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.

[pt] Leitura de traços convergentes que atualizam a questão da modernidade na obra de Miguel Torga. A escolha dos tópicos, centrada na análise de determinados marcadores textuais, objetiva evidenciar os elementos agenciadores do discurso, bem como estabelecer os mecanismos que levam à busca de um sentido de identidade e de um sentimento de universalidade, propiciadores da descoberta da unidade, que se fundamenta na redescoberta de si mesmo e da terra portuguesa. / [en] A reading of converging outlines that bring up to date the question of modernity in Miguel Torga work. The choice of themes, centered on the analisys of certain textual traits, has the purpose of turning clea the structural elements of the discourse, as well as establishing the mechanism that lead to the search of a sense of identy and a communal feeling, facts that favour the unveiling of unity, which is based in the rediscovery of self and of the portuguese land.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / Esta tese investiga, através das produções do Instituto Histórico e Geográfico Brasileiro entre 1838 e 1889, sobretudo o seu periódico, os limites para a escrita da história do presente no século XIX. Os sócios, imbuídos de um discurso que por vezes legitimava essa prática por considerá-la pertinente em uma associação próxima ao imperador D. Pedro II, em geral a desqualificavam em prol de uma concepção moderna de história na qual o afastamento temporal, combinado à imparcialidade, era condição fundamental para se chegar à verdade dos fatos, e de contingências políticas do próprio tempo. Assim, a partir da análise do cotidiano da associação extraiu-se uma sequência de procedimentos que levou à consideração de que a força da censura foi muito maior do que a da permissividade em relação ao tratamento de fatos coetâneos naquele momento. A utilização de outras fontes de pesquisa, como memórias históricas e autobiografias produzidas fora do grêmio, além da análise das produções do Institut Historique de Paris, permitiu a ampliação da problemática e a conclusão de que se não havia um único conjunto de regras a partir do qual o historiador devia se pautar, mesmo no IHGB, fora dele essa diversidade era ainda maior e nisso incluía-se o problema da história contemporânea. / This thesis investigates, through the production of the Instituto Histórico e Geográfico Brasileiro between 1838 and 1889, especially its journal, the limits for the writing of presents history in the nineteenth century. The associates, imbued with a speech that sometimes legitimized this practical, considering the relevance of the close association between the institution and the Emperor D. Pedro II, generally disqualified it on favor of a historys modern conception at which the temporal distance, combined with impartiality, was an essential condition to get to the truth of the facts, in addition to the political contingencies of the own time. Therefore, starting from the associations daily analysis it was possible to extracted a sequence of procedures that led to the consideration that the strength of censorship was much bigger than the permissiveness of regarding the treatment of recents facts at this proper time. The use of other sources of research, such as historical memories and autobiographies produced outside the society, as well as analysis of the productions of the Institut Historique of Paris, allowed the enlargement of the question and the conclusion that if it wasnt one single rule which could be guide the historian, even in IHGB, out of this association, the diversity would be even bigger and the problem of contemporary history would be included on it.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / Esta tese investiga, através das produções do Instituto Histórico e Geográfico Brasileiro entre 1838 e 1889, sobretudo o seu periódico, os limites para a escrita da história do presente no século XIX. Os sócios, imbuídos de um discurso que por vezes legitimava essa prática por considerá-la pertinente em uma associação próxima ao imperador D. Pedro II, em geral a desqualificavam em prol de uma concepção moderna de história na qual o afastamento temporal, combinado à imparcialidade, era condição fundamental para se chegar à verdade dos fatos, e de contingências políticas do próprio tempo. Assim, a partir da análise do cotidiano da associação extraiu-se uma sequência de procedimentos que levou à consideração de que a força da censura foi muito maior do que a da permissividade em relação ao tratamento de fatos coetâneos naquele momento. A utilização de outras fontes de pesquisa, como memórias históricas e autobiografias produzidas fora do grêmio, além da análise das produções do Institut Historique de Paris, permitiu a ampliação da problemática e a conclusão de que se não havia um único conjunto de regras a partir do qual o historiador devia se pautar, mesmo no IHGB, fora dele essa diversidade era ainda maior e nisso incluía-se o problema da história contemporânea. / This thesis investigates, through the production of the Instituto Histórico e Geográfico Brasileiro between 1838 and 1889, especially its journal, the limits for the writing of presents history in the nineteenth century. The associates, imbued with a speech that sometimes legitimized this practical, considering the relevance of the close association between the institution and the Emperor D. Pedro II, generally disqualified it on favor of a historys modern conception at which the temporal distance, combined with impartiality, was an essential condition to get to the truth of the facts, in addition to the political contingencies of the own time. Therefore, starting from the associations daily analysis it was possible to extracted a sequence of procedures that led to the consideration that the strength of censorship was much bigger than the permissiveness of regarding the treatment of recents facts at this proper time. The use of other sources of research, such as historical memories and autobiographies produced outside the society, as well as analysis of the productions of the Institut Historique of Paris, allowed the enlargement of the question and the conclusion that if it wasnt one single rule which could be guide the historian, even in IHGB, out of this association, the diversity would be even bigger and the problem of contemporary history would be included on it.

Neurodegenerative disorders are heterogenous in nature and include a range of ataxias with oculomotor apraxia, which are characterised by a wide variety of neurological and ophthalmological features. This family includes recessive and dominant disorders. A subfamily of autosomal recessive cerebellar ataxias are characterised by defects in the cellular response to DNA damage. These include the well characterised disorders Ataxia-Telangiectasia (A-T) and Ataxia-Telangiectasia Like Disorder (A-TLD) as well as the recently identified diseases Spinocerebellar ataxia with axonal neuropathy Type 1 (SCAN1), Ataxia with Oculomotor Apraxia Type 2 (AOA2), as well as the subject of this thesis, Ataxia with Oculomotor Apraxia Type 1 (AOA1). AOA1 is caused by mutations in the APTX gene, which is located at chromosomal locus 9p13. This gene codes for the 342 amino acid protein Aprataxin. Mutations in APTX cause destabilization of Aprataxin, thus AOA1 is a result of Aprataxin deficiency. Aprataxin has three functional domains, an N-terminal Forkhead Associated (FHA) phosphoprotein interaction domain, a central Histidine Triad (HIT) nucleotide hydrolase domain and a C-terminal C2H2 zinc finger. Aprataxins FHA domain has homology to FHA domain of the DNA repair protein 5’ polynucleotide kinase 3’ phosphatase (PNKP). PNKP interacts with a range of DNA repair proteins via its FHA domain and plays a critical role in processing damaged DNA termini. The presence of this domain with a nucleotide hydrolase domain and a DNA binding motif implicated that Aprataxin may be involved in DNA repair and that AOA1 may be caused by a DNA repair deficit. This was substantiated by the interaction of Aprataxin with proteins involved in the repair of both single and double strand DNA breaks (XRay Cross-Complementing 1, XRCC4 and Poly-ADP Ribose Polymerase-1) and the hypersensitivity of AOA1 patient cell lines to single and double strand break inducing agents. At the commencement of this study little was known about the in vitro and in vivo properties of Aprataxin. Initially this study focused on generation of recombinant Aprataxin proteins to facilitate examination of the in vitro properties of Aprataxin. Using recombinant Aprataxin proteins I found that Aprataxin binds to double stranded DNA. Consistent with a role for Aprataxin as a DNA repair enzyme, this binding is not sequence specific. I also report that the HIT domain of Aprataxin hydrolyses adenosine derivatives and interestingly found that this activity is competitively inhibited by DNA. This provided initial evidence that DNA binds to the HIT domain of Aprataxin. The interaction of DNA with the nucleotide hydrolase domain of Aprataxin provided initial evidence that Aprataxin may be a DNA-processing factor. Following these studies, Aprataxin was found to hydrolyse 5’adenylated DNA, which can be generated by unscheduled ligation at DNA breaks with non-standard termini. I found that cell extracts from AOA1 patients do not have DNA-adenylate hydrolase activity indicating that Aprataxin is the only DNA-adenylate hydrolase in mammalian cells. I further characterised this activity by examining the contribution of the zinc finger and FHA domains to DNA-adenylate hydrolysis by the HIT domain. I found that deletion of the zinc finger ablated the activity of the HIT domain against adenylated DNA, indicating that the zinc finger may be required for the formation of a stable enzyme-substrate complex. Deletion of the FHA domain stimulated DNA-adenylate hydrolysis, which indicated that the activity of the HIT domain may be regulated by the FHA domain. Given that the FHA domain is involved in protein-protein interactions I propose that the activity of Aprataxins HIT domain may be regulated by proteins which interact with its FHA domain. We examined this possibility by measuring the DNA-adenylate hydrolase activity of extracts from cells deficient for the Aprataxin-interacting DNA repair proteins XRCC1 and PARP-1. XRCC1 deficiency did not affect Aprataxin activity but I found that Aprataxin is destabilized in the absence of PARP-1, resulting in a deficiency of DNA-adenylate hydrolase activity in PARP-1 knockout cells. This implies a critical role for PARP-1 in the stabilization of Aprataxin. Conversely I found that PARP-1 is destabilized in the absence of Aprataxin. PARP-1 is a central player in a number of DNA repair mechanisms and this implies that not only do AOA1 cells lack Aprataxin, they may also have defects in PARP-1 dependant cellular functions. Based on this I identified a defect in a PARP-1 dependant DNA repair mechanism in AOA1 cells. Additionally, I identified elevated levels of oxidized DNA in AOA1 cells, which is indicative of a defect in Base Excision Repair (BER). I attribute this to the reduced level of the BER protein Apurinic Endonuclease 1 (APE1) I identified in Aprataxin deficient cells. This study has identified and characterised multiple DNA repair defects in AOA1 cells, indicating that Aprataxin deficiency has far-reaching cellular consequences. Consistent with the literature, I show that Aprataxin is a nuclear protein with nucleoplasmic and nucleolar distribution. Previous studies have shown that Aprataxin interacts with the nucleolar rRNA processing factor nucleolin and that AOA1 cells appear to have a mild defect in rRNA synthesis. Given the nucleolar localization of Aprataxin I examined the protein-protein interactions of Aprataxin and found that Aprataxin interacts with a number of rRNA transcription and processing factors. Based on this and the nucleolar localization of Aprataxin I proposed that Aprataxin may have an alternative role in the nucleolus. I therefore examined the transcriptional activity of Aprataxin deficient cells using nucleotide analogue incorporation. I found that AOA1 cells do not display a defect in basal levels of RNA synthesis, however they display defective transcriptional responses to DNA damage. In summary, this thesis demonstrates that Aprataxin is a DNA repair enzyme responsible for the repair of adenylated DNA termini and that it is required for stabilization of at least two other DNA repair proteins. Thus not only do AOA1 cells have no Aprataxin protein or activity, they have additional deficiencies in PolyADP Ribose Polymerase-1 and Apurinic Endonuclease 1 dependant DNA repair mechanisms. I additionally demonstrate DNA-damage inducible transcriptional defects in AOA1 cells, indicating that Aprataxin deficiency confers a broad range of cellular defects and highlighting the complexity of the cellular response to DNA damage and the multiple defects which result from Aprataxin deficiency. My detailed characterization of the cellular consequences of Aprataxin deficiency provides an important contribution to our understanding of interlinking DNA repair processes.

Made available in DSpace on 2015-03-26T13:44:35Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1003783 bytes, checksum: 3778c74afb684b7a8992a6d39ccd6939 (MD5) Previous issue date: 2013-03-11 / This dissertation aims to study the group of the poems The Ark of the Covenant , part of the work The livings , by Carlos Lejar (2011), a Brazilian contemporary poet and literary critic for the bias of studies about post-modern intertextuality. The Ark of the Covenant is a representative work of Brazilian view of modern poetry and this text investigates the discursive intersections between literature and Bible because it dialogues with the two texts. Therefore, it is possible to study about the poetical productivity and the post-modern parody. Furthermore, it is considerate a peculiar literary production because it was made and incorporated to the book during twenty years of poetic growing and maturing. The first work edition was published in 1979 and the 1995 s edition includes 300 new poems as the ones will be studied. Basing on a theoretical support that offers investigative and contemplative about the work possibilities, we intend examine how the poet redesign the religious discourse by transposing it to the literary discourse in the contemporaneity, even though the contemporary poetry study is far from the criticism. Thereby, this subject is interesting once it makes possible a research about how the world is recreated by God s salvation, in a certain faith language heretofore , can express itself in a poetical language?, or how, using poetical initiative, the artist of word recreates and resets a new mythical world order of holy scripture. Thus, researching this subject is motivating and relevant in Literary Studies, which aims to contribute for the amplitude of scientific literary research. / Esta dissertação pretende um estudo acerca do conjunto de poemas A Arca da Aliança , parte integrante da obra Os Viventes, de Carlos Nejar (2011), poeta e crítico literário brasileiro contemporâneo, pelo viés dos estudos sobre intertextualidade pós-moderna. A Arca da Aliança é uma obra representativa do panorama brasileiro da poesia atual e se faz profícuo texto para a investigação das intersecções discursivas entre o literário e o bíblico, por possuir correspondências muito claras entre os dois textos, o que nos leva ao estudo sobre produtividade poética e paródia pós-moderna. Além disso, se configura como ímpar na produção literária do momento por ter sido confeccionada e incorporada ao livro no decorrer de um período de aproximadamente 20 anos de crescimento e amadurecimento poético. A primeira edição da obra foi em 1979, sendo que a edição de 1995 consta de acréscimos de cerca de 300 novos poemas, dentre eles, o conjunto a que nos propomos estudar. Com base em um suporte teórico que nos oferece possibilidades investigativas e contemplativas sobre o tema, almejamos perscrutar como o poeta reelabora o discurso religioso transpondo-o para o literário na contemporaneidade, ainda que o estudo de poesia contemporânea se limite a uma falta de distanciamento crítico. Deste modo, este tema nos interessa a partir do momento em que possibilita uma pesquisa sobre como o mundo recriado pela iniciativa salvífica de Deus, outrora numa certa linguagem da fé, pode exprimir-se, por sua vez, na linguagem de iniciativa poética?, ou como por meio da iniciativa poética, o artista da palavra recria e reconfigura uma nova ordem do mundo mítico da sagrada escritura. Assim, sondar este tema se faz, para nós, um instigante e pertinente trabalho na área dos Estudos Literários, que busca contribuir para o alargamento dos horizontes da pesquisa literária científica.

The genetic systems involved in the utilisation of aromatic β-glucosides in E. coli consist of the bgl, asc, and chb operons and the locus bglA encoding phospho-β-glucosidase A. The bgl and asc operons are known as cryptic or silent systems since their expression is not sufficient for utilisation of these sugars in wild type strains of E. coli. Their transcriptional activation by different classes of mutations confers a Bgl+ phenotype to the mutant. The maintenance of cryptic genes without accumulating deleterious mutation in spite of being silent is an evolutionary puzzle. Several observations have suggested the possibility that these genes may be expressed under specific physiological conditions conferring a fitness advantage to the organism. The main aim of this study was to investigate the possible role of aromatic β-glucoside catabolic systems of E. coli in combating nutrient stress and microaerobic growth conditions. The results presented in Chapter 2 address the evolution of aromatic β-glucoside catabolic systems when exposed to a novel β-glucoside as the sole substrate. The results indicate that the bgl opeon, the primary system involved in the utilisation of the aromatic β-glucosides arbutin and salicin, is also involved in esculin utilisation. In the absence of bglB encoding the enzyme phospho-β-glucosidase B, activation of the silent asc operon enables esculin utilisation. The bglA gene encoding phospho-β-glucosidase A specific for arbutin, can undergo successive mutations to evolve the ability to hydrolyse esculin and salicin sequentially when bglB and ascB are absent. The Esc+ and Sal+ mutants retain their arbutin+ phenotype, indicating that the mutations enhance the promiscuity of the enzyme. Sequencing data indicate that the first step Esc+ mutant carries a four base insertion within the promoter of the bglA gene that results in enhanced transcription of bglA. RT-PCR studies confirm that both the steady-state levels as well as the half-life of the bglA mRNA are enhanced in the mutant. This is further corroborated by the observation that overexpression of wild type bglA in the parent strain using a multicopy plasmid confers an Esc+ phenotype. The second step Sal+ mutant carries a point mutation within bglA ORF, a thymine to guanine transversion at position 583 (T583G) of the bglA gene, resulting in an amino acid change from cysteine to glycine at position 195 (C195G) of the BglA ORF close to the active site. Presence of a plasmid carrying the T583G mutation, introduced by site-directed mutagenesis, results in a Sal+ phenotype, confirming the role of the transversion in conferring the Sal+ phenotype. Based on docking studies, the positioning of salicin into the substrate binding site of the mutant BglA enzyme is different compared to wild type BglA due to the loss of stearic hindrance for the binding of salicin when C195 is replaced by the smaller amino acid glycine in the mutant protein. These observations indicate that under conditions of nutrient deprivation, exposure to novel substrates can result in the evolution of new metabolic capabilities by the sequential modification of a pre-existing genetic system. In the case of one novel substrate, the mutation results in the overexpression of the hydrolytic enzyme, while in the case of the second substrate, a mutation close to its active site increases its substrate specificity. Results presented in Chapter 3 specifically deal with the involvement of the bgl operon under low levels of oxygen. Earlier observations have shown that there is a 22 fold enhancement in the expression of the bgl operon under anaerobic condition. The present results provide evidence that bgl expression has a physiological role under low levels of oxygen and in addition suggest a possible mechanism for the overexpression of the bgl operon that involves the ArcAB two component system known to mediate regulation under microaerobic and static conditions. Transcription studies using a lacZ reporter fused to the wild type bgl promoter show that there is enhanced transcription from the bgl promoter under microaerobic and static conditions in the presence of arcA encoding the response regulator compared to that in its absence. The positive effect of arcA on the expression of the bgl operon is dispensable in the absence of H-NS since presence or absence of arcA does not change the expression of the bgl operon in an hns-null background, implying that the involvement of ArcA is via antagonizing H-NS. Competition experiments indicate that there is growth advantage associated with the activated allele of the bgl operon under low levels of oxygen since Bgl+ strains carrying the activated allele of the bgl operon as well as strains expressing BglG constitutively can out-compete wild-type strains. Presence of the wild type arcA allele results in a strong growth advantage compared to its absence under static conditions but not aerobic condition. The bgl operon seems to be one of the possible downstream targets of ArcA under static condition since absence of the bgl operon results in a modest reduction of the growth advantage (GASP) phenotype conferred by arcA. The up-regulation of the bgl operon is likely to enable the cells to scavenge available nutrients from their niche more efficiently. These experiments also show that the GASP phenotype associated with BglG constitutive strains under static conditions involves downstream genes that are different from oppA known to be one of the downstream targets during aerobic growth. It is possible that under low level of oxygen, the bgl operon is regulating a different set of downstream genes involving a different mechanism. In summary, the results of this investigation show that the aromatic β-glucoside catabolic systems in E. coli play a role in the generation of new metabolic capabilities via mutations in pre-existing genetic systems as well as through changes in gene expression patterns. The mechanisms outlined in this study are likely to be of broader significance applicable to microbial evolution under stress in general.