane elasticity and in vitro skin permeation and deposition. The deformable liposomes consisted of remifentanil, soybean phosphatidylcholine, surfactant tween 80 or tween 20, and ethanol at 10% or 20%. They have been prepared by two methods: sonication or conventional mechanical dispersion and sonication. The size was determined by light scattering, the entrapment efficiency by dialysis, the membrane elasticity by filtration and size determination. Franz diffusion cells and ear porcine skin wore used to evaluate the in vitro skin permeation and the skin deposition. The deformable liposomes showed small size (40 a 71 nm) compared with others from literature and their sizes wore dependent of the formulation. All deformable liposomes showed good membrane elasticity and showed permeation that was dependent of the formulation and diameter size. The remifentanil hidroetanolic solution and the excipients physical mixture with remifentanil did not have any permeation and retention. The deformable liposomes entrapment efficiency has been coherent with others literature results and also was dependent of the formulation and preparation method. The permeation of deformable liposomes dispersions wore evaluated to get information about the deformable liposome action mechanisms. The transport of encapsulated substances on liposome was the action mechanism of major importance.

Transcription initiation is an important step in the process of gene regulation in prokaryotes. Promoters are stretches of DNA sequence that are present in the upstream region of transcription start sites (TSSs), where RNA polymerase and other transcription factors bind to initiate transcription. Recent advancement in sequencing technologies has resulted in huge amount of raw data in the form of whole genome sequences. This sequence data has to be annotated, in order to identify coding, non-coding and regulatory regions. Computational tools are useful for a quick and fairly reliable annotation of many genome sequences. Promoter prediction is an important step in genome annotation process which is needed, not only for the validation of predicted genes, but also for the identification of novel genes, especially those coding for non-coding RNA, which are missed by gene prediction programs. DNA sequence dependent structural properties such as DNA duplex stability, bendability and intrinsic curvature have been found to be associated with promoter regions in all domains of life. The work presented in this thesis focuses on the analysis of these structural features in the promoter regions of published prokaryotic transcriptome data. Furthermore, promoters were predicted using these structural features and their role in gene expression were studied. The organization of thesis is as follows. An overview of transcription machinery of prokaryotes, promoter architecture, available promoter prediction programs and sequence dependent structural features is presented in chapter 1. Chapter 2 describes the datasets and methods used in entire study. Structural features of promoters associated with primary and operon TSSs of H.pylori26695 genes and their orthologs (chapter 3) Promoter regions in genomic sequences from all domains of life show similar trends in their structural properties such as stability, bendability, curvature. This chapter dis-cuss the DNA duplex stability and bendability of various classes of promoter regions (based on the identification of different classes of transcription start sites, viz. primary, secondary, internal, operon TSSs etc, in transcriptome study) of Helicobacter pylori 26695 strain. It is found that the primary TSS and operon associated TSS promoters show significantly strong structural features in their promoter regions. DNA free energy based promoter prediction tool PromPredict has been used to annotate promoters of different classes and very high recall values (80%) are obtained for primary TSS. Orthologous genes from 10 different strains of H. pylori show conservation of structural properties in promoter regions as well as coding regions. PromPredict annotates promoters of orthologous genes with very high recall and precision values. DNA duplex stability of promoter region is conserved in the orthologous genes in 10 different strains of Helicobacter pylori genome. Sequence dependent structural features of promoters in prokaryotic transcriptome (chapter 4) Next-generation sequencing studies have revealed that a wide range of transcripts such as primary, internal, antisense and non-coding RNA, are present in the prokaryotic transcriptome and a large fraction of them are functionally involved in various regulatory activities. Identification of promoters associated with different transcripts is important for characterization of transcriptome. The current chapter discusses DNA sequence dependent structural properties like stability, bendability and curvature in the promoter region of six different prokaryotic transcriptomes (Helicobacter pylori, Anabaena, Synechocystis, Escherichia coli, Salmonella and Klebsiella). Using these structural features, promoters associated with different category of transcripts were predicted, which constitute an integral part of the transcriptome. Promoter annotation using structural features is fairly accurate and reliable as compared to motif-based approach since different category of transcripts show poor sequence conservation in the promoter region. Most importantly, it is universal in nature unlike sequence-based approach that is generally organism specific. Role of sequence dependent structural properties in gene expression in prokaryotes (chapter 5) DNA duplex stability, bendability and intrinsic curvature play crucial roles in the process of transcription initiation. Hence, in order to understand the relationship be-tween these structural features and gene expression, the relative differences in stability, bendability and curvature in the promoter regions of high and low expressed genes were studied. It is found that these features are relatively accentuated in the promoter regions associated with high gene expression as compared to low gene expression. Promoter regions associated with high gene expression are annotated more reliably using DNA structural features, compared to those for low gene expression. Sequence dependent structural properties in the promoter region of essential and non-essential genes of the prokaryotes (chapter 6) Essential genes are the minimal possible set of genes required for the survival of organism. These sets of genes can be identified by experiments such as single gene deletion and transposon mediated inactivation. Here, the analysis of DNA duplex stability and bendability in the promoter regions of essential and nonessential genes of prokaryotes is reported. It is found that the average free energy and bendability pro-files are distinct in the promoters regions of essential and nonessential genes. Whole genome promoter predictions using in-house program, PromPredict, for essential and nonessential genes has also been carried out. Chapter 7 present the summary and conclusion of the entire thesis work followed by future perspectives in the field. Optimization of PromPredict algorithm and updating PromBase with newly sequenced genomes (Appendix A) PromPredict is an in-house program, which is based on the relative stability of the DNA in flanking regions. It was found to perform well in predicting promoters across all organisms. In previous studies, it was observed that for organisms having low genomic GC content (<35%), promoter prediction resulted in low precision values, which indicates higher false positive rate. Threshold values of PromPredict algorithm were re-vised in order to optimize the algorithm with low false positive rate. PromBase is a comparative genomics database of microbial genomes. It stores different genomic and structural properties of the microbial genomes. It also displays the predictions obtained from PromPredict in a graphical as well as tabular format. Newly sequenced genomes were downloaded from NCBI and processed using in-house programs and added to the mysql database (back end of the PromBase). Stability profiles for predictions were also added for the RNA coding genes, earlier only profiles for protein coding genes were displayed. Comparative genomics of asymmetric gene orientation in prokaryotes (Appendix B) Transcription proceeds in 5’ to 3’ direction on the template strand, hence it provides directionality. Prokaryotic genomes show asymmetry in gene orientation on leading and lagging strands. The different phyla of prokaryotes were analyzed in terms of asymmetry in gene orientation. It is found that organisms belonging to a particular phyla known as “Firmicutes”, show high asymmetry in gene orientation, which are known to have different DNA polymerase systems for replication.

L’objectif de cette thèse de doctorat a consisté à déterminer l’influence des conditions réactionnelles du vaporeformage de l’éthanol (ESR), de la dispersion du catalyseur et de la promotion par le potassium sur l’état de la surface. Ce travail a aussi aidé à comprendre l’influence de ces facteurs sur les propriétés catalytiques. Nous avons utilisé les catalyseurs à base de cobalt (promus et non promus par le potassium) supportés à l’oxyde de cérium et à l’oxyde de zirconium à faible et à forte dispersion. Les changements de l’état de la surface des catalyseurs pendant la réaction d’ERS ont été étudiés à travers la spectrométrie photoélectronique X (XPS), alors que les changements des produits ont été analisés en utilisant la spectrométrie de masse et la chromatographie en phase gazeuse. Le catalyseur supporté sur oxyde de cérium à forte dispersion a été caractérisé sous une basse pression (0.2-20 mbar) avec le rapport molaire eau/éthanol de 3/1 (420ºC). Les autres tests ont été faits sur tous les catalyseurs sous une pression totale de 1 atm avec les rapports molaires de 3/1, 9/1, 12/1 (420ºC). Nous avons utilisé un mélange eau/éthanol dans un rapport molaire de 12/1 pour étudier les changements de l’état de la surface de tous les catalyseurs dans le temps. Il a été démontré que la sélectivité d’ESR des catalyseurs pour produire des gaz et pour déposer le carbone est réglée par la concentration des groupes hydroxyles sur la surface. Quant aux catalyseurs promus, elle dépend aussi de la concentration Kδ+–Osurfδ-. / The aim of the thesis was determination the influence of the ethanol steam reforming (ESR) reaction conditions, catalyst’s dispersion and potassium promotion on a surface’s composition and understanding the influence of these changes on catalysts’ performance. Cobalt-based catalysts (unpromoted and promoted with potassium) with low- and high-dispersed ceria and zirconia supports were used. The changes of the surface state of catalysts during the ESR were studied by means of X-ray photoelectron spectroscopy, whereas the reaction products evolution was followed by mass spectrometer or gas chromatograph. Highly-dispersed ceria-supported catalyst was characterized under low pressure conditions (0.2–20 mbar) with the water/ethanol molar ratio equal to 3/1 (at 420ºC). The other tests were carried out over all catalysts under total pressure of 1 atm with 3/1, 9/1 and 12/1 molar ratios (at 420ºC). The water/ethanol ratio of 12/1 was chosen for studies of the surface state of all catalysts with time-on-stream. It was found that the ESR selectivity to gaseous products and carbon deposition is governed mainly by surface hydroxyl species concentration; in the promoted catalysts together with Kδ+–Osurfδ- surface sites.

Proper development of skeletal muscle occurs through a highly complex process where activation and repression of genes are essential. Control of this process is regulated by timely and spatial expression of specific transcription factors (TFs). Six1 and Six4 are homeodomain TFs known to be essential for skeletal muscle development in mice. Using the C2C12 cell line, a model for skeletal muscle differentiation, I used a functional genomics approach, employing siRNA specific to both these TFs, to characterize their role in skeletal myogenesis. To identify the genes that are regulated by both these TFs, gene expression profiling by microarray of cells treated with siRNA against Six1 and/or Six4 was performed. The knock-down of these TFs caused lower expression of markers of terminal differentiation genes in addition to an impairment of myoblast fusion and differentiation. Interestingly, transcript profiling of cells treated with siRNA against myogenin revealed that several of the Six1 and Six4 target genes are also regulated by myogenin. Through a combination of bioinformatic analyses it was also found that specific knock-down of Six4 causes an up-regulation of genes involved in mitosis and the cell cycle. In summary, these results show that Six1 and Six4 can both independently regulate different genes, but can also cooperate together with other TFs where they play an important role in the proper regulation of skeletal myogenesis.

O colágeno XVIII é um componente das membranas basais com diversos domínios funcionais, como a endostatina e o domínio frizzled, que têm importante papel em processos celulares como proliferação e diferenciação. COL18A1 possui dois promotores alternativos: o promotor 1, que regula a síntese da variante NC11-303, e o promotor 2 responsável pelas variantes NC11- 728 e NC11-493, expressas por hepatócitos. Existe uma variação interindividual da endostatina circulante e da expressão do colágeno XVIII no fígado. A expressão do colágeno XVIII/endostatina foi correlacionada com a progressão tanto do hepatocarcinoma (HCC), quanto da fibrose/cirrose hepática. Elucidar a regulação da expressão de COL18A1 pode auxiliar na compreensão dessa variação interindividual e da progressão dessas doenças. Neste trabalho demos início a caracterização do promotor 2 do COL18A1. Identificamos na seqüência predita como promotora cinco regiões conservadas entre humanos e camundongos. A análise in silico e funcional dessas regiões revelou que os fatores de transcrição, Sp1, Sp3, YY1, Oct-1, C/EBP&#945; e C/EBP&#946;, interagem com as mesmas. Demonstramos que C/EBP&#946;&#61472;aumenta a taxa de transcrição do promotor 2 em hepatócitos, e que existe uma correlação positiva da expressão de NC11-493 com C/EBP&#945;&#61472;e C/EBP&#946;&#61472;em tecido hepático cirrótico e tumoral. As expressões de C/EBP&#945;&#61472;em tecido hepático cirrótico e tumoral estão diretamente correlacionadas, enquanto que os níveis de NC11-493 nos tumores estão inversamente correlacionados com o tamanho dos mesmos. Mostramos a existência de diversos SNPs no promotor 2. O SNP-700T/G, funcional in vitro, afeta a interação de Sp3 e YY1 com essa região regulatória. A deleção da região do SNP indicou que ela possui elementos importantes para a transcrição em hepatócitos, apesar deste SNP não estar relacionado com o nível de expressão do colágeno XVIII em fígado fibrótico ou com susceptibilidade a HCC. O SNP- 700T/G está em desequilíbrio de ligação com o SNPc.1135C/T, no domínio frizzled do colágeno XVIII. Não foi possível elucidar a funcionalidade do SNPs c.1135C/T in vitro, mas os haplótipos formados por esses dois SNPs têm diferentes frequências entre descendentes de europeus e de africanos. Nosso trabalho traz importantes contribuições e abre novas perspectivas para a compreensão da regulação do colágeno XVIII em fígado humano, tanto em situações fisiológicas, quanto em processos fibrogênicos e tumorigênicos¶ / Collagen XVIII is a basal membrane component with several funcional domains, such as endostatin and frizzled domains, which have important roles in cellular processes such as proliferation and differentiation. COL18A1 has two promoter regions: promoter 1, that regulates the synthesis of NC11-303 isoform, and promoter 2, localized in intron 2, responsible for NC11-728 and NC11-493 isoforms expressed by hepatocytes. There is a large interindividual variation in circulating endostatin and in collagen XVIII liver expression. Collagen XVIII/endostatin levels were correlated with hepatocellular carcinoma (HCC) progression, as well as liver fibrosis/cirrhosis, conditions that precede HCC. Elucidating the mechanisms that regulate COL18A1 expression in hepatocytes may help understanding its variation among individuals and liver disease stages, as well as contribute to new treatment strategies. In this work we began to characterize COL18A1 promoter region 2. We identified in the predicted promoter sequence five conserved regions between human and mouse. The in silico and functional analysis of these regions revealed that transcription factors Sp1, Sp3, YY1, Oct-1, C/EBP&#945; and C/EBP&#946; interact with them. We have demonstrated that C/EBP&#946; increases promoter 2 transcription rate in hepatocytes, and that there is a positive correlation of NC11-493 expression with that of C/EBP&#945; and C/EBP&#946; in cirrhotic and tumor liver samples. Non-tumor and tumor C/EBP&#945; expressions positively correlate between themselves, while NC11-493 tumor expression inversely correlates with tumor size. We also showed that there are several SNPs in COL18A1 promoter 2 region. SNP-700T/G, functional in vitro, affects Sp3 and YY1 interaction with the promoter 2 region and deletion of the SNP region indicated that this sequence has important hepatocyte regulatory elements. Our results suggest that this SNP does not significantly affects COL18A1 expression in fibrotic/cirrhotic liver and is not associated with HCC susceptibility. SNP-700T/G is in linkage disequilibrium with SNPc.1135C/T, at collagen XVIII frizzled domain. We could not elucidate SNPc.1135C/T functionality in vitro, but the haplotypes formed by these two SNPs have different frequencies in European and African descendants. In conclusion, our work brings important contributions and opens new perspectives for the comprehension of collagen XVIII regulation in human liver in physiological situations, as well as in fibrotic/cirrhotic and tumorigenic process.

O amadurecimento dos frutos é um processo caracterizado pela ocorrência de diversas alterações bioquímicas que ocorrem em um curto intervalo de tempo e que são importantes para a qualidade desses alimentos. Na banana uma das características mais importantes é o adoçamento do fruto, que ocorre como resultado da degradação do amido e acúmulo de sacarose. Resultados do nosso grupo apontam a &#946;-amilase como uma enzima importante no processo de mobilização do amido, o que também é visto em estudos recentes utilizando Arabidopsis thaliana como modelo, os quais mostram que a principal via de degradação do amido transitório presente nas folhas ocorre pela ação da &#946;-amilase. Entretanto, em bananas, faltam evidências quanto à funcionalidade de um gene de &#946;-amilase, parcialmente isolado da polpa do fruto, e que é expresso durante o amadurecimento e que parece ser modulado por hormônios vegetais. Em vista disso, esse trabalho objetivou realizar a caracterização funcional desse gene, a qual permitiu constatar que esse gene codifica, de fato, para uma proteína capaz de ser endereçada aos cloroplastos. Também foi observado que o promotor desse gene contém motivos regulatórios para os mesmos hormônios previamente relacionados com a modulação da expressão desse gene em bananas. Essas novas evidências reforçam a idéia de que o produto desse gene de &#946;-amilase tem um importante papel no processo de degradação do amido durante o amadurecimento da banana. / Fruit ripening is characterized by several biochemical changes that occur in a short time. These changes account for the color, taste and texture of the edible fruits, which are important postharvest characteristics for the fruit commercialization. In bananas, one of the most important features is the fruit sweetness which is the result of the starch degradation and sucrose accumulation. Results of our research group point &#946;-amylase as an important enzyme in starch degradation process in bananas which is in agreement to recent studies using Arabidopsis thaliana as plant model. These studies show that the main degradation pathway of the transitory starch present in leaves on Arabidopsis plants occurs due &#946;-amylase action. However, in bananas there are no evidences about the functionality of the expression product of a &#946;-amylase gene, which was demonstrated to be modulated by plant hormones and expressed during ripening. In view of this, the aim of this work was to proceed the functional characterization of this gene which was showed to encode for an protein targeted to chloroplasts. It was also observed that its promoter region contains regulatory motifs related to the same plant hormones previously reported to modulate &#946;-amylase expression. These new evidences support the idea that expression of &#946;-amylase gene has an important role in starch degradation during banana ripening.