O Brasil tem a maior produção de cana-de-açúcar do mundo. O cultivo de cana-de-açúcar no Brasil está voltado principalmente para a produção de açúcar ou Etanol e nos últimos anos para a produção de bioeletricidade através da utilização da biomassa do bagaço e da palha. Apesar da importância econômica e do potencial sustentável que a cana-de-açúcar apresenta, o genoma de referência para esta cultura ainda não está disponível na literatura. A principal justificativa para isso está na complexidade do mesmo, em especial pela alopoliploidia e autopoliploidia. De fato esta característica é a principal barreira para o desenvolvimento de novas variedades comerciais. Na literatura há diferentes estratégias que visam contribuir com o conhecimento genômico de cana-de-açúcar sendo mais prevalente dados de transcriptoma e pouca informação sobre o processo de regulação gênica. Além disso, diferente do que é observado em outras culturas comerciais, em cana-de-açúcar não há trabalhos associados com a caracterização in silico da região Promotora, assim como na identificação de sítios de ligação para Fatores de Transcrição (TFBSs). Por esta razão, o nosso trabalho foi direcionado para a caracterização in silico de regiões regulatórias em cana-de-açúcar. Para esta tarefa nós realizamos apenas a rotulação de sequências de DNA não codificante que estavam a upstream de cada gene anotado em cana-de-açúcar. Todos os genes foram selecionados de dados de transcriptoma e a sequência de DNA da região Promotora foi isolada do Genespace de cana-de-açúcar SP80-3280 gerado pelo projeto de sequenciamento do genoma de referência do nosso grupo. A rotulação da região regulatória em cana-de-açúcar foi executada em duas subsequências: Core Promoter e Promotor Proximal. Na região Core Promoter nós realizamos a identificação do sítio de inicio de transcrição (TSS), a estimativa do tamanho da região 5\' UTR e a classificação da região Core Promoter em TATA-box ou TATA-less. Todos os processos foram realizados através da ferramenta TSSPlant. A utilização da ferramenta TSSPlant motivou o desenvolvimento de uma nova ferramenta para predição do sinal de TSS que aqui chamamos de TSSFinder. A ferramenta TSSinder apresentou resultados de predição do sinal de TSS superior aos seus pares, além disso esta ferramenta foi bem sucedida em diferentes organismos como Arabidopsis thaliana, Gallus gallus e Saccharomyces cerevisiae. Na região Promotora Proximal nós realizamos a identificação de TFBSs através de duas metodologias: predição de novo e mapeamento de matrizes de TFBS (PSSM). O processo de predição de novo foi realizada por meio de dois modelos: Maximização da expectativa e Gibbs Sampler e esse processo foi executado apenas para o subgrupo de genes co-expressos ou apenas para o conjunto de sequências homeólogas de cada gene de cana-de-açúcar selecionado. Para o restante das sequências foi realizado apenas o mapeamento das matrizes de TFBSs identificadas durante o processo de predição de novo. Em paralelo todos TFBSs identificados no nosso trabalho foram comparados com o banco de TFBS para plantas. Através desse procedimento foi possível estimar qual classe de Fator de Transcrição está interagindo com o TFBS identificado na região Promotora Proximal dos genes Scdr1, ScSuSy, ScPAL. Com este trabalho, nós cobrimos parte da lacuna observada em estudos in silico paras regiões regulatórias de cana-de-açúcar. Além disso, nós aperfeiçoamos o processo de identificação do sinal de TSS para diferentes organismos; inclusive para plantas Dicotiledôneas e Monocotiledôneas. / Brazil has the highest production of sugarcane in the world. Its cultivation in Brazil is aimed at producing of sugar or ethanol and in recent years, biomass for bioenergy from bagasse and straw. Despite the economic importance and the sustainable potential that sugarcane presents, a reference genome for this crop is not yet available in the literature. One justification for this absence lies in the sugarcane genome complexity, allopolyploidy and autopolyploidy. In fact these characteristics are the main barrier for the development of new commercial varieties. In the literature different strategies aimed at contributing to genomic sugarcane mostly on the transcriptome and little information on the process of gene regulation. Furthermore, unlike other commercial crops, sugarcane has no reported in silico characterization of its promoter regions and identification of Transcription Factor binding sites. For this reason, our work was directed to an in silico characterization of regulatory regions in sugarcane. For this task we performed the labeling of non-coding DNA sequences that were upstream of each gene annotated in sugarcane. All genes were using from transcriptome data and the promoter region DNA sequence was isolated from Genespace of the SP80-3280 reference genome obtained of our group. The labeling of the regulatory region in sugarcane was carried out in two subsections: Core Promoter and Proximal Promoter. In the Core Promoter region we performed the identification of the TSS signal, the estimation of the size of the 5\' UTR region and the classification of the Core Promoter region in TATA-box or TATA-less. All processes were performed using the TSSPlant tool. The use of the TSSPlant tool motivated the development of a new tool to predict the TSS signal that we call TSSFinder. The TSSinder tool presented TSS signal prediction results superior to its peers, moreover this tool was successful in different organisms - Arabidopsis thaliana, Gallus gallus and Saccharomyces cerevisiae. In the Proximal Promoter region we performed the identification of TFBSs through two methodologies: de novo prediction and mapping of TFBS matrices (PSSM). The de novo prediction process was performed using two models: Expectancy Maximization and Gibbs Sampler and this process was performed only for subgroups of coexpressed genes or only for the set of homeologues sequences from each sugarcane gene. For the rest of the sequences only the mapping of the matrices of TFBSs identified during the de novo prediction process was conducted. In parallel all TFBSs identified in our work were compared with the TFBS database for plants. Through this procedure it was estimated which class of Transcription Factor is interacting with the TFBS identified in the Proximal Promoter region of the Scdr1, ScSuSy, ScPAL genes.With this work, we cover part of the gap observed in in silico studies for the regulatory region of sugarcane. In addition, we improved the process of identification the TSS signal for different organisms including dicotyledonous and monocotyledonous plants.

No description available.

Corynebacterium glutamicum is a Gram-positive non-sporulating soil bacterium which is used in biotechnology as a producer of amino acids, nucleotides, biofuels and alcohols. The aim of this thesis was to create a hybrid σ factor of RNA polymerase which would be able to recognize a matching hybrid promoter without effect on expression of the host genes. Based on the σD and σH amino acid sequence, two types of hybrid factors, σDH and σHD , were designed by the sequence combination of sigD and sigH. As an alternative approach, based on the in silico homology modeling, mutations of wild-type σH in the region recognizing the -35 promoter element of the σH -dependent promoter were introduced. Hybrid promoters were constructed by combining the -35 and -10 promoter regions that were derived from the σD - and σH - dependent promoters. Promoter activity was determined by using gfpuv reporter gene under the control of hybrid promoter. The expression of gfpuv in strains with hybrid sigma factors σDH / σHD and hybrid promoters was rather low compared to strains that carried wild-type σ factor and the respective promoter. The aim of the thesis was achieved by using one of the mutant σH factor (σmutH_6A ) with alterations in the region recognizing the -35 element of the σH -dependent promoter. This mutant σ...

In Drosophila, XX embryos express Sxl from the early promoter, SxlPe, and become females. At the same time, XY embryos with only one X chromosome become males. I investigated the role of repression in the establishment of the strict regulation of SxlPe. I found that the co-repressor Groucho, is responsible for amplification of the two-fold difference in X-encoded activator genes into an all-or-nothing difference in Sxl expression. Three new basic helix-loop-helix repressors of Sxl were identified: Hey, Cwo and the prooncogene Myc, all of which are maternally supplied. I have shown that Myc specific repression is important as early as cycle 10, which is 2 cycles earlier than the onset of normal Sxl expression.

An exceptionally effective transformation procedure has been established by using class I embryo-derived rice callus. Every treated callus clump yielded multiple independently transformed plants (average 40 plantlets). Analysis of genomic DNA blots and pollen expressing green fluorescent protein (GFP) from T0 plants revealed that 64% bore a single locus T-DNA insertion in which half had one T-DNA copy. Additive transgene expression was observed fromT0 plants with GFP driven by mUbi1 promoter. Transgenic plants could be rapidly characterized by analyzing GFP pollen from T0 plants without the need for further generations or genomic DNA blot analysis. Agrobacterium tumefaciens-mediated transformation of microspore-derived callus for generating large numbers of T-DNA haploid and doubled haploid(DH) plants has also been investigated. The established transformation procedure resulted in 100% transformation frequency for class I microspore-derived rice callus. Each callus typically yields multiple independent transgenic plants. Genomic DNA blot analysis suggested 98% of the transgenic plants are independent events. About half of the transgenic plants were identified as haploid plants, whereas half are DH hemizygous or homozygous transgenic plants. DH homozygous transgenic plants were obtained from T0plants and confirmed by pollen GFP expression and genomic blot analysis in T0transgenic DH plants. In this study, about 60% ofT0transgenic DH plants had a single locus T-DNA insertion of which 45% bore one T-DNA copy. Furthermore, in a population of over 2,000 haploid and doubled haploid T-DNA plants , about 25% showed phenotypic differences from non-transformed haploid plants. Approximately 5% were seriously phenotypically abnormal including lethal or semi-lethal mutants. This highly efficient transformation procedure using microspore-derived callus could be valuable in speeding up plant breeding and in new gene discovery. Diversification of the mUbi1 promoter led to a minimal promoter that has a similar function as the original mUbi1. Transient and stable transformation as measured from gene expression driven by the minimal promoter suggested that it has a similar function as the original wild type promoter.

名古屋大学博士学位論文 学位の種類:博士(医療技術学) (課程) 学位授与年月日:平成23年3月25日

Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / "December 2002." Typescript. Vita. Includes bibliographical references (leaves 179-198). Also issued on the Internet.

Chlorate and perchlorate pollutions have become a problem in the environment in the last decades. Studies have shown that some bacteria can degrade these substances into unharmful substances such as chloride and molecular oxygen. One of these chlorate degrading bacteria is Ideonella dechloratans that uses chlorate reductase and chlorite dismutase to process chlorate. In the promoter gene sequence of chlorite dismutase there might be regulator sequences such as fumarate and nitrate reductase regulator (FNR) and aerobic respiration control protein (ArcA) that might control the transcription of this enzyme. This promoter sequence was placed in a pBBR1MCS-4-LacZ reporter vector and the possible regulatory sequences were changed through site-directed mutagenesis and tested on activity through beta-galactosidase assays. The changes in the FNR binding sequence gave beta-galactosidase activity that was close to a negative control which might give conclusions that either FNR has an important role or an important part of the promoter was hit. The changes in the ArcA regulator binding sequence did not give such big differences and no certainty can be given if this made important changes to the promoter.

Metabolic engineering has enabled the advancement of biotechnology over the past few decades through the use of cells as biochemical factories. Cellular factories have now provided many new safe and sustainable routes to fuels, pharmaceuticals, polymers, and specialty chemicals. Many of these successes have been achieved using the yeast Saccharomyces cerevisiae, which has been used and shaped by humans for millennia for the production of food and drink. Consequently, S. cerevisiae is one of the most studied eukaryotic organisms in existence, and has established genetic tools for engineering efforts. However, despite the many achievements in metabolic engineering of S. cerevisiae, it is still significantly more difficult to engineer than its prokaryotic counterpart, Escherichia coli. As a result, there is an unmet need to further develop genetic tools in yeast and to do so in the context of metabolic pathway engineering. The work presented here addresses this need through the study and engineering of heterologous gene expression. First, a new biosynthetic pathway is engineered for the production of muconic acid in yeast. Muconic acid is a dicarboxylic acid that can serve as a platform chemical for the production of several bio-polymers. The final muconic acid producing strain was developed through significant metabolic engineering efforts and reached a titer of nearly 141 mg/L muconic acid, a value nearly 24-fold higher than the initial strain. Second, a new method of engineering promoters is presented that allows for increased expression of native promoters and the de novo design of synthetic promoters. The highest expression synthetic promoter is within the top 6th percentile of native yeast promoters. Third, a study of native and synthetic terminators for heterologous gene expression is completed for the first time. This study demonstrates that terminators can tune heterologous expression by as much as an order of magnitude. Fourth, a comparative study of plasmid components dissects the different contributions to plasmid burden, copy number, and gene expression level. Collectively, this work represents a significant step forward in the metabolic engineering of yeast through the establishment of a new pathway and the study and engineering of new tools for heterologous gene expression. / text

The creative potential and work of the performer in new music extends from the moment of conceptualising a concert to the moment of presenting it on stage and comprises many areas between and around those two points. In this thesis I explore the nature of this activity, from the act of playing itself to the commissioning and creating of new pieces, curatorial and collaborative tasks, and the actual concert presentation. I deliberately include interrelations between performer and music promoters, composers and the audience. This leads me to further areas of investigation, namely the question of the performer’s leadership, the charismatic bond with the audience and the creation of what I call “concert aura”. I do not strive to offer all-purpose formulae for the “perfect concert” or for the ideal collaboration. I investigate performance practice not as an absolute art but rather as something embedded in and shaped by social relations and society. Accordingly I understand this thesis as an empirically based study of the questions performers could ask, as well as processes in which they might want to engage, to find meaningful solutions for each new situation. Not all of the questions I raise will be new to each performer, but in their collaborative and concert practice many performers rely on a random, unsystematic, empirical understanding that has been gained by chance. In contrast, I attempt to draw a theoretical basis for my investigation from the fields of psychology, philosophy, media science and sociology, together with the evaluation of my own and other artists’ performance practice. In this way I hope to develop an academic foundation and a comprehensive, systematic approach that can be applied to different collaboration and concert situations. Part 1 of my thesis is concerned with theories and concepts relating to creativity, collaboration and presentation (concert aura and charisma) and aims to establish a firm theoretical basis for application in practice. Part 2 presents, discusses and analyses a selection of case studies from my own practice, considered in relation to the theories discussed in the first part. I conclude by offering guidelines to collaboration and giving a model example of how one might plan a future performance, aiming to create a Gesamtkunstwerk through the totality of the preparation and presentation, its social and psychological connotations. The thesis includes two DVDs with Quicktime Movies and two CDs with recordings of the compositions, commissioned as part of this research and discussed throughout the thesis. The Appendix contains three sample-CDs with an accompanying commentary which give an introduction to contemporary playing techniques for the acoustic and electronic violin and acoustic viola. This CD is intended as a guide for composers to get acquainted with the instruments and was given to each composer involved in this research.