Stem cells have great potential value for treating a number of diseases and conditions, including diabetes, Parkinson's, and spinal cord injuries. Applying stem cells for therapeutic purposes will require an in-depth understanding of their biology, not only of the genes they express, but also the functions of the proteins encoded by the genes. The goal of the project presented in this thesis was to develop a method for high-throughput analyses of protein localization in mouse stem cells. Localization information can provide insight into the functions and biological roles of proteins. <br /><br /> One means of studying protein localization involves creating proteins with a green fluorescent protein (GFP) reporter gene and analyzing their localization using fluorescence microscopy. The research outlined in this thesis focused on developing a system to create a large number of GFP-tagged proteins by constructing a cDNA?GFP fusion library. This involved exploring methods for optimizing cDNA synthesis, designing a retroviral vector (pBES23) for the expression of cDNA?GFP fusions in mouse stem cells, and constructing a cDNA?GFP fusion library in this vector using R1 mouse embryonic stem cell mRNA. The library constructed was not successfully delivered to target cells for GFP-tagged protein expression; it was therefore not possible to characterize protein localization in mouse stem cells. Suggestions are given as to how the methods used in this thesis might be optimized further.

The relatively high level of palmitic acid (22 mol%) in cottonseeds may be due in part to the activity of a palmitoyl-acyl carrier protein (ACP) thioesterase (PATE). In embryo extracts, PATE activity was highest at the maximum rate of reserve accumulation (oil and protein). The cotton FatB mRNA transcript abundance also peaked during this developmental stage, paralleling the profiles of PATE enzyme activity and seed oil accumulation. A cotton FatB cDNA clone was isolated by screening a cDNA library with a heterologous Arabidopsis FatB probe (Pirtle et al., 1999, Plant and Cell Physiology 40: 155-163). The predicted amino acid sequence of the cotton PATE preprotein had 63% identity to the Arabidopsis FatB thioesterase sequence, suggesting that the cotton cDNA clone probably encoded a FatB-type thioesterase. When acyl-CoA synthetase-minus E. coli mutants expressed the cotton cDNA, an increase in 16:0 free fatty acid content was measured in the culture medium. In addition, acyl-ACP thioesterase activity assays in E. coli lysates revealed that there was a preference for palmitoyl-ACP over oleoyl-ACP in vitro, indicating that the cotton putative FatB cDNA encoded a functional thioesterase with a preference for saturated acyl-ACPs over unsaturated acyl-ACPs (FatA). Overexpression of the FatB cDNA in transgenic cotton resulted in elevated levels of palmitic acid in transgenic somatic embryos compared to control embryos. Expression of the anti-sense FatB cDNA in transgenic cotton plants produced some plants with a dwarf phenotype. These plants had significantly smaller mature leaves, all with smaller cells, suggesting that these plants may have less palmitic acid available for incorporation into extraplastidial membrane lipids during cell expansion. Thus manipulation of FatB expression in cotton directly influenced palmitic acid levels. Collectively, data presented in this dissertation support the hypothesis that there indeed is a palmitoyl-ACP thioesterase in cotton, encoded by the isolated FatB cDNA, which plays a major role in regulating palmitic acid content of extraplastidial complex glycerolipids. This work forms the basis for future studies of the influence of palmitic acid content on plant membrane function and provides a key target for the metabolic engineering of palmitic acid levels in storage oils of developing cottonseeds.

Os hormônios glicocorticóides causam uma completa reversão tanto \"in vitro\" quanto \"in vivo\" do fenótipo tumoral para normal, na linhagem celular ST1 de glioma de rato (Armelin & Armelin, 1983) que é um variante da linhagem C6 de glioma de rato. Neste trabalho, foram construídas bibliotecas normalizadas de cDNAs subtraídos e normalizadas, através da metodologia de PCR supressivo, para identificar genes regulados por glicocorticóides, potencialmente envolvidos na reversão fenotípica de células ST1. A construção e análise de duas bibliotecas de cDNAs subtraídos, permitiu o isolamento de 7 genes diferencialmente expressos, alguns dos quais são conhecidos efetores do controle da proliferação celular e/ou diferenciação: trombospondina-1, ciclina G, tirosina fosfatase CL100 e NRP/B. Foi isolado, também, um clone similar a tenascina-X, que apresenta um transcrito de tamanho diferente. O isolamento e caracterização de um cDNA completo correspondente a esse transcrito revelou que é constituído apenas por motivos de fibronectina tipo III e fibrinogênio. A dramática indução deste transcrito por hidrocortisona sugere um papel potencial para esse variante de TN-X no controle da proliferação celular. A análise da expressão destes genes em células P7, um variante de C6 que não sofre reversão fenotípica mediada com glicocorticóides, e em células U87 de glioblastoma humano, revelou que são expressos em níveis muito baixos ou indetectáveis, nestas células indicando que o produto destes genes pode ter um papel importante na reversão fenotípica tumoral-normal. A prevalência de transcritos raros e diferencialmente expressos nestas bibliotecas, mostra que são adequadas para o rastreamento, através de métodos automatizados de microarranjos de DNA, para a validação de clones diferencialmente expressos, podendo revelar, rapidamente, um painel de genes candidatos a estarem envolvidos nas respostas anti-proliferativa e anti-tumoral dos glicocorticóides. / Treatment of the C6 rat glioma cell variant, ST1 cells, with glucocorticoids, leads to a complete reversion of their transformed phenotype and loss of tumorigenic potential. In this work, two subtracted and equalized cDNA libraries were constructed, based on suppressive PCR, in order to identify glucocorticoid-regulated genes related to the ST1 phenotypic reversion. Analysis of both subtracted cDNA libraries revealed 7 differentially expressed genes, some of which are well known negative growth regulators: thrombospondin-1, cyclin G, tyrosine phosphatase CL 100 and NRP/B. In addition, a cDNA clone similar to TN-X, but exibiting a different transcript size was also isolated. Isolation and characterization of a full-length cDNA corresponding to this transcript, revealed that it is composed of fibronectin type III repeats and one single fibrinogen domain. Glucocorticoid induction of this transcript may suggest a different role of TN-X variants in controling cell proliferation. Expression analysis of the C6-derived glucocorticoid-resistant, P7 variant, and of the human glioblastoma U87 cells, revealed very low or undectable levels of these transcripts, further suggesting that their expression is related to the ST1 cells\' phenotypic reversion. Prevalence of rare and differentially expressed transcripts in these libraries suggests that they are likely to be useful in automated systems (DNA microarrays) to rapidly validate differentially expressed clones and to reveal a panel of candidate genes that mediate the anti-proliferative and anti-tumor effects of glucocorticoids.

Submitted by (ana.araujo@ufrpe.br) on 2017-02-10T14:18:44Z No. of bitstreams: 1 Gabriela de Morais Guerra Ferraz.pdf: 1062151 bytes, checksum: b0123f5d7101341f981de975826dc25b (MD5) / Made available in DSpace on 2017-02-10T14:18:44Z (GMT). No. of bitstreams: 1 Gabriela de Morais Guerra Ferraz.pdf: 1062151 bytes, checksum: b0123f5d7101341f981de975826dc25b (MD5) Previous issue date: 2008-02-29 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The Northeast of Brazil, major national producer of bananas, presents as a major limiting factor the stretch of saline soil. The urgency in the development of tolerant cultivars to salinity has led to breeding programs with the aimed to differ the bananas cultivars in diploid genotypes tolerant and sensitive, to be considered viable genetic material for pollination of triploid and tetraploid cultivars. Generally, the present study aimed to characterize diploid genotypes belonging to the AA banana genomic group on salinity and identify genes differentially expressed in contrasting genotypes. At first, this research assessed the growth and accumulation of inorganic ions in 19 diploid genotypes (AA) of bananas subjected to salt stress. The genotypes were grown in a greenhouse and submitted to irrigation with no saline water (0 mM NaCl) or with saline water (100 mM NaCl) for a period of 21 days, when the experiment was collected. To the physiological evaluation, were considered the parameters of growth:leaf area, height, number of leaves, diameter of the pseudostem, weight of the tax burden of fresh and dry, while the chemical assessment observed the concentration of sodium ions, potassium, chloride, magnesium and calcium in limbo leaf, and root pseudostem / subroot. The addition of NaCl to the cultivar solution has, in general, reduced the growth expressed by height, formation of new leaves, leaf area, diameter of the pseudostem and production of fresh and dry materials. This reduction in growth probably due to factors such as: the toxic effect of ions that have been absorbed, the low osmotic and water potential of the cells as well as the use of metabolic energy in the process of osmotic adjustment. In assessing chemical factors, it was possible to observe that the ions concentration has been preferentially presented in root tissue, showing that it is a culture moderately tolerant to salinity.The sodium and chlorine ions increased significantly with the salt increase in differentparts of the plant, while potassium suffered reduction in the leaf lamina and the pseudostem probably because it is associated to the competition with the sodium ion leading to the conclusion that the differential accumulation of ions potentially toxic (sodium and chlorine) and the maintenance of potassium, contribute to the tolerance to salinity in banana. From the physiological analysis and by means of the toxicity symptoms caused by NaCl, it was possible to observe that the effects were less intense in Birmania and Khai Nai On genotypes, than in Sowmuk, its indicates the possibility of use of this plants in programmes for cultivars improvement as tolerant and sensitive, respectively. Seven genotypes were selected for characterization by means of molecular markers RAPD, with the aim to link the physiological responses to salt stress with the formation of groups. We tested 16 random primers. The resultsshow a broad molecular genetic variability among seven genotypes studied. The formation of groups, in part is related to the data obtained in the physiological assessment, keeping in the same group the Birmania and Khai Nai On genotypes.These genotypes that showed higher tolerance to salt stress, when compared to the more salt-sensitive genotype (Showmuk), became distant genetically, so, it can be noticed the possibility of use of this molecular marker in the study of genetic diversity for this species. For the genome functional study, or transcriptoma, this study aimed the detection of possible changes in the pattern of genes expressed in the three diploid genotypes (AA) of banana, Khai Nai On, Burma and Sowmuk with contrasting results when in the absence and presence of high salt concentrations. The Differential Display PCR - ddPCR technique was used to identify and compareregions of bands from fragments of cDNA on agarose gel. A total of 43 fragments of differentially expressed cDNA was generated from the combination of four primers anchors and six random primers. Among the transcripts, 30 were once expressed by the genotype Burma and Kha Nai On, and 13 only by Sowmuk genotype. Theregions of bands with greater consistency of formation, is between 4000 bp and 150 bp. By the results of this research, it was possible to identify some fragments potentially involved to the condition of salinity in banana. The isolation, purification and sequencing of these - Expressed sequence tags - ESTs can assist in the development of new varieties of banana, more adapted to salt stress, in addition to enriching the database of public functional sequences of genomes banks. / O Nordeste do Brasil, maior produtor nacional de banana, apresenta como fator limitante a grande extensão de solos salinos. A urgência no desenvolvimento de cultivares tolerantes a salinidade tem levado programas de melhoramento genético da cultura a classificar os genótipos diplóides de bananeira em tolerantes e sensíveis, por serem considerados materiais genéticos viáveis para polinização de cultivares triplóides e tetraplóides. De modo geral, o presente trabalho buscou caracterizar genótipos diplóides pertencentes ao grupo genômico AA de bananeira quanto a salinidade além de identificar genes diferencialmente expressos nos genótipos contrastantes. Na primeira fase desta pesquisa, foi avaliado o crescimento vegetativo e o acúmulo de íons inorgânicos em 19 genótipos diplóides (AA) de bananeiras submetidas a estresse salino. Os genótipos foram cultivados em casa de vegetação e submetidos à irrigação com água não salina (0 mM de NaCl) ou águasalina (100 mM de NaCl) durante um período de 21 dias, quando foi coletado o experimento. Para a avaliação fisiológica, foram considerados os parâmetros de crescimento: área foliar, altura, nº de folhas, diâmetro do pseudocaule, peso da matéria fresca e peso da matéria seca; enquanto que a avaliação química observou a concentração dos íons sódio, potássio, cloro, magnésio e cálcio no limbo foliar, pseudocaule e raiz/rizoma. A adição de NaCl à solução de cultivo, provocou, de modo geral, redução no crescimento expresso pela altura, formação de novas folhas, área foliar, diâmetro do pseudocaule e produção de matéria fresca e seca, provavelmente devido a fatores como: o efeito tóxico dos íons que foram absorvidos; o baixo potencial osmótico e hídrico das células; bem como a utilização de energia metabólica no processo de ajustamento osmótico. Na avaliação química, foi possívelobservar que a concentração dos íons foi preferencialmente no tecido radicular, reafirmando tratar-se de uma cultura moderadamente tolerante a salinidade. Os íonssódio e cloro aumentaram significativamente frente ao incremento salino nas diferentes partes da planta, enquanto o potássio sofreu redução no limbo foliar e no pseudocaule, possivelmente por estar associado a competição com o íon sódio levando a conclusão de que o acúmulo diferencial de íons potencialmente tóxicos (sódio e cloro) e a manutenção do potássio, contribuem para a tolerância à salinidade em bananeira. A partir da análise fisiológica e por meio da sintomatologia de toxidez provocada pelo NaCl, foi possível observar que os efeitos foram menos intensos nos genótipos Birmânia e Khai Nai On, do que no Sowmuk, indicando possíveis plantas a serem utilizadas nos programas de melhoramento da cultura como tolerantes e sensível, respectivamente. Sete genótipos foram selecionados para caracterização por meio de marcadores moleculares RAPD, buscando-se relacionar as respostas fisiológicas ao estresse salino com a formação de grupos.Foram testados 16 primers randômicos. Os resultados moleculares mostram uma ampla variabilidade genética entre os sete genótipos estudados. A formação dos agrupamentos, em parte correspondeu aos dados obtidos na avaliação fisiológica, mantendo em um mesmo grupo os genótipos Birmânia e Khai Nai On. Estes genótipos que apresentaram maior tolerância ao estresse salino, quando comparados com a mais sensível ao sal (Showmuk), mostraram-se distantes geneticamente, o que vem a demonstrar a possibilidade de utilização deste marcador molecular no estudo da diversidade genética para esta espécie. Para o estudo do genoma funcional, ou transcriptoma, o presente trabalho objetivou a detecção das possíveis alterações no padrão de genes expressos nos três genótipos diplóides (AA) de bananeira, Khai Nai On, Birmânia e Sowmuk, com respostascontrastantes quando na ausência e presença de alta concentração salina. A técnica de Differential Display PCR – ddPCR foi utilizada para identificar e comparar regiões de bandas de fragmentos de cDNA em gel de agarose. Um total de 43 fragmentosde cDNA diferencialmente expressos foram gerados a partir da combinação de quatro primers âncoras e seis primers aleatórios. Dentre os transcritos, 30 foram expressos unicamente pelos genótipos Birmânia e Kha Nai On, e 13 apenas pelo genótipo Sowmuk. As regiões de bandas com maior consistência de formação, encontraram-se entre 4000 pb e 150 pb. Pelos resultados deste trabalho, foi possível identificar alguns fragmentos potencialmente envolvidos em respostas à condição de salinidade em bananeira. O isolamento, purificação e sequenciamento destes –Expressed sequence tags - ESTs poderá auxiliar no desenvolvimento de novos cultivares de bananeira, mais adaptados ao estresse salino, além de enriquecer a base de dados de bancos públicos de seqüências de genomas funcionais.

No description available.

Major recent improvements in biotechnology have led to an accelerated production of DNA sequences. The completion of the human genome sequence, along with the genomes of more than two hundred other species, has marked the arrival of the genome era. The ultimate goal is to understand the structure and function of genomes and their genes. This thesis has focused on the computational analysis of complementary DNA (cDNA) sequences. These are copies of mRNA transcripts that correspond to the coding regions of genomes. Studying the expression patterns of genes is essential for understanding gene function. Many gene expression profiling techniques generate short sequence tags that derive from transcripts. A pilot study was performed to assess the feasibility of using the pyrosequencing platform for gene expression analysis. The sequences generated by pyrosequencing in most cases (≈ 85%) were long enough (&gt; 18 nucleotides) to uniquely identify the corresponding transcripts through database searches. Aspects of transcript identification by short sequence tags were further investigated in a number of public databases, revealing that a tag length 16-17 nucleotides was sufficient for unique identifi- cation. Longer transcript representations are obtained from expressed sequence tag (EST) sequencing. Method development for the analysis and maintenance of large EST data sets has been performed on data from poplar, which is a tree of commercial interest to the forest biotechnology industry. In 2003 a large ESTsequencing project reached &gt; 100 000 reads, providing a unique resource for tree biology research. ESTs have been grouped into clusters and singletons that represent potential genes. Preliminary analyses have estimated gene content in Populus to be very similar to that of model organism Arabidopsis thaliana. EST data collections provide a rich source for mining polymorphisms. A software application was developed and applied to EST data from two Populus species, and candidate single nucleotide polymorphisms (SNPs) were recorded. A study of genetic variation between the species revealed a striking similarity, with orthologous pairs being &gt; 98% identical on the protein level. Keywords: cDNA, EST, gene expression, SNP, SAGE, polymorphism, assembly, clustering, DNA sequencing, pyrosequencing, mRNA transcript, orthology, tree biotechnology, restriction enzyme

Promoter hypermethylation mediated gene silencing is a frequent epigenetic finding in many cancers that affects genes known to have important roles in several aspects of cell biology. Hematological malignancies have been reported to harbor multiple genes aberrantly silenced by promoter hypermethylation and as a result, cytosine analogs known to inhibit the DNA methylation machinery are currently being evaluated in clinical trials. As such, the general goal of this thesis was to identify genes silenced by promoter hypermethylation in human acute myeloid leukemia (AML) and to study the mechanism of promoter hypermethylation mediated gene silencing. Interestingly, the cyclin dependent kinase inhibitor p15 was found to be methylated at a high frequency in AML patients and cell lines in association with a lack of detectable p15 mRNA. Treatment with the cytosine analog 5-Aza-2-deoxycytidine (5-Aza-dC) in vitro resulted in promoter demethylation and p15 mRNA re-expression, which was associated with a release of a transcriptionally repressive complex at the p15 promoter. Importantly, 5-Aza-dC treatment also reversed specific histone amino-terminal modifications at the p15 promoter which are normally associated with transcriptionally inactive chromatin regions, implicating chromatin remodeling in promoter hypermethylation mediated gene silencing. The recently discovered DNA methylation inhibitor, zebularine considered more stable than 5-Aza-dC was also able to reconstitute p15 mRNA in vitro in association with promoter demethylation, regional enrichment of histone acetylation, and growth inhibition. To identify novel genes silenced by promoter hypermethylation in AML, cDNA microarray analysis was employed following in vitro pharmacological inhibition of DNA methylation and histone deacetylation. Of note, four genes from the metallothionein family of cysteine rich small molecules were consistently upregulated following drug treatment and further evaluation identified the gene MT1H to be hypermethylated at a high frequency in AML patients and cell lines. Taken together, the data suggests that aberrant promoter hypermethylation mediated gene silencing occurs in multiple genes from different gene families during the molecular pathogenesis of human AML. Furthermore, the mechanism of promoter methylation mediated transcriptional silencing acts in concert with specific histone modifications which, importantly, can be reversed by treatment with pharmacological inhibitors of DNA methylation.

Stem cells have great potential value for treating a number of diseases and conditions, including diabetes, Parkinson's, and spinal cord injuries. Applying stem cells for therapeutic purposes will require an in-depth understanding of their biology, not only of the genes they express, but also the functions of the proteins encoded by the genes. The goal of the project presented in this thesis was to develop a method for high-throughput analyses of protein localization in mouse stem cells. Localization information can provide insight into the functions and biological roles of proteins. <br /><br /> One means of studying protein localization involves creating proteins with a green fluorescent protein (GFP) reporter gene and analyzing their localization using fluorescence microscopy. The research outlined in this thesis focused on developing a system to create a large number of GFP-tagged proteins by constructing a cDNA?GFP fusion library. This involved exploring methods for optimizing cDNA synthesis, designing a retroviral vector (pBES23) for the expression of cDNA?GFP fusions in mouse stem cells, and constructing a cDNA?GFP fusion library in this vector using R1 mouse embryonic stem cell mRNA. The library constructed was not successfully delivered to target cells for GFP-tagged protein expression; it was therefore not possible to characterize protein localization in mouse stem cells. Suggestions are given as to how the methods used in this thesis might be optimized further.

Promoter hypermethylation mediated gene silencing is a frequent epigenetic finding in many cancers that affects genes known to have important roles in several aspects of cell biology. Hematological malignancies have been reported to harbor multiple genes aberrantly silenced by promoter hypermethylation and as a result, cytosine analogs known to inhibit the DNA methylation machinery are currently being evaluated in clinical trials. As such, the general goal of this thesis was to identify genes silenced by promoter hypermethylation in human acute myeloid leukemia (AML) and to study the mechanism of promoter hypermethylation mediated gene silencing. Interestingly, the cyclin dependent kinase inhibitor p15 was found to be methylated at a high frequency in AML patients and cell lines in association with a lack of detectable p15 mRNA. Treatment with the cytosine analog 5-Aza-2-deoxycytidine (5-Aza-dC) in vitro resulted in promoter demethylation and p15 mRNA re-expression, which was associated with a release of a transcriptionally repressive complex at the p15 promoter. Importantly, 5-Aza-dC treatment also reversed specific histone amino-terminal modifications at the p15 promoter which are normally associated with transcriptionally inactive chromatin regions, implicating chromatin remodeling in promoter hypermethylation mediated gene silencing. The recently discovered DNA methylation inhibitor, zebularine considered more stable than 5-Aza-dC was also able to reconstitute p15 mRNA in vitro in association with promoter demethylation, regional enrichment of histone acetylation, and growth inhibition. To identify novel genes silenced by promoter hypermethylation in AML, cDNA microarray analysis was employed following in vitro pharmacological inhibition of DNA methylation and histone deacetylation. Of note, four genes from the metallothionein family of cysteine rich small molecules were consistently upregulated following drug treatment and further evaluation identified the gene MT1H to be hypermethylated at a high frequency in AML patients and cell lines. Taken together, the data suggests that aberrant promoter hypermethylation mediated gene silencing occurs in multiple genes from different gene families during the molecular pathogenesis of human AML. Furthermore, the mechanism of promoter methylation mediated transcriptional silencing acts in concert with specific histone modifications which, importantly, can be reversed by treatment with pharmacological inhibitors of DNA methylation.

Natural products have long furnished medical science playing a significant role in drug discovery and development. Their importance notwithstanding, it is estimated that less than 1% of microorganisms can be cultivated from environmental sources using standard laboratory techniques. It is therefore necessary to develop biochemical and genetic techniques to access these uncultivable genomes. Here as a point of departure toward this goal, two cDNA libraries of two microorganisms were constructed along with five fosmid libraries with DNA isolated from marine environmental samples. We describe the construction of cDNA libraries from marine microbial species and detail experiments to exploit these libraries for their natural product biosynthetic pathways and other metabolic enzymes they harbor. However, no useful biosynthetic pathways were detected within the cDNA libraries. Genetic selection by complementation was additionally explored as a method to identify and localize biosynthetic gene clusters within marine microbial DNA libraries. Genetic selection is a fast and economic method which utilizes selection of a part of a pathway to represent the presence of an entire pathway for the complementation of known mutant strains. We describe genetic selection to localize biotin biosynthetic pathways of Hon6 (Chromohalobacter sp.) as a proof of principle experiment for the identification and localization of biosynthetic pathways in general. Instead of developing purification methods or manipulating cultivation conditions, large fragments of non-culturable bacterial genomes can be cloned and expressed using recombinant DNA technology. A strong transcriptional promoter to control high-level gene expression is required in recombinant expression plasmids. We aimed to develop new tools to control gene expression through the use of riboswitches. Riboswitches are metabolite-sensing ribonucleic acid (RNA) elements that possess the remarkable ability to control gene expression. The thiamine pyrophosphate (TPP) riboswitch system was chosen as it will enable use of E. coli as a suitable host strain. This system is particularly attractive because it has one of the simplest structures among the riboswitches elucidated to date. The use of the TPP riboswitch will also enable modulation of pathway gene expression by varying the TPP coccentration as many gene products are toxic. The violacein gene cluster from Chromobacterium violaceum was selected and placed under the control of this riboswitch. We describe modulation of heterologous gene expression by the ThiC/Riboswitch and detail experiments to investigate the expression and manipulation of the gene cluster under various promoters.