Opções terapêuticas para tratar infecções por Pseudomonas aeruginosa são limitadas por seus diversos mecanismos de resistência, que podem ou não ser detectados no laboratório clínico. Um fenótipo observado na rotina laboratorial é o surgimento de subpopulações resistentes a partir de uma população sensível aos antimicrobianos – heteroresistência. Em P. aeruginosa esse fenômeno já foi investigado para carbapenêmicos, porém, em relação à polimixina B, não há dados literários. Objetivamos avaliar a heteroresistência à polimixina B em dois grupos de P. aeruginosa, um sensível e outro resistente aos carbapenêmicos. Cento e vinte e quatro isolados de P. aeruginosa foram obtidos, aleatoriamente, no Hospital de Clínicas de Porto Alegre em 2011. O perfil de susceptibilidade aos antimicrobianos, disco difusão e microdiluição em caldo – CIM (determinação da concentração inibitória mínima) para polimixina B, foi realizada conforme o Clinical Laboratory Standard Institute (CLSI) 2011. Isolados resistentes aos carbapenêmicos foram avaliados para CIM dos carbapenêmicos e cefalosporinas, e para pesquisa fenotípica e genotípica de metalo-β-lactamase (MβL). Um total de 24/124 isolados foi separado em dois grupos, um sensível (grupo S) e outro resistente (grupo R) aos carbapenêmicos (imipenem e/ou meropenem) para investigação da heteroresistência a polimixina B. Realizou-se ensaio de heteroresistência em duplicata através de diluições seriadas, partindo de uma suspensão de 0,5 de MacFarland e inoculadas em Agar Mueller Hinton com concentrações crescentes de polimixina B (0; 0,5; 1; 2; 4 e 8μg/mL). Após 5 dias de passagem em meio sem antibiótico, foi determinada a CIM dos isolados que cresceram na concentração mais alta de polimixina B. O perfil de análise populacional (PAP) foi definido pela razão do número de unidades formadoras de colônia (UFC) da placa com maior concentração de polimixina B onde houve crescimento bacteriano, pelo número de UFC da placa sem antibiótico. Foram consideradas heteroresistentes amostras que apresentaram subpopulações com crescimento em concentração de polimixina B ≥ 2 μg/mL. Amostras com subpopulações com crescimento em concentração de polimixina B superiores duas vezes ao CIM original, mas < 2 μg/mL, foram classificadas como heterogêneas. O resultado do disco difusão indicou heterogeneidade de suscetibilidade, sendo que gentamicina e imipenem foram os antibióticos com maior percentual de resistência e aztreonam e ciprofloxacino apresentaram os maiores perfis de sensibilidade. Todos os isolados foram sensíveis à polimixina B, com CIM50 e CIM90 de 1μg/mL e 2μg/mL, respectivamente. Trinta e sete isolados (30%; 37/124) apresentaram resistência aos carbapenêmicos. Quatro amostras foram positivas para MβL no teste fenotípico, sendo que o gene blaIMP foi idenficado nestas amostras. O grupo S não apresentou subpopulação heteroresistente, porém 3 isolados apresentaram subpopulação heterogênea. A freqüência do PAP no grupo S variou entre 2,1x10-4 a 4,0x10-7. O grupo R apresentou uma amostra heteroresistente e 6 isolados apresentaram subpopulação heterogênea, a freqüência do PAP variou entre 2,6x10-4 a 2,0x10-7. Os resultados deste estudo indicam baixa ocorrência de heteroresistência à polimixina B em amostras de P. aeruginosa tanto resistentes quanto sensíveis aos carbapenêmicos. No entanto, diversas amostras apresentaram subpopulações heterogêneas (CIM aumentada para a polimixina B), o que poderia explicar eventuais falhas terapêuticas durante o tratamento. / Therapeutic options to treat infections caused by Pseudomonas aeruginosa are limited because of their different resistance mechanisms that can be or don't be detected in the clinical laboratory. A phenotype that has been observed in our laboratory is the emergence of resistant subpopulations from a population sensitive to antibiotics - a phenomenon named heteroresistance. In P. aeruginosa this phenomenon has been investigated for carbapenems, however, in relation to the polymyxin B no data in the literature. We investigate the heteroresistance and polymyxin B into two groups P. aeruginosa, one sensitive and second resistant to carbapenems. One hundred twenty-four strains of P. aeruginosa were obtained randomly at the Hospital de Clinicas de Porto Alegre in 2011. The Antimicrobial Susceptibility Testing (Disk-difusion and the microdiluition broth, with determination of minimum inhibitory concentration (MIC) for polymyxin B, was performed according the Clinical Laboratory Standards Institute (CLSI), 2011. Isolates resistant to carbapenems were evaluated for MIC of carbapenems and cephalosporins, also for phenotypic and genotypic metallo-β-lactamase (MβL). A total of 24/124 strains were separated in two groups, one sensitive (S group) and other resistant (R group) to carbapenems (imipenem and / or meropenem) for investigation of heteroresistance polymyxin B. The assay was performed in duplicate heteroresistance through serial dilutions, starting from a 0.5 MacFarland suspension and inoculated into Mueller Hinton Agar with increasing concentrations of polymyxin B (0; 0,5; 1; 2; 4 e 8μg/mL). After 5 days of passage in medium without antibiotics, was determined the MIC of the isolates that grew at the highest concentration of polymyxin B. The population analysis profile (PAP) was defined as the ratio of the number of colony forming units (CFU) on the card with the highest concentration of polymyxin B in which bacterial growth, the number of CFU plate without antibiotic. We considered heteroresistant samples that showed subpopulations with growth in concentration of polymyxin B ≥ 2 mg / mL. Samples with subpopulations growing at higher concentration of polymyxin B twice CIM original, but <2 mg / mL were classified as heterogeneous. The result of AST indicated heterogeneity of susceptibility, and gentamicin and imipenem were the highest percentage with antibiotic resistance and aztreonam and norfloxacin showed the highest sensitivity profiles. All isolates were susceptible to polymyxin B, with CIM50 and CIM90 of 1μg/mL and 2μg/mL, respectively. Thirty-seven isolates (30%; 37/124) were resistant to carbapenems. Four samples were positive for the phenotypic test to MβL and the blaIMP gene was indentificated in this samples. The S group showed no subpopulation heteroresistente, but 3 isolates showed heterogeneous subpopulation. The frequency of PAP in group S varied between 2,0x10-4 to 4,0x10-7. The group R provided a sample heteroresistant and 6 isolates showed heterogeneous subpopulation, the frequency of PAP varied between 2,6x10- 4 a 2,0x10-7. The results of this study indicate a low occurrence of heteroresistance to polymyxin B in samples of P. aeruginosa so resistant assensitive to carbapenems. However, several samples showed heterogeneous subpopulations (MIC increased to polymyxin B) which could explain possible treatment failure during treatment.

A doença de Chagas possui uma importante relevância clínica, sendo uma das principais causas de mortalidade e morbidade na América Latina. As relações entre a lesão tecidual miocárdica e os defeitos na inervação autonômica na doença de Chagas são pouco conhecidas. Este trabalho descreve o desenvolvimento e aplicação de métodos de segmentação, corregistro e análise de imagens capazes de prover uma análise integrada das lesões teciduais através do imageamento de ressonância magnética (MRI), perfusão miocárdica e inervação autonômica, disponíveis através da tomografia de emissão de fótons (SPECT). O método proposto é baseado na segmentação e corregistro entre as imagens MRI e imagens SPECT usando 99mTc-MIBI e 123I-MIBG. Para realizar a segmentação do miocárdio, foi utilizada a técnica de Contorno Ativo Geodésico. A segmentação de fibrose em imagens MRI foi realizada com base na maximização da entropia de Tsallis. O corregistro não-rígido foi realizado através do método B-Spline. Os resultados de quantificação indicam correlações entre a presença de fibrose, desnervação e isquemia, além de mostrar a presença de regiões de miocárdio vivo, isquêmico e desnervado. Assim, a ferramenta desenvolvida fornece uma análise integrada de informação, permitindo uma melhor compreensão da relação entre o dano ao tecido do miocárdio e defeitos de inervação autonômica causadas pela doença de Chagas. / Chagas disease is of major clinical relevance, and a major cause of morbidity and mortality in Latin America. The relations between the myocardial tissue damage, myocardial perfusion and defects in autonomic innervations are poorly understood. This study proposes the development and application of image analysis methods capable of providing an integrated visualization and analysis of tissue injuries through enhanced magnetic resonance imaging (MRI), autonomic innervations and myocardial perfusion, available through photon emission tomography (SPECT). The proposed method is based on segmentation and registration between MRI images and SPECT images using 99mTc-MIBI and 123I-MIBG. To perform the segmentation of myocardium, we used Geodesic Active Contour. Fibrosis segmentation in MRI images was performed based on the algorithm of maximum Tsallis entropy. Nonrigid registrations was performed based on B-Spline method. The quantification results showed correlations between the presence of fibrosis, denervation and ischemia, as well as showing the regarded presence of regions of healthy myocardium, ischemic and denervated. Thus, the developed tool provides an integrated analysis of information contributing to a better understanding of the relationship between myocardial tissue damage and autonomic innervations injuries caused by Chagas disease.

As estrelas Be compreendem uma grande faixa de massas e temperaturas. Por definição, são objetos de tipo B com classe de luminosidade entre V e III que apresentam, ou apresentaram alguma vez, linhas de Balmer em emissão (eventualmente metais uma vez ionizados) e/ou linhas com padrões de absorção shell, possivelmente formadas em um envelope circunstelar. Embora se saiba há muito tempo que esses objetos são rodadores rápidos e que giram pelo menos 1,5 a 2 vezes mais rápido do que as estrelas B normais, ainda é incerto se esses objetos são ou não em média rodadores crticos, não obstante as recentes observações interferométricas de HD 10144 (Achernar) (uma estrela Be tpica) indicarem se tratar de um rodador crtico. Devido às suas altas taxas de rotação, as quais originam distorções geométricas e distribuições não uniformes de temperatura dependentes da latitude estelar, os valores de velocidade de rotação derivados por métodos clássicos são sistematicamente subestimados. Além disso, os efeitos da rotação, aliados à presença do envelope circunstelar, mascaram as condições fsicas desses objetos, resultando em diferenças significativas em seus estágios evolutivos na seqüência principal. Neste trabalho apresentamos os resultados do estudo espectroscópico de estrelas Be em duas vertentes. Na primeira tratamos o tema da formação e estrutura do envelope circunstelar de estrelas Be, através das análises de duas estrelas, HD 127972 ( Cen) e HD 10144 (Achernar). Nesse estudo identificamos e caracterizamos seus modos de pulsação, os quais se constituem em um possvel mecanismo de perda de massa e formação do envelope. Além disso, estudamos a estrutura de seus envelopes circunstelares através da modelagem de perfis de Balmer em emissão. Na segunda vertente quantificamos as condições fsicas de 141 estrelas de campo, onde 114 são de tipo Be e 27 estrelas são B normais. Nesse estudo, comparamos os estágios evolutivos desses objetos obtidos através de métodos clássicos com os estágios evolutivos corrigidos dos efeitos da rotação elevada. Concluimos que o \"fenômeno Be\" pode ocorrer em todas as fases da evolução estelar na seqüência principal. / Be stars encompass a large mass and temperature range. By definition, they are B-type objects with luminosity classes V to III that have, or have shown at least once, Balmer lines in emission (eventually single-ionized metals) and/or lines with shell absorption patterns possibly formed in a circumstellar envelope. Though it has long been known that these objects are fast rotators and that they rotate at least 1.5 to 2 times faster than normal B stars, it is still uncertain whether or not these objects are in average critical rotators, although recent interferometric observations on Achernar (a typical Be star) pointed it out to be a critical rotator. Due to their high rotation rates which originate geometrical distortions and non-uniform temperature distributions dependent on the stellar latitude, the rotation velocity values derived from classical methods are systematically underestimated. Moreover, the rotation effects allied to the continuum emission due to the presence of a circumstellar envelope disguise the physical conditions of these objects, resulting in significative differences of their main-sequence evolutionary stages. In this work we present the results of the spectroscopic study of Be stars in two approaches. In the first one we treat the subject of formation and structure of the circumstellar envelope of Be stars through the analyses of two stars, HD 127972 ( Cen) and HD 10144 (Achernar). In this study we identify and characterize their pulsation modes, which constitute in a possible mechanism of mass loss and envelope formation. Moreover we study the structure of their circumstellar envelopes through the modeling of Balmer profiles in emission. In the second approach we quantify the physical conditions of 141 field stars, where 114 are Be-type stars and 27 stars are normal B stars. In this study, we compared the evolutionary stages of these objects obtained through classical methods with evolutionary stages corrected for high rotation effects. We conclude that the \"Be phenomenon\" can occur at whatever stage of the stellar evolution on the main sequence.

Submitted by Celso Magalhaes (celsomagalhaes@ufrrj.br) on 2017-08-28T14:10:07Z No. of bitstreams: 1 2014 - Karla Emanuelle Campos Araujo.pdf: 681239 bytes, checksum: 370286856e74db8636cdc28b6d35e0b6 (MD5) / Made available in DSpace on 2017-08-28T14:10:07Z (GMT). No. of bitstreams: 1 2014 - Karla Emanuelle Campos Araujo.pdf: 681239 bytes, checksum: 370286856e74db8636cdc28b6d35e0b6 (MD5) Previous issue date: 2014-07-29 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / ARAUJO, Karla Emanuelle Campos. Isotopic fractionation of 15N in nitrogen fixation in soybean due to light intensity and Bradyrhizobium spp. strain inoculated. 2014. 40f. Dissertation (Master in Plant Science). Institute of Agronomy, Department of Plant Science, Federal Rural University of Rio de Janeiro, Serop?dica, RJ, 2014. This work aimed to study the isotopic fractionation of N from BNF in stems function and light intensity in soybean plant symbiosis with Bradyrhizobium. The 'B' value was evaluated for ten Bradyrhizobium strains, as well as their interaction with the soybean plant (Glycine max L.). The experiment was conducted at Embrapa greenhouse Agrobiologia, Serop?dica, RJ. The soybean plants inoculated with the different strains of Bradyrhizobium and a control not inoculated, were grown under conditions similar to Leonard vessels using substrate of sand and vermiculite in a 2: 1 (v / v). In the second experiment to assess the value 'B' of the resulting symbiosis BNF among cultivars (cv) of soybean BRS 133, BRS 184, MONSOY9144 nine Bradyrhizobium. Soybean plants in all the treatments, were grown under the same conditions, the soybean cultivation was performed in vessels Leonard with sand and perlite substrate in the proportion 1: 1 (v / v). In both experiments after 47 days of planting the plants were harvested and calculated the value 'B'. In the third experiment evaluated the effect of different strains of Bradyrhizobium and the reduction in the intensity of light on natural abundance of 15N N2 fixed by the strains in symbiosis with soybean cv BRS 133. An experiment was conducted in the experimental field of Embrapa Agrobiology. The soybean plants inoculated with 11 Bradyrhizobium and treatment uninoculated were grown under similar conditions in plastic pots with sand substrate and pearlite in the proportion 1: 1 (v / v) .After 75 days after planting the plants were collected and calculated the value 'B'.No experiment 1, the values of ?Bpa? ranged between -2 and -4 ?, according to the strain used. In experiment 2, the values of ?Bpa? showed no difference between the soybean cultivars inoculated with Bradyrhizobium as the isotopic abundance of 15N in plants grown entirely dependent on FBN. There was a tendency for the abundance of 15N values in ?Bpa? treatment of plants inoculated with strains of B. elkanii to be less negative than in the case of plants inoculated with B. japonicum. In experiment 3 the values of ?B?PA showed natural 15N abundance values significantly more negative than the shaded plants. There was a tendency for the abundance of 15N in the shoot (and values of ?Bpa?) treatment of the plants inoculated with B. japonicum strains to be less negative than in the case of plants inoculated with B. elkanii. For the determination of the 'B' value, the plants should be in the same stage of maturity that being used to sample the plants in the field, to assess the value 'B' in low light intensity conditions (green house) can lead to the result of a 'B' value less negative than that given in full sun and so probably less appropriate uses it to calculate the contribution of BNF in legume under field conditions. / ARAUJO, Karla Emanuelle Campos. Fracionamento isot?pico do 15N na fixa??o biol?gica de nitrog?nio na soja em fun??o da intensidade de luz e estirpe de Bradyrhizobium spp. inoculada. 2014. 40f. Disserta??o (Mestrado em Fitotecnia). Instituto de Agronomia, Departamento de Fitotecnia, Universidade Federal Rural do Rio de Janeiro, Serop?dica, RJ, 2014. Este trabalho teve como objetivo estudar o fracionamento isot?pico do N proveniente da FBN, em fun??o de estipes e intensidade de luz na simbiose da planta de soja com Bradyrhizobium. O valor ?B? foi avaliado para dez estirpes de Bradyrhizobium, assim como sua intera??o com a planta de soja (Glycine max, L.). Foi conduzido o experimento na casa de vegeta??o da Embrapa Agrobiologia, Serop?dica, RJ. As plantas de soja inoculadas com as diferentes estirpes de Bradyrhizobium e um controle, n?o inoculado, foram crescidas em condi??es semelhantes em vasos Leonard, utilizando substrato de areia e vermiculita na propor??o 2:1 (v/v). No segundo experimento para avaliar o valor ?B? da FBN resultante da simbiose entre as cultivares (cv) de soja BRS 133, BRS 184, MONSOY9144 e nove estirpes de Bradyrhizobium. As plantas de soja para todos os tratamentos, foram crescidas em condi??es iguais, o cultivo da soja foi realizado em vasos Leonard, utilizando substrato de areia e perlita na propor??o 1:1 (v/v). Nos dois experimentos ap?s 47 dias do plantio as plantas foram colhidas e calculou o valor ?B?. No terceiro experimento avaliou-se o efeito de diferentes estirpes de Bradyrhizobium e da redu??o na intensidade de luz sobre abund?ncia natural de 15N do N2 fixado pelas estirpes em simbiose com a cv. de soja BRS 133. Foi conduzido um experimento no campo experimental da Embrapa Agrobiologia. As plantas de soja inoculadas com 11 estirpes de Bradyrhizobium e um tratamento n?o inoculado, foram crescidas em condi??es semelhantes em vasos pl?sticos, utilizando substrato de areia e perlita na propor??o 1:1 (v/v). Ap?s 75 dias do plantio as plantas foram colhidas e calculou-se o valor ?B?. No experimento 1, os valores de ?Bpa? variaram entre -2 e -4 ?, de acordo com a estirpe usada. No experimento 2, os valores de ?Bpa? n?o apresentaram diferen?a entre as cultivares de soja inoculadas com os Bradyrhizobium quanto a abund?ncia isot?pica de 15N nas plantas crescida inteiramente dependente da FBN. Houve uma tend?ncia para a abund?ncia de 15N nos valores de ?Bpa? das plantas dos tratamentos inoculados com estirpes de B. elkanii a serem menos negativo do que no caso de plantas inoculadas com B. japonicum. No experimento 3 os valores de ?Bpa? apresentaram valores de abund?ncia natural de 15N significativamente mais negativos do que as plantas sombreadas. Houve uma tend?ncia para a abund?ncia de 15N na parte a?rea (e os valores de ?Bpa?) das plantas dos tratamentos inoculados com estirpes de B. japonicum a serem menos negativo do que no caso de plantas inoculadas com B. elkanii. Para ? determina??o do valor ?B?, as plantas devem estar no mesmo est?gio de maturidade que as que est?o sendo utilizadas para amostrar as plantas no campo, a avalia??o do valor ?B? em condi??es de intensidade de luz reduzida (casa de vegeta??o) pode levar ao resultado de um valor ?B? menos negativo do que aquele determinado em pleno sol e assim provavelmente menos apropriado utiliza-lo, para calcular a contribui??o da FBN em leguminosa em condi??es de campo.

Plusieurs données expérimentales suggèrent que la protéine Core du virus de l’Hépatite B (HBV), en plus de ses fonctions structurales pour la formation des nucléocapsides dans le cytoplasme, pourrait avoir des fonctions régulatrices importantes dans le noyau des hépatocytes infectés. En effet, Core s’associe à l’ADNccc et aux promoteurs de certains gènes cellulaires dans le noyau des hépatocytes infectés et pourrait ainsi contrôler leur régulation transcriptionnelle. De plus, de par sa capacité à lier les ARN, elle pourrait également participer au métabolisme post-transcriptionnel de gènes viraux et/ou cellulaires. Pour caractériser ces fonctions, nous avons réalisé une analyse protéomique des facteurs cellulaires qui interagissent avec la protéine Core dans le noyau d’hépatocytes humains. Cet interactome a mis en évidence un grand nombre de protéines de liaison aux ARN (RBP), qui participent au métabolisme des ARN et en particulier aux mécanismes d’épissage. Deux interactants majeurs de Core ont été plus particulièrement étudiés, SRSF10 et RBMX, impliqués notamment dans l’épissage et la réparation de l’ADN. Une analyse fonctionnelle effectuée par une approche siRNA a montré que SRSF10 et RBMX affectent différemment le niveau des ARN viraux, vraisemblablement en agissant à des étapes différentes du cycle viral. De même, un composé ciblant l’activité de certaines RBP diminue fortement la réplication d’HBV en affectant l’accumulation des ARN viraux. Ainsi, ces résultats suggèrent que Core pourrait interagir avec certaines RBP pour contrôler le destin des ARN viraux et/ou cellulaires, une piste intéressante pour le développement de nouvelles stratégies antivirales ciblant l’hôte / Converging evidences suggest that the Hepatitis B virus (HBV) core protein, beside its well-known structural role to form nucleocapsids in the cytoplasm, could have important regulatory functions in the nucleus of infected hepatocytes. Indeed, nuclear Core was shown to associate with the cccDNA and to the promoters of some cellular genes, suggesting that Core may control viral and/or cellular gene expression. In addition, Core has the capacity to bind RNA, and may thus regulate HBV RNA metabolism. To elucidate these functions, we performed a proteomic analysis of the cellular factors interacting with nuclear Core in human hepatocytes. This interactome revealed a majority of highly interconnected RNA-binding proteins (RBPs), which participate in several steps of mRNA metabolism, including transcription, splicing and nuclear egress. We focused on two major Core-interacting factors, SRSF10 and RBMX that were previously involved in splicing and DNA repair. Functional analyses performed by a siRNA approach indicated that RBMX and SRSF10 were able to differentially regulate the levels of all viral RNAs most likely by acting at different steps of the viral life-cycle. Similarly, a small compound, affecting the activity of selected RBPs, severely impaired HBV replication by strongly reducing viral RNA accumulation. Altogether, these results strongly suggest that Core interacts with some selected RBPs to control the fate of viral and/or cellular RNAs and provide new critical information for the development of novel host-targeting antiviral agents (HTA)

A utilização de produtos naturais para a terapêutica do câncer foi iniciada com a actinomycina D, obtida a partir de culturas de Streptomyces e desde então, a busca por compostos bioativos de origem natural constitui uma importante linha de pesquisa. Estima-se que aproximadamente 60% dos agentes antineoplásicos, introduzidos para a terapia do câncer nas últimas décadas, tem origem vegetal ou microbiana. Dentre os micro-organismos proeminentes para produção de compostos ativos, as actinobactérias se destacam pela versatilidade metabólica, praticidade para cultivo in vitro e eficiência para produção de compostos com atividade anticâncer. Em seu último relatório, a Organização Mundial da Saúde reportou 8,8 milhões de mortes em decorrência de câncer, no ano de 2017. O índice representa um em cada seis óbitos em todo o mundo, sendo mais expressivo em países de média e baixa renda. Vale ressaltar que avanços significativos foram alcançados nos últimos anos para o tratamento de leucemia aguda infantil e tumores derivados de células germinais. Contudo, tumores sólidos de pulmão, próstata, mama e cólon ainda representam altos índices de mortalidade. Frente a isso, torna-se evidente a necessidade de identificar e desenvolver estratégias para o tratamento da doença. Com intuito de acessar novos recursos microbianos com potencial biotecnológico, a prospecção avança para áreas pouco exploradas, como por exemplo, o Continente Antártico. A Antártica foi o último dos continentes a ser acessado pelo homem e apresenta características edafoclimáticas favoráveis ao endemismo. Em vista da problemática e da potencialidade do Continente Antártico, os recursos microbiológicos associados à rizosfera de Deschampsia antarctica Desv. foram acessados e avaliados para a produção de compostos com propriedade antitumoral. Em resultado foram obtidos 42.528 clones metagenômicos e 72 linhagens de actinobactérias, dentre as quais Streptomyces sp. CMAA 1527, que apresentou pronunciada atividade antiproliferativa in vitro, para tumores de mama, pulmão, rim e sistema nervoso central, através da produção de cinerubina B. A análise taxonômica das actinobactérias isoladas revelou a presença de linhagens com baixo índice de similaridade, com as linhagens tipo conhecidas, o que pode significar a presença de novas espécies para os gêneros Nocardia, Rhodococcus e Streptomyces, reconhecidos pela capacidade de produzir metabólitos ativos e enzimas de interesse biotecnológico. A análise taxonômica polifásica da linhagem CMAA 1533 possibilitou a descrição da espécie Rhodococcus psychrotolerans sp. nov. (TaxoNumber TA00191; NRRL B-65465T = DSM 104532T), grupo bacteriano promissor como agente de biorremediação e produção de compostos bioativos. Com isso, o Continente Antártico foi considerado um ambiente promissor para a busca de novos micro-organismos, dentre eles actinobactérias, eficientes na produção de compostos antitumorais e outras substâncias com potencial biotecnológico. / The use of natural products for cancer therapy was initiated with the actinomycin D, obtained from Streptomyces. Since then, the search of bioactive from natural sources represent an essential line of research. It is estimated that approximately 60% of the antineoplasic agents inserted for the cancer therapy in recent decades have vegetal and microbial origin. Among the prominent microorganisms used to produce active compounds, actinobacterias are known by their metabolical versatility, convenience related to in vitro culture, and efficiency on the production of anticancer compounds. The Health World Organization, on its last review, reported 8.8 million of deaths in 2017, caused by cancer. Those numbers represent one out of six deaths worldwide, being more expressive in middle and low income countries. It is worth pointing out that meaningful advances were established in recent years for the treatment of childhood acute leukemia and germ cell-derived tumors. However, solid tumors of the lung, prostate, breast and colon still represent high mortality rates. For this reason, it is necessary to identify and develop strategies for the treatment of the disease. With the aim of accessing new microbial resources that contain biological potential, the prospection advance to areas barely explored, such as the Antarctic Continent. Antarctica was the last of the continents to be accessed by man and presents edaphoclimatic characteristics favorable to endemism. In light of the problematic and the potentiality of the Antarctic Continent, the microbiological resources associated with the rhizosphere of Deschampsia antarctica Desv. were accessed and evaluated for the production of compounds with antitumor properties. The results obtained had shown 42,528 metagenomic clones and 72 strains of actinobacteria, among them Streptomyces sp. CMAA 1527, which had presented anti-proliferative activity in vitro to breast, lung, kidney and central nervous system tumors, through the production of cinerubin B. The taxonomic analysis of the actinobacteria isolated revealed the presence of strains with low rate of similarity, with known type strains, which may mean the presence of new species for the genera Nocardia, Rhodococcus and Streptomyces, recognized for the ability to produce active metabolites and enzymes of biotechnological interest. The polyphasic approach of the CMAA 1533 strain made possible the description of the species Rhodococcus psychrotolerans sp. nov. (TaxoNumber TA00191; NRRL B-65465T = DSM 104532T), promising bacterial group as a bioremediation agent and production of bioactive compounds. As a result, the Antarctic Continent was considered a promising environment to search new microorganisms, among them, the actinobacteria, which is efficient on the production of antitumor compounds and other substances with biotechnological potential.

L'infection par le virus de l'hépatite B est un problème de santé publique avec 250 millions de porteurs chroniques et cela malgré l'existence d'un vaccin préventif. Les traitements actuellement utilisés sont les analogues de nucléos(t)ide et/ou l'interféron a. Bien qu'ils permettent une diminution de la charge virale, ils ne permettent pas d'éradiquer la maladie du fait de la persistance de l'ADNccc, le minichromosome de l'hépatite B. Cet ADN sert de matrice à la transcription virale, et la présence d'une seule copie permet la réactivation de l'infection. En prenant en compte la longue demi-vie des hépatocytes et de la stabilité de l'ADNccc dans leur noyau, un modèle mathématique suggère que de nombreuses années de traitement seraient nécessaires pour éliminer complètement cet ADN du foie des patients infectés chroniquement. Les techniques utilisées en routine pour la quantification de l'ADNccc ne sont pas assez sensibles pour pouvoir détecter des faibles concentrations de cet ADN, notamment dans des biopsies de patients infectés chroniquement et traités à long terme. Il est nécessaire de développer de nouvelles stratégies permettant de cibler directement l'ADNccc afin d'éliminer le virus. Ainsi les travaux de cette thèse reposent sur le développement d'une nouvelle technologie : la Droplet Digital PCR (ddPCR) pour permettre la quantification de l'ADNccc dans les biopsies de patient. Cette technique permet un gain de 2 log au niveau de la sensibilité par rapport à la qPCR, technique utilisée actuellement en routine. Elle nous a ainsi permis de constater la présence de cet ADN chez des patients traités à long terme par des analogues de nucléos(t)ides et même en présence d'interféron. La présence d'ARNpg et les expériences de ChIP ont également confirmé que l'ADNccc était encore transcriptionnellement actif. Ces résultats confirment d'autant plus la nécessité d'élaborer de nouvelles thérapeutiques pour permettre l'inactivation voire l'élimination de l'ADNccc. L'une des stratégies envisagées est le système CRISPR/Cas 9. Ainsi le dernier axe de cette thèse a été de développer ce système dans des modèles d'infection du virus de l'hépatite B. Pour vérifier l'efficacité de ce système sur le VHB, nous avons testé 8 ARN guide différents incorporer via des ribonucléoprotéines dans des cellules HepG2-NTCP. Les résultats préliminaires ont ainsi démontré que ce système pouvait réduire le pool d'ADNccc dans ces cellules et ouvre des perspectives intéressantes pour le développement de nouveaux traitements / Hepatitis B virus {HBV) is a major health problem with 250 million chronic carriers, despite the existence of a preventive vaccine. Currently the treatments used are nucleos{t)ide analogues and / or interferon a. Although they efficiently reach a decrease of the viral load, they do not allow the eradication the disease due to the persistence of the cccDNA, the minichromosome of the hepatitis B. This DNA serves as a template for the viral transcription and only a single copy suffice for the infection rebound. However, the techniques used routinely for the quantification of the cccDNA are not sensitive enough to be able to detect low concentrations of this DNA, in particular in biopsies of patients chronically infected and long term treated. ln addition, it is necessary to develop new strategies to target the cccDNA in order to clear the infection. Thus, my thesis work is based on the development of a new technology: the Droplet Digital PCR {ddPCR) to allow the quantification of cccDNA in patient biopsies. This technique allows a gain of 2 log in sensitivity compared to the qPCR technique currently used in routine. lt allowed us to see the presence of this DNA in long-term treated patients even in the presence of interferon. The presence of pgRNA and ChlP experiments also confirmed that the cccDNA was still transcriptionally active.These results confirm the requirement to develop new therapeutics to allow the inactivation or even the elimination of the cccDNA. One of the strategies envisaged is the CRlSPR / Case 9 system. Thus, the following part of this thesis was to develop this system in hepatitis B virus infection models. To reduce off-target effect we tested 8 different guide RNAs incorporated via ribonucleoproteins into HepG2- NTCP cells. Preliminary results have shown that this system can reduce the pool of cccDNA in these cells and open up the possibilities to test this model on PHH and opens interesting perspectives for the development of new treatments

Wong, Yee Man Melody. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 148-174). / Abstract also in Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / Abbreviations --- p.v / Review of the literature --- p.1 / Overview of liver fibrosis --- p.2 / Pathophysiology of liver fibrosis --- p.3 / Histological classification of liver fibrosis --- p.4 / Gold standard for fibrosis assessment - Liver biopsy --- p.6 / Biomarker in blood - non-invasive method for assessing diseases --- p.7 / Significance of non-invasive markers of liver fibrosis --- p.9 / Biomarkers of liver fibrosis --- p.10 / Direct markers --- p.10 / Indirect markers --- p.11 / Proteomics / Why proteomics? --- p.16 / Clinical values of proteomics in biomarker discovery --- p.17 / Challenges in proteomics --- p.18 / Current proteomics technologies in biomarker discovery --- p.21 / Gel based --- p.21 / Gel free approach - MS based --- p.23 / Quantitative proteomics --- p.29 / Application of proteomics to discovery of biomarkers for diagnosis of liver fibrosis --- p.33 / Rationale and Objectives of the Project --- p.35 / Chapter Section 1 --- Method development of magnetic beads-based proteomic profiling for quantitative proteomic profiling and micro- purification in parallel --- p.36 / Chapter 1.1 --- Introduction --- p.36 / Chapter 1.2 --- Materials and methods --- p.38 / Chapter 1.3 --- Results / Chapter 1.3.1 --- Serum proteome profiles obtained with different types of chromatographic magnetic beads --- p.46 / Chapter 1.3.2 --- Performance of PCS 4000 ProteinChip reader --- p.49 / Chapter 1.3.3 --- Reproducibility of magnetic beads-based serum proteomic profiling --- p.54 / Chapter 1.3.4 --- Gel electrophoresis of the eluted proteins --- p.58 / Chapter 1.3.5 --- Identification of the protein peaks --- p.58 / Chapter 1.4 --- Discussion --- p.60 / Chapter 1.5 --- Conclusion --- p.64 / Chapter Section 2 --- Development of a proteome-based fingerprinting model for detecting liver fibrosis in patients with chronic hepatitis B infection --- p.65 / Chapter 2.1 --- Introduction --- p.65 / Chapter 2.2 --- Materials and methods --- p.68 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Patients characteristics --- p.75 / Chapter 2.3.2 --- Correlation between biochemical/serological markers and the degrees of liver fibrosis --- p.81 / Chapter 2.3.3 --- Serum proteomic profiling by linear MALDI-TOF MS --- p.81 / Chapter 2.3.4 --- Correlation of proteomic features with Ishak score --- p.81 / Chapter 2.3.5 --- Correlation of significant proteomic features with serological markers --- p.89 / Chapter 2.3.6 --- Construction of diagnostic model in detecting liver fibrosis and cirrhosis --- p.91 / Chapter 2.3.7 --- Cross-validation of the diagnostic model using pre- treatment samples in detecting liver fibrosis and cirrhosis --- p.91 / Chapter 2.3.8 --- Independent validation of the diagnostic model using post-treatment samples in detecting liver fibrosis and cirrhosis --- p.95 / Chapter 2.3.9 --- Comparison against other non-invasive models in detecting liver fibrosis and cirrhosis --- p.98 / Chapter 2.4 --- Discussion --- p.103 / Chapter 2.5 --- Conclusion --- p.112 / Chapter Section 3 --- Identification of proteomic features to form diagnostic fingerprint for the detection of liver fibrosis in patients with chronic hepatitis B infection --- p.113 / Chapter 3.1 --- Introduction --- p.113 / Chapter 3.2 --- Materials and methods --- p.115 / Chapter 3.3 --- Results --- p.121 / Chapter 3.3.1 --- Protein identification of the protein marker in the diagnostic model --- p.121 / Chapter 3.3.2 --- Immunodepletion of apolipoprotein C-III --- p.125 / Chapter 3.3.3 --- Serum levels of apolipoprotins and their association with liver fibrosis --- p.127 / Chapter 3.4 --- Discussion --- p.130 / Chapter 3.5 --- Conclusion --- p.139 / General discussion --- p.141 / Reference --- p.148 / Original Data --- p.175

Further functional investigation revealed that knockdown of GRP78 expression by RNA interference resulted in a significant increase of both intracellular and extracellular HBV virions in the transient HBV-producing HepG2 cells, concomitant with enhanced levels of hepatitis B surface antigen and e antigen in the culture medium Conversely, overexpression of GRP78 in HepG2 cells led to HBV suppression concomitant with induction of the positive regulatory circuit of GRP78 and interferon-beta 1 (IFN-beta1). In this connection, IFN-beta1-mediated 2', 5'-oligoadenylate synthetase (OAS) and ribonuclease L (RNase L) signaling pathway was noted to be activated in GRP78-overexpressing HepG2 cells. Moreover, GRP78 was significantly down-regulated in the livers of chronic hepatitis B patients after effective anti-HBV treatment (p= 0.019) as compared with their counterpart pre-treatment liver biopsies. / Hepatitis B virus (HBV) infection is a global public health problem, which plays a crucial role in the pathogenesis of chronic hepatitis, cirrhosis and hepatocellular carcinoma. Although considerable progress has been made over the past decade, the pathogenesis of HBV infection and the mechanisms of host-virus interactions are still elusive. / In conclusion, the present study demonstrates for the first time that GRP78 functions as an endogenous anti-HBV factor via IFN-beta1-OAS-RNase L pathway in hepatocytes. Induction of hepatic GRP78 may provide a novel therapeutic approach in treating HBV infection. / In this study, we applied a two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomic approach to globally analyze the host early response to HBV by using an inducible HBV-producing cell line HepAD38. Twenty-three proteins were identified as differentially expressed, with glucose-regulated protein 78 (GRP78) as one of the most significantly up-regulated proteins induced by HBV replication. This induction was further confirmed in both HepAD38 and HepG2 cells transfected with HBV-producing plasmids by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, as well as in HBV-infected human liver biopsies by immunohistochemistry. / Ma, Yan. / Adviser: Ming-Liang He. / Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 111-129). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Lau, Chi Chiu. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 133-142). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.