Yes / This chapter evaluates the consistency of the United Kingdom (UK) regulatory framework on shale gas with Commission Recommendation 2014/70/EU on minimum principles for the exploration and production of unconventional oil and gas. In the absence of European-wide legislation, European Union (EU) Member States have the right to determine the conditions for exploiting their unconventional energy sources. However, due to the environmental and human health risks associated with hydraulic fracturing, the EU has expressed its interest in ensuring adequate protection of the environment and to creating clear and transparent common standards for the benefit of operators, investors and the public while promoting the interests of those Member States which are currently exploring unconventional energy. It can be argued that the UK regime has been designed to address the environmental risks arising from hydraulic fracturing operations and as such it sets a high environmental threshold for operations. In fact, the UK legislation appears to be more comprehensive than in many other jurisdictions commercially exploiting shale gas, and therefore it has a potential to inform the content of any future harmonising measures on the exploration and extraction of such resources at the EU level.

Atualmente, a Leucemia Linfocítica Crônica (LLC) pode ser dividida em dois grupos: um com mutações somáticas no gene da região variável da cadeia pesada da imunoglobulina (MIgVH) e outro sem mutações (NMIgVH). Alguns estudos mostraram que a expressão de CD38 na superfície das células B de LLC pode estar correlacionada com o estado mutacional do gene VHIg, entretanto, esses controversos. Estudos recentes mostraram que a expressão da proteína tirosina quinase Zap-70 está melhor associada com o estado mutacional do gene IgVH. O objetivo deste estudo foi avaliar a expressão de Zap-70 e CD38, por citometria de fluxo, nas células CD19+ de pacientes com LLC e correlacioná-los com o estádio clínico (EC), sobrevida livre de tratamento (SLT) e sobrevida global (SG). A expressão de Zap-70 e CD38 foi avaliada, em 144 de pacientes com LLC classificados nos estádios clínicos A, B e C de acordo com os critérios de Binet: 59 (41%) do EC-A, 38 (26%) do EC-B e 47 (33%) do EC-C. Foi observada menor positividade para Zap-70 e CD38 nos pacientes do EC-A do que nos EC-B e C. Quando avaliada a SLT nos pacientes do EC-A, os casos Zap-70+ assim como os CD38+ apresentaram menor SLT. A média de SG dos pacientes Zap-70+ e CD38+ foi menor quando comparado com os Zap-70- e CD38- entretanto quando correlacionada com o EC não foi observada diferença estatisticamente significante entre a expressão desses marcadores e o EC-A, B ou C. Pela analise combinada de CD38 e Zap-70, dividimos os pacientes em dois grupos (Zap-70-/CD38- e Zap-70+ ou CD38+). Observamos que a expressão positiva desses dois marcadores estava associada ao EC, uma vez que a grande maioria dos pacientes dos estádios B (74%) e C (66%) expressam Zap-70 ou CD38. Entretanto, os pacientes do EC-A, Zap-70+ ou CD38+, apresentaram SG menor quando comparado com os Zap-70-/CD38-. Essa diferença não foi observada nos pacientes do EC-B e do EC-C. Também foi observada menor SLT nos pacientes no EC-A, Zap-70+ ou CD38+. Esses resultados sugerem que análise combinada de Zap-70 e CD38 podem ser empregadas na avaliação dos pacientes do EC-A para se acompanhar a evolução clinica desse grupo de pacientes. Porém, estudos adicionais devem ser realizados para se validar a utilização clínica desses marcadores. / Actually, chronic lymphocytic leukemia (CLL) can be divided in two subsets: one with somatically mutated immunoglobulin heavy-chain variable-region genes (MIgVH) and other with unmutated sequences. (UMIgVH). Some studies have shown that CD38 expression in CLL cells are correlated with IgVH mutational status. However, the value of CD38 as surrogate IgVH mutational status is controversial. Recent studies, have found that Zap-70 protein tyrosine kinase expression is strongly associated with the mutational status IgVH. The aim of this study was to evaluate the Zap-70 and CD38 expression, for flow cytometry, in CD19+ LLC cells and correlate with the Binet’s staging system, treatment-free survival (TFS) and a overall survival (OS). Zap-70 and CD38 was evaluated, in 144 CLL patients that was classified in A, B and C Binet’s staging system: 59 (41%) in stage A, 38 (26%) in B and 47 (33%) in C. We observed low Zap-70 and CD38 expression in stage A patients than in stage B and C cases. When we analyzed the TFS in stage A patients Zap-70+ and CD38+ patients showed shorter TFS than Zap-70- and CD38-. Then we observed that the OS of Zap-70+ and CD38+ patients was, also, shorter than Zap-70- and CD38- cases. However, statistical differences was not found when Zap-70 and CD38 expression was correlated with stage A, B or C Binet’s staging system. To understand the associated Zap-70 and CD38 expression, we divided the CLL patients in two subgroups (Zap-70-/CD38 - and Zap-70+ or CD38+). We observed that CD38+ or Zap-70+ was associated Binet’s staging system, once most of stage B (74%) and C (66%) patients are Zap-70+ or CD38+. However, stage A patients, Zap-70+ or CD38+, showed shorter OS than Zap-70-/CD38-. These differences were not observed in stage B and C patients. Shorter TFS was also observed in the Zap-70+ or CD38+ stage A patients. These results suggest that combined analysis of Zap-70 and CD38 can be used to evaluate stage A patients to observe the clinical evolution of the disease. Nevertheless, other studies must be carried to confirm the clinical use of these markers.

INTRODUÇÃO: A limpeza prévia de endoscópios rígidos (ER) seguida de desinfecção de nível intermediário com álcool etílico a 70% p/v após o exame de endoscopia nasal é uma prática adotada em muitos serviços de otorrinolaringologia. A literatura atual, no entanto, recomenda a esterilização ou desinfecção de alto nível como o método de descontaminação mais aceito para produtos para saúde classificados como semicríticos. No entanto, há que se fazer distinção entre equipamentos de alta complexidade e sua invasividade como os endoscópios flexíveis com lumens longos e estreitos utilizados na endoscopia digestiva, daqueles de conformação simples sem lumens de baixa invasividade como os endoscópios rígidos utilizados em otorrinolaringologia. OBJETIVO: Avaliar a segurança da desinfecção de nível intermediário com álcool etílico 70% p/v, após limpeza prévia dos endoscópios rígidos utilizados em procedimentos clínicos de endoscopia nasal considerando a carga microbiológica recuperada após o uso. MÉTODO: Imediatamente após a realização do exame, uma gaze úmida foi utilizada para o arraste da carga biológica do endoscópio rígido, gerando as amostras do Controle Positivo e, após a aplicação do POP, um novo arraste para constituir as amostras do Grupo Experimental. Estas gazes foram inicialmente submetidas à sonicação e agitação imersas em soro fisiológico e em seguida a solução foi submetida a uma técnica de extração de carga microbiológica por filtragem por meio de uma Membrana de Celulose de 0,22um de poro que foi, em seguida, semeada nos meios de ágar Sangue, Chocolate, Sabouraud, Löwenstein-Jensen e Tioglicolato. Estes meios ficaram incubados em estufa a 37ºC ± 2ºC e avaliados, no máximo, até por 60 dias conforme o perfil de crescimento dos diferentes microrganismos de interesse; foram analisados de maneira quantitativa e qualitativa para identificação e classificação dos micro-organismos recuperados após as semeaduras. RESULTADO: Os resultados da análise estatística evidenciaram diferença significativa entre Controle Positivo e Grupo Experimental quando comparados em relação à presença de Streptococcus coagulase negativa (p < 0,001), Bacillus spp (p < 0,001) e Staphylococcus aureus (p=0,001). No Controle Positivo, foram encontradas presença desses micro-organismos respectivamente na seguinte frequência: 63,2%, 28,9% e 28,9%, enquanto que, no Grupo Experimental, não foi houve recuperação microbiana alguma. CONCLUSÃO: Os resultados desta pesquisa demonstram a eficiência, na prática diária, da desinfecção de nível intermediário dos endoscópios utilizados na otorrinolaringologia por meio da fricção com álcool etílico 70% p/v por 90 segundos, com protocolo de limpeza prévia / INTRODUCTION: Prior cleaning of rigid endoscopes (REs) followed by intermediate-level disinfection with 70% ethyl alcohol (w/v) after nasal endoscopy is a common practice in many otolaryngology services. Current literature, in turn, recommends high-level sterilization or disinfection as the most accepted decontamination method for health products classified as semi-critical. However, it is necessary to distinguish highly complex equipment according to their invasiveness, e.g., flexible endoscopes with long and narrow lumens used in digestive endoscopy and those with a simple conformation without lumens of low invasiveness, such as rigid endoscopes used in otorhinolaryngology. OBJECTIVE: To evaluate the safety of intermediate-level disinfection with 70% ethyl alcohol (w/v) after cleaning of REs used in clinical procedures of nasal endoscopy considering the microbiological load recovered after use. METHOD: Immediately after the test, a wet gauze was used to drag the biological load from the RE, generating positive control samples; after applying POP, dragging was carried out again to generate samples of the experimental group. These gasses were initially subjected to sonication and shaking while immersed in physiological saline; the solution was then subjected to the microbiological loading technique by filtration through a 0.22-um pore cellulose membrane and then cultivated on blood, chocolate, Sabouraud, Löwenstein-Jensen, and thioglycolate agar media. These media were incubated at 37ºC ± 2ºC and evaluated for up to 60 days, according to the growth profile of the different microorganisms of interest. A quantitative and qualitative analysis was performed for the identification and classification of microorganisms recovered after cultivation. RESULTS: The results of statistical analysis showed a significant difference between the positive control and experimental groups for the presence of coagulase-negative Streptococcus (p < 0.001), Bacillus spp (p < 0.001), and Staphylococcus aureus (p=0.001). In the positive control group, these microorganisms were found in the following proportions: 63.2%, 28.9%, and 28.9%, respectively, whereas in the experimental group, no microorganisms were recovered. CONCLUSION: The results of this study demonstrate the efficiency of the daily practice of intermediate-level disinfection of endoscopes used in otorhinolaryngology by means of treatment with 70% ethyl alcohol (w/v) for 90 seconds, using a previous cleaning protocol

O estudo de características de isospin na excitação do estado 2 POT.+IND.1 foi implementado nos núcleos ANTPOT.70, 72, 74 Ge com a realização de medidas de espalhamento inelástico na região de interferência nuclear coulombiana (INC) com ANTPOT.6 Li de 28 MeV. A análise da INC, utilizando projéteis leves de interação isoescalar, na excitação de estados coletivos é uma ferramenta poderosa para evidenciar mudanças de estrutura em cadeias de núcleos, e fornecer resultados robustos para testes de modelos. A principal vantagem do método é a extração simultânea dos parâmetros de deformação de carga e massa das transições, minimizando as incertezas na razão entre as probabilidades reduzidas de transição elétrica e de massa, B(El) e B(ISl), indicadores importantes da coletividade dos estados nucleares. A análise foi realizada na aproximação de Born com ondas distorcidas e potencial óptico deformado (DWBA-DOMP), usando um conjunto de parâmetros de potencial óptico global. Os ajustes das previsões à distribuições angulares, empregando o método interativo de Gauss, exibiram excelente qualidade. Os parâmetros correlacionados extraídos na minimização do chi-quadrado, delta IND.n(comprimento de deformação nuclear) e C=delta IND.c/delta IND.n (razão entre os comprimentos de deformação de carga e nuclear), e suas incertezas, foram submetidos a testes estatísticos que os validaram. Dentre as grandezas determinadas a partir desses parâmetros, os valores de B(IS2), inéditos nos isótopos de Ge, mostraram uma mudança de estrutura não evidenciada pelos valores de B(E2). A probabilidade reduzida de transição isoescalar cresceu com o incremento do número de nêutrons, com um aumento abrupto no ANTPOT.74 Ge. Já a probabilidade reduzida de transição elétrica aumentou de maneira mais suave. Essa diferença de comportamento foi um reflexo da variação do parâmetro extraído C, que no ANTPOT.74 Ge indicou uma ) contribuição maior dos nêutrons frente aos prótons na excitação do primeiro estado quadrupolar. Os isótopos ANTPOT.70, 72 Ge, por outro lado, revelaram contribuições equivalentes e homogêneas para essas transições. Com a determinação precisa das contribuições de massa e de carga para a excitação do primeiro estado quadrupolar, novos subsídios estão disponíveis para futuros cálculos. / The analysis of the isospin character of the 2 POT.+ IND.1 excitation in the ANTPOT.70,72,74 Ge nuclei, with 28 MeV ANTPOT.6 Li, was applied to the inelastic scattering in the coulomb nuclear interference (CNI) region. The CNI measurements in the exitation of collective states with light isoscalar projectiles are powerful tools to evidence structure changes in nuclei chains, allowing tests of nuclear models. In this method the simultaneous extraction of the charge and mass deformation parameters, minimizes the uncertainties in the ratio of charge B(El) to mass B(ISl) trasition reduced probabilities, wich are important indicators of the nuclear collectivity. The present analysis was developed in the framework of the distorted wave Born approximation within a deformed optical potential approach (DWBA-DOMP), using a set of global optical model parameters. Excellent quality fits of the predictions to the experimental angular distributions, employing the Gauss iterative method, were obtained. The correlated parameters delta IND.N (nuclear deformation length) and C = delta IND.C/delta IND.N (ratio of charge to nuclear deformation lenghts), and their uncertainties extracted in the least squares method, were validated by statistical tests. The B(IS2) values for germanium isotopes, not previously reported, were determined in the analysis and revealed a structure change not evidenced by the B(E2) values. The isoscalar transition reduced probability raises when the neutron number increases, showing in the ANTPOT.74 Ge an abrupt enhancement. Nevertheless, the eletric transition reduced probability grows smoothly. This behavior was detected following the experimental extracted C parameters, indicating in the heavier isotope a predominance of neutrons in the first quadrupolar excitation. The ANTPOT.70,72 Ge isotopes, on the other hand, displayed equivalent and homogeneous contributions of protoons and neutrons. New information on mass and charge contributions for the 0 POT.+ IND.1 -> 2 POT.+ IND.1 transition, in the germanium isotopes, is now available for future theoretical interpretation.

As suspensões de estearato de eritromicina, de procedência nacional, apresentam a inconveniência que reside na elevada viscosidade ou flutuação do fármaco disperso. Foram estudados diferentes adjuvantes farmacotécnicos, a fim de verificar fatores interferentes que poderiam melhorar a estabilidade da suspensão de estearato de eritromicina. Como agentes suspensores foram utilizados BoniasolR2600 (carboximetilcelulose sódica), veegumR HV (silicato de alumínio e magnésio) e ViscosolR RV (glicolato de amido sódico) e como edulcorantes a glicerina, sorbitol a 70% e sacarose. As determinações analíticas para avaliar a estabilidade das suspensões foram: pH, densidade, viscosidade, aspecto da suspensão, volume do fármaco disperso, características de ressuspensão e doseamento do antibiótico. As suspensões contendo BoniasolR 260 e glicerina ou sorbitol não apresentaram o fenômeno da flutuação. As preparações contendo VeegumRRV e glicerina ou sorbitol na concentração de 30% (p/v) apresentaram estabilidade adequada. / Suspensions of erithromycin stearate of national origin present the inconvenience of the high viscosity or of the flotation of the dispersed drug. Different pharmaceutical helpers were studied in order to verify of the erythromycin stearate suspension. BoniasolR2600 (sodium carboxymethyl cellulose), VeegumR HV (aluminum magnesium silicate) and ViscosolR RV (sodium starch glycollate) were used as suspending agents and glycerin, sorbitol at 70% and sucrose were used as sweeteners. Analytic determination to evaluate the suspensions stability were: pH, density, viscosity, aspecto of suspension, volume of the dispersed drug, resuspension characteristics and determination of the antibiotic dose. Suspensions containing BoniasolR 2600 and glycerin or sorbitol didn\'t present floatation. Preparation with veegumR HV and glycerin or sorbitol in a concentration of 30% (p/v) presented adequate stability.

The subject of this thesis is a genre analysis of Soviet film comedies of the late 60s and the 70s that is based on the reception and production history data. The aim of this research is to grasp this type of comedy as a specific political and socially-cultural phenomenon of the Eastern bloc and its relation to Soviet popular culture. A methodology of the genre analysis will derive from Rick Altman's semantic-syntactic approach, which will allow us to describe the chosen genre group as a complex genre trend set in the unique social, cultural, economic and in this particular case also ideological context, to follow the development and fluidity of the genre trend, as well as to find and to compare mutual attributes of the chosen films. The analysis will not be based solely on the group of the most watched films, but also on the production data focusing mostly on the films' budget, as well as dramaturgic supervision and censorship. Another part of the diploma thesis will be based on the gathered reception data such as contemporary media response and viewer ratings, focusing on the important role that Soviet film comedies played in the period and partially continue to play in the present-day Russian popular culture. Fusion of all analytic parts shall bring us to the basic definition of the Soviet film...

Made available in DSpace on 2016-04-27T19:30:19Z (GMT). No. of bitstreams: 1 Valeria Aparecida Alves.pdf: 11158129 bytes, checksum: 197fdc3394b6120655436bb40ebb7714 (MD5) Previous issue date: 2011-06-10 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The object of study of this research has singled out Torquato de Araújo Neto, who experienced political, economical, and cultural transformations in the late 1950 s and the early 1970 s. He participated actively in the tropicalism movement in music and in the marginal or underground movie production. He also used the press, a privileged medium, to debate and explicit the context in which he was living. From the analysis of Torquato Neto s life s path and discourse, this work aimed at problematizing the Brazilian cultural production during the period of authoritarianism and at discussing the proposals for a renewed Brazilian popular music, the debate among artists and intellectuals, and the various forms of resistance. The research was carried out using the following sources of information: song lyrics, three newspaper columns - Música Popular, published in the Sports newspaper in 1967, Plug, published in Correio da Manhã in 1970, and Geléia Geral, published in Última Hora also in 1970. Other sources were letters, notes, poems, and images about the tropicalism movement found on record covers, in O Estado de São Paulo, Folha de São Paulo and Folha da Tarde newspapers, and in Veja magazine. Videos which registered the stage performance of the movement participants were also used. It is possible to conclude that, during the period of time when increasing measures were taken to curtail freedoms and to repress, a number of artists and intellectuals resisted that arbitrariness and protested against the established order, using the defense that Creating is resisting. They marginally produced, searched for alternative spaces and a new aesthetic language. They displayed anarchic behavior in relation to political and cultural order. They revendicated freedom in every aspect of life, and Torquato Neto was the spokesman of this defense movement / A pesquisa ora apresentada privilegiou como objeto de estudo Torquato de Araújo Neto. Sujeito que vivenciou as transformações políticas, econômicas e culturais ocorridas entre o final dos anos 50 e início da década de 1970. Participante ativo do tropicalismo musical e da produção do cinema marginal ou underground, ocupou espaço privilegiado a imprensa para debater e explicitar o contexto vivido. Buscou-se a partir da análise da trajetória e discurso de Torquato Neto, problematizar a produção cultural brasileira durante o período de vigência do autoritarismo e discutir as propostas de renovação para a Música Popular Brasileira, o debate entre artistas e intelectuais e as diversas formas de resistência. As fontes utilizadas para o desenvolvimento da pesquisa foram: as letras das canções, as colunas Música Popular , publicada no jornal dos Sports, no ano de 1967, Plug , no Correio da Manhã e Geléia Geral , no jornal Última Hora, ambos na década de 1970, além de cartas, anotações e poemas, as imagens produzidas sobre o movimento tropicalista, encontradas nas capas dos discos, nas fotos publicadas na imprensa, nos jornais O Estado de São Paulo, Folha de São Paulo e Folha da Tarde e na revista Veja, além dos vídeos que registraram a performance de seus integrantes nos palcos. Através desta pesquisa, conclui-se que durante o período de ampliação das medidas de cerceamento das liberdades e repressão, uma parcela de artistas e intelectuais resistiu ao arbítrio e protestou contra a ordem estabelecida, assumindo a defesa de que Criar é resistir . Produziram à margem, buscaram espaços alternativos e uma nova linguagem estética. Assumiram uma postura anárquica em relação à ordem política e cultural. Reivindicavam liberdade, em todos os sentidos, e Torquato Neto foi porta-voz dessa defesa

As suspensões de estearato de eritromicina, de procedência nacional, apresentam a inconveniência que reside na elevada viscosidade ou flutuação do fármaco disperso. Foram estudados diferentes adjuvantes farmacotécnicos, a fim de verificar fatores interferentes que poderiam melhorar a estabilidade da suspensão de estearato de eritromicina. Como agentes suspensores foram utilizados BoniasolR2600 (carboximetilcelulose sódica), veegumR HV (silicato de alumínio e magnésio) e ViscosolR RV (glicolato de amido sódico) e como edulcorantes a glicerina, sorbitol a 70% e sacarose. As determinações analíticas para avaliar a estabilidade das suspensões foram: pH, densidade, viscosidade, aspecto da suspensão, volume do fármaco disperso, características de ressuspensão e doseamento do antibiótico. As suspensões contendo BoniasolR 260 e glicerina ou sorbitol não apresentaram o fenômeno da flutuação. As preparações contendo VeegumRRV e glicerina ou sorbitol na concentração de 30% (p/v) apresentaram estabilidade adequada. / Suspensions of erithromycin stearate of national origin present the inconvenience of the high viscosity or of the flotation of the dispersed drug. Different pharmaceutical helpers were studied in order to verify of the erythromycin stearate suspension. BoniasolR2600 (sodium carboxymethyl cellulose), VeegumR HV (aluminum magnesium silicate) and ViscosolR RV (sodium starch glycollate) were used as suspending agents and glycerin, sorbitol at 70% and sucrose were used as sweeteners. Analytic determination to evaluate the suspensions stability were: pH, density, viscosity, aspecto of suspension, volume of the dispersed drug, resuspension characteristics and determination of the antibiotic dose. Suspensions containing BoniasolR 2600 and glycerin or sorbitol didn\'t present floatation. Preparation with veegumR HV and glycerin or sorbitol in a concentration of 30% (p/v) presented adequate stability.

In bacteria, the RNA polymerase (RNAP) consists of the following subunits: α2, β, β’, ω and σ. The core RNAP (α2ββ’ω) possesses the polymerising activity and it associates with one of the sigma factors to initiate transcription from a promoter region on the DNA template. All bacteria carry an essential housekeeping sigma factor and a number of extra cytoplasmic function (ECF) sigma factors. During alternate physiological states, a major part of transcriptional regulation is carried out by sigma factors, which act as transcriptional switches, thus, making it possible for bacteria to adapt to varied environmental signals by transcribing the necessary set of genes. Bacteriophages utilise various mechanisms for subverting the bacterial biochemical machinery for their advantage. One such example in E. coli is AsiA protein encoded by an early gene of T4 bacteriophage. Because of its property of binding to σ70, AsiA can inhibit transcription from E. coli promoters bearing –10 and –35 DNA sequences leading to inhibition of growth. σ70 of E. coli is also regulated by a stationary phase specific protein, Rsd, whose major function seems to be helping the cell in switching the transcription in favour of stationary phase genes. In this study we have investigated the mechanism of interaction of T4 AsiA and E. coli Rsd to σ70 of E. coli and also tried to determine the basis of differential inhibition of E. coli growth by AsiA and Rsd. In chapter one we have reviewed the published literature on regulation of transcription in bacteria. Some of the well known mechanisms of regulating gene expression are: DNA supercoiling, two component signal transduction system (TCS), regulation by alarmone ppGpp and 6S RNA, and sigma-antisigma interactions. Most bacteria carry a number of sigma factors and each of them is dedicated to transcribing genes in response to environmental signals. Intracellular levels of sigma factors and their binding affinity to core RNAP are deciding factors for initiating transcription from specific subsets of genes. In addition, sigma factor activity is also controlled by specific proteins, which bind to sigma factors (anti-sigma factors) under certain environmental conditions. A number of anti-sigma factors have been isolated from a variety of bacteria and the mechanisms of action of binding to cognate sigma factors have been worked out by using genetic, biochemical and structural tools. In chapter two, using yeast two hybrid assay (YTH), we have identified the regions of σ70 which interact with AsiA, and it was observed that amino acid residues from 547-603, encompassing region 4.1 and 4.2 are involved in binding to σ70. Interestingly, we found that truncated σ70 fragments lacking the N-terminal regions, apparently bound to AsiA with higher affinity compared to full length σ70. As AsiA expression, because of its transcription inhibitory activity, is inhibitory to E.coli growth, co-expression of the truncated C-terminal σ70 fragments (e.g. residues 493-613, σ70C121), which bind to σ70 with high affinity, could relieve growth inhibition. The complex of GST:AsiA-σ70C121 could be purified from E. coli cells. GST:AsiA purified from E .coli cells was found to be associated with RNAP subunits. Since further studies on this interaction required GST:AsiA preparation devoid of RNAP subunits, we decided to express this protein in S. cerevisiae. Bioinformatics analysis indicated the absence of a σ70 homologue in S.cerevisiae. As expected, GST:AsiA purified from the yeast was found to be free from any RNAP like proteins. The protein purified from yeast was used for in-vitro binding experiments. Our YTH analysis had indicated that deletion a part of region 4.1 or 4.2 of σ70 leads to loss of binding to AsiA. However, the published NMR structure of AsiA in complex with peptides corresponding to region 4 of σ70, showed that either region 4.1 or 4.2 alone can bind to AsiA indicating at the possible existence of two binding sites for AsiA. In order to confirm the physiological significance of this finding, we studied the interaction of truncated σ70 fragments lacking either region 4.1 or 4.2 with AsiA in-vivo in E. coli and in-vitro by affinity pull down assays. It was observed that σ70 fragments lacking either region 4.1 (σ70∆4.1) or 4.2 (σ70∆4.2), did not neutralize the GST:AsiA toxicity, indicating lack of interaction. The affinity purified GST:AsiA from these E. coli cells did not have σ70∆4.1 or σ70∆4.2 associated with it. Similar results were obtained from pull down assays in-vitro, where we found that σ70∆4.1 or σ70∆4.2 do not show any observable interaction with AsiA. This clearly established that the minimum region of σ70 required for physiologically relevant interaction with AsiA consists of both the regions 4.1 and 4.2. Chapter 3 of this thesis has been devoted to this aspect of AsiA-σ70 interaction. Having defined the minimum region of σ70 interacting with AsiA, we sought to identify the regions and amino acid residues of AsiA, which are critical for interaction with σ70. The approach for identification of mutants and their characterisation has been discussed in chapter 4. For this purpose, we made systematic deletions in the N and C-terminal regions of the protein and also isolated random mutants of AsiA, which lack binding to σ70 and thus are non-inhibitory to E. coli growth. It was found that deletion of 5 amino acids from N-terminus and 17 amino acids from C-terminus did not alter the inhibitory activity of AsiA. In contrast, deletion of N-terminal 10 amino residues led to complete loss of activity, while in the C-terminus, a gradual loss of activity was observed when amino acid residues beyond 17 amino acids were deleted. A 34 amino acids C-terminal deletion mutant was found to be completely inactive. E10K mutant was found to be inactive, but changes of E to other amino acids such as S, Y, L, A and Q were tolerated, indicating that negative charge at E10 is not a crucial element for interaction with σ70. Inactive mutants could be overexpressed in E. coli and showed reduced binding in YTH assay and were also poor inhibitors of in-vivo transcription in E. coli. We concluded that the primary σ70 binding site of AsiA is present in the N-terminus, yet C-terminal 64-73 amino acid residues are required for effective binding in-vivo. These studies also correlate the inhibitory potential of AsiA with its σ70 binding proficiency. In chapter 5, we have made a comparative analysis of mechanism of interaction of AsiA and Rsd to E. coli RNAP. Overexpression of Rsd was found to be less inhibitory to E. coli cell growth than that of AsiA. The affinity purified GST-AsiA from E. coli was found to have all the RNAP subunits associated with it, whereas, only σ70 was found to be associated with similarly purified GST:Rsd, pointing towards differences in binding to RNAP. In affinity pull down assays, in-vitro, it was found that both AsiA and Rsd do not show any observable binding to core RNAP. Binding of AsiA to σ70 in holo RNAP led to the formation of a ternary complex, whereas no ternary complex was observed when Rsd was made to interact with holo RNAP. Analysis of protein-protein interaction by YTH showed that region 4.1 and 4.2 are critical for binding of both AsiA and Rsd to σ70. However, in the case of Rsd, the surface of interaction is not limited to this region only and other regions of σ70 make significant contribution to this binding. Possibly, the interaction of Rsd with the core binding regions of σ70 prevents its association with core RNAP. Kinetic analysis of binding by surface plasmon resonance (SPR) showed that binding affinities (Kd) of AsiA and Rsd to σ70 are in similar range. Therefore, we concluded that the ability of AsiA to trap the holo RNAP is, probably, responsible for higher inhibitory activity of this protein compared to that of Rsd. Thus, T4 AsiA and E. coli Rsd, which share regions of interaction on σ70, have evolved differences in their mechanism of binding to RNAP such that T4 AsiA, by trapping the holo RNAP subverts the complete bacterial transcription machinery to transcribe its own genes. Rsd, on the other hand, has evolved to interact primarily with σ70, which favours the utilisation of core RNAP by other sigma factors.

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