A compensação da dosagem gênica entre fêmeas XX e machos XY em mamíferos é adquirida através de um complexo mecanismo epigenético que resulta na inativação de grande parte de um dos cromossomos X nas células femininas. O processo de inativação do cromossomo X (XCI) se inicia cedo durante a embriogênese, concomitantemente à diferenciação celular, e envolve a aquisição de modificações epigenéticas características do cromossomo X inativo (Xi). Uma estabelecido, o padrão de inativação é estavelmente mantido através de todas as mitoses celulares subsequentes e por toda a vida do organismo (exceto para células germinativas que sofrem reativação do Xi). Os mecanismos envolvidos na iniciação e estabelecimento da XCI foram extensivamente estudados, especialmente em camundongos. Embora algumas características epigenéticas associadas à manutenção da XCI tenham sido descritas, a identidade e modo específico de ação de fatores envolvidos durante essa fase da XCI são aspectos ainda não bem compreendidos. Além disso, o processo de XCI apresenta diferenças importantes entre humanos e camundongos e estudos direcionados para a identificação de novos componentes envolvidos na manutenção da XCI em humanos tornam-se de fundamental importância. Triagens funcionais genômicas por bibliotecas de shRNAs constituem uma ferramenta poderosa para a identificação de genes envolvidos em diferentes mecanismos celulares e vias bioquímicas. Sendo assim, utilizamos essa ferramenta para triar genes envolvidos na manutenção da XCI em humanos. Células somáticas femininas primárias HPRT+/-/HPRT- foram transduzidas com uma biblioteca lentiviral de shRNAs e posteriormente tratadas em meio de cultura contendo a droga HAT para seleção de células HPRT+ nas quais esperava-se que o cromossomo Xi presente tivesse sofrido reativação em decorrência do knockdown de genes envolvidos na manutenção da XCI. Essa estratégia nos permitiu identificar 20 novos genes candidatos a estarem envolvidos na manutenção da XCI. Esses candidatos deverão ser avaliados individualmente para confirmar seu papel no processo de controle epigenético do cromossomo X / Transcriptional dosage compensation between mammalian XX females and XY males is acquired through a complex epigenetic mechanism that leads to the inactivation of most part of one of the X chromosomes in the female cells. The X chromosome inactivation (XCI) process takes place early during embryogenesis and involves the acquisition of epigenetic modifications that are characteristic of the inactive X chromosome (Xi). Once silencing is established, the inactivation pattern is maintained in through all the subsequent mitosis and the same X chromosome remains stably silenced in all the descendant cells and throughout the life of the organism (except for the germ line cells that undergo X chromosome reactivation). The initiation of XCI has been studied extensively, especially in mice. Although some epigenetic features associated with the maintenance of XCI have already been described, the identity and specific mode of action of the factors involved in this phase of XCI are largely unknown. Moreover, the XCI process presents important differences between mice and humans, and studies directed to the identification of new players involved in the maintenance of human XCI are fundamentally important. Functional genome-wide screens using multiplex shRNA libraries are a powerful tool for the identification of genes involved in different cellular mechanisms and biochemical pathways. In order to screen for genes involved in the maintenance of XCI in humans, a population of HPRT+/-/HPRT- primary somatic female cells were transduced with a multiplex lentiviral shRNA library and subsequently treated in HAT medium to select for HPRT+ cells in which we expected that the Xi would undergo reactivation as a result of the knockdown of genes involved in the maintenance of XCI. As a result, we identified 20 new candidate genes that could potentially be involved in the maintenance of XCI. These candidates should be individually evaluated in order to confirm their role in the epigenetic control of the X chromosome

A soja vem ganhando destaque, pois alguns estudos apontam uma série de potenciais benefícios para a saúde. Estão presentes na soja dois grupos de inibidores de proteases. Alguns estudos demonstram que o tratamento térmico pode não ser suficiente para a inativação completa desses inibidores. Esses inibidores podem afetar a digestibilidade da proteína e a absorção e biodisponibilidade de alguns minerais, como o ferro. Realizaram-se cinco tratamentos: T1 e T2 - com secagem prévia + 10 e 15 minutos de forno, respectivamente; T3, T4 e T5 - sem secagem prévia + 45, 50 e 60 minutos de forno. O controle foi seco em estufa (55ºC) durante 31 horas. A quantidade de ferro nas amostras analisadas variou de 83,67 a 58,8 mg/kg e a porcentagem de ferro dialisado foi de 0,95% a 1,38%, estando de acordo com o encontrado na literatura. Os valores da digestibilidade da proteína in vitro variaram de 91,21% a 93,14%, sem que houvesse correlação com os inibidores de tripsina, sendo a melhora da digestibilidade devida ao tratamento térmico. A atividade dos inibidores de tripsina para os tratamentos com secagem prévia variou de 25,24 a 23,94 UTI/mg de proteína e para os sem secagem prévia de 46,39 a 38,33 UTI/mg de proteína. A atividade dos inibidores para o controle foi de 55,81 UTI/mg de proteína. Os valores estão de acordo com a literatura, sendo, portanto, considerados como adequados, mostrando que a tostagem em forno convencional é bastante eficaz quanto à inativação do inibidor de tripsina. Após a proteólise in vitro, verificou-se reativação dos inibidores de tripsina em todos os tratamentos analisados. Os valores variaram de 5,48 UTI/mg de proteína (controle) a 7,65 UTI/mg de proteína (T1). As maiores porcentagens foram para os tratamentos com secagem prévia, 30,31% e 28,45%, enquanto que para os sem secagem prévia foi de 14,68%, 14,12% e 19,38%. O controle apresentou 9,82% de reativação. Concluiu-se que a tostagem em forno convencional inativou os inibidores de tripsina, sendo o tratamento sem secagem prévia o mais eficaz, pois, com esse tratamento, houve menor reativação dos inibidores após a proteólise in vitro. A tostagem foi responsável pela melhora da digestibilidade da proteína e da diálise de ferro in vitro. O tratamento que promoveu a maior aceitabilidade dos grãos de soja tostados foi o que recebeu o tempo intermediário de 50 minutos, sem secagem prévia. / The soy comes gaining prominence, therefore some studies point a series of potential benefits with respect to the health. Two groups of inhibitors of proteases are present in the soy. Some studies demonstrate that the thermal treatment can not be enough for the complete inactivation of these inhibitors. These inhibitors can affect the digestibility of the protein and the absorption and availability of some minerals, as the iron. Five treatments had been do: T1 and T2 - with previous drying + 10 and 15 minutes of oven, respectively; T3, T4 and T5 - without previous drying + 45, 50 and 60 minutes of oven. The control was dry in oven (55ºC) during 31 hours. The amount of iron in the analyzed samples varied of 58,8 the 83,67 mg/kg and percentage of dialysed iron was of 0,95% the 1.38%, being in accordance with the found one in literature. The values of the digestibility of protein in vitro had varied of 91,21% the 93.14%, without that it had correlation with trypsin inhibitors, being the improvement of the digestibility due to the thermal treatment. The trypsin inhibitors activity for the treatments with previous drying varied of 25,24 the 23,94 UTI/mg of protein and for the ones without previous drying varied of 38,33 the 46,39 UTI/mg of protein. The inhibitors activity for the control was of 55,81 UTI/mg of protein. The values are according with the literature, being, therefore, considered as adjusted, showing that the roasting in conventional oven is sufficiently efficient how much to the inactivation of trypsin inhibitors. After proteolysis in vitro, verified reactivation of trypsin inhibitors in all the analyzed treatments. The values had varied of 5,48 UTI/mg of protein (control) the 7,65 UTI/mg of protein (T1). The biggest percentages had been for the treatments with previous drying, 30.31% and 28.45%, while that for the ones without previous drying it was of 14,68%, 14.12% and 19,38%. The control presented 9.82% of reactivation. The treatment the most efficient without previous drying was concluded that the roasting in conventional oven inactivated trypsin inhibitors, being, therefore, with this treatment, proteolysis had minor after reactivation of inhibitors in vitro. The roasting was responsible for the improvement of the digestibility of protein and dialyse of the iron in vitro. The treatment that promoted the biggest acceptability of the roasted grains of soy was what it received the time intermediate from 50 minutes, without previous drying.

Resíduos de Serviços de Saúde (RSS), ainda que tratados e dispostos em aterros sanitários, podem ser causas de impactos ambientais por apresentarem algum indicador de periculosidade. Segundo o apêndice IV da Resolução da Agência Nacional de Vigilância Sanitária (Anvisa) RDC nº 306/2004, para as tecnologias de tratamento desses resíduos é necessário que se atinja pelo menos o Nível III de inativação microbiana. Perante a ausência de dados na literatura que revelassem o tempo de exposição, temperatura e pressão ideais para a inativação microbiana no tratamento de RSS por autoclavagem e se a fração ocupada dos resíduos na autoclave poderia interferir na eficiência da desinfecção definiu-se o objetivo do trabalho para otimização do processo. Para os testes foram utilizados endósporos de Geobacillus stearothermophilus como bioindicadores e instalados cinco termopares na autoclave para aferição da temperatura dentro de todo o espaço da câmara. O RSS foi sintetizado diante das caracterizações como composição gravimétrica, distribuição granulométrica, densidade específica aparente, massa específica aparente e teor de umidade. Apresentou-se uma proposta metodológica para a avaliação do tratamento de RSS frente às dificuldades encontradas. Realizou-se ensaios com 116ºC, 125ºC e 134ºC, observando a fração de inativação em seis tempos de exposição diferentes. Sabendo-se que é padronizada em estabelecimentos de saúde a temperatura ideal para esterilização de materiais de 121ºC, apesar de não terem sido encontrados trabalhos científicos que comprovem a eficiência da esterilização nessa circunstância, foram realizados testes a 50 e 60 minutos de exposição para avaliar essa condição. Inoculou-se 106 endósporos nas amostras e a recuperação foi feita com filtração após a lavagem dos resíduos, sendo realizada a técnica de pour plate para contagem das unidades formadoras de colônias (UFC). O mesmo procedimento foi feito com a amostra retirada da autoclave. A fração de inativação dos endósporos atingiu 100% no tempo de 30 minutos de exposição à temperatura máxima de 134ºC e pressão absoluta de 2,3 kgf/cm2, resultado obtido através da relação do número de micro-organismos recuperados considerados como inoculados e o número de micro-organismos sobreviventes ao tratamento. A 121ºC houve recuperação de UFC após tratamento. A fração de ocupação não foi um fator delimitante para a inativação de endósporos, pois a temperatura se manteve a mesma independentemente da quantidade de resíduo submetido à autoclavagem. Um outro objetivo era avaliar experimentalmente a reprodução dos micro-organismos frente às condições operacionais de autoclavagem e do tempo de permanência do rejeito em condições ambientais. Esse estudo comprovou que a inativação nessas condições otimizadas esteriliza o resíduo, sendo que nenhum micro-organismo voltou a se reproduzir após dias expostos em temperatura ambiente, ou seja, não haveria riscos de contaminação em aterro sanitário quando depositados os rejeitos. Ter encontrado essas condições ideais e avaliado o processo de autoclavagem pode significar um grande avanço nas próprias unidades de tratamento, que terão um parâmetro estabelecido para trabalho. / Healthcare waste, even when treated and disposed of in landfills, can be causes of environmental impacts because they present some hazard indicator. According to appendix IV of Resolution of the National Health Surveillance Agency of Brazil (Anvisa) RDC nº 306/2004, for technologies of healthcare waste treatment, it is necessary to achieve at least Level III of microbial inactivation. The purpose of this research to optimize the process was defined taking into account the absence of data in the literature that revealed the optimal exposure time, temperature and pressure for microbial inactivation through the healthcare treatment by autoclaving and whether the fraction occupied by waste in the autoclave could interfere with the effectiveness of the disinfection. For the tests, endospores of Geobacillus stearothermophilus were used as bioindicators and five thermocouples were installed in the autoclave for temperature measurement throughout the chamber space. The healthcare waste was synthesized by characterizations such as gravimetric composition, granulometric distribution, apparent specific density, apparent specific mass and moisture content. A methodological proposal for the evaluation of waste treatment was introduced taking into consideration the difficulties encountered. Tests were performed at 116ºC, 125ºC and 134ºC, observing the fraction of inactivation at six different exposure times. Since the sterilization of materials at 121ºC is standardized in health establishments, even though no scientific studies were found to prove the sterilization efficiency in these conditions, tests were performed at 50 and 60 minutes of exposure to evaluate this condition. The concentration of 106 endospores were inoculated in the samples and the recovery was done with filtration after washing the waste, and the \"pour plate\" technique was used to count the colony forming units. The same procedure was done with the sample removed from the autoclave. The inactivation fraction of the endospores reached 100% in the time of 30 minutes of exposure to the maximum temperature of 134ºC and absolute pressure of 2.3 kgf/cm2, a result obtained by the ratio of the number of recovered microorganisms considered as inoculated and the number of microorganisms surviving the treatment. At 121ºC there was recovery of colony forming units after treatment. The occupation fraction was not a limiting factor for the inactivation of endospores, since the temperature remained the same regardless of the amount of waste submitted to autoclaving. Another objective was to experimentally evaluate the reproduction of the microorganisms taking into consideration the operational conditions of autoclaving and the amount of time that the waste remains under environmental conditions. This study proved that the inactivation under these optimized conditions sterilizes the waste, and that no microorganism would reproduce again after days exposed at room temperature, therefore, there would be no risk of contamination in a landfill when the waste is deposited. Having found these ideal conditions and evaluated the autoclaving process can mean a major advance in the treatment units themselves, which will have an established parameter for work.

Experimentos com vista à avaliação das propriedades físico-quimica, fitoquímicas e biológica, bem como a sua estabilidade, em raízes tuberosas maduras, caules e folhas jovens de duas cultivares, RBS Amélia e RBS Cuia, e dois acessos de Ipomoea batatas, um de polpa púrpura com manchas brancas (C) e outro de polpa púrpura (D), cultivados ecologicamente e em condições de sequeiro na Horta Comunitária de Lomba do Pinheiro (30º 06’ 46’’ S; 51º 06’ 34’’ O) no Município de Porto alegre, RS no período de 2013 a 2015 foram efetuados nos laboratórios de Higiene, Qualidade e Microbiologia de alimentos e de Química e Bromatologia do Instituto de Ciência e Tecnologia de Alimento (ICTA) da Universidade Federal do Rio Grande do Sul (UFRGS). A avaliação quantita-tiva das propriedades físico-químicas e fitoquímicas seguiu os métodos descritos no Manual das Normas Técnicas para Determinação das Propriedades Físico-químicas em alimentos do Instituto Adolfo Lutz. O teor de polifenóis totais foi determinado pelo método de Folin & Ciocalteu, o con-teúdo de antocianinas pelo método de pH diferencial, os flavonoides, taninos e vitamina C avalia-dos por métodos espectrofotométricos. A atividade biológica consistiu na determinação das pro-priedades antioxidantes dos materiais usando o método fotocolorimétrico in vitro, empregando o radical livre estável 2,2-difenil-1-picrilhidrazila (DPPH) e na avaliação da atividade antimicrobi-ana, expressa como intensidade de atividade de inibição bacteriana (IINIB) e intensidade de ativi-dade de inativação bacteriana (IINAB), usando sistema de tubos múltiplos frente a diferentes con-centrações de Salmonella Enteritidis (ATCC 11076), Enterococcus faecalis (ATCC 19433), Es-cherichia coli (ATCC 11229) e Staphylococcus aureus (ATCC 25923). Foi igualmente analisada a estabilidade físico-química, fitoquímica e biológica das raízes tuberosas, caules e folhas estoca-dos a temperatura ambiente por períodos de 90, 180 e 270 dias, através da mensuração e avaliação das concentrações dos componentes físico-químicos, fitoquímicos e atividade biológica naqueles pontos de estocagem, Foram encontrados teores de proteínas variando de 10,5 a 26,3%, carboidra-tos totais oscilando de 15,7 a 61,1% com valores altos observados na cultiva RBS Amélia. Maiores teores de fenólicos totais e antocianinas foram observados em acessos de polpa púrpura com valo-res até 709,3 e 78,7mg/100g, respectivamente. Elevada atividade antioxidante foi também verifi-cada na maioria dos extratos testados que, igualmente, evidenciaram atividade antibacteriana frente a todos os inóculos testados ainda que a sua intensidade tenha sido diferente. Porém, nenhuma atividade antibacteriana foi verificada em extratos preparados com base no calor, quais sejam in-fusão e decocção. A estabilidade das propriedades de todos esses materiais foi presenciada embora algumas características tenham reduzido seus teores aos 270 dias de estocagem. O trabalho conclui que as raízes tuberosas, caules e folhas das cultivares RBS Amélia e RBS Cuia e dos acessos de batata-doce estudados se constituem em hortaliças vegetais com alto potencial físico-químico, fi-toquímico e biológico, a avaliar pelos altos teores de carboidratos totais, considerável concentração de proteínas, elevados valores de polifenóis totais e, ou antocianinas, principalmente nos acessos de polpa púrpura. Os mesmos materiais têm um indicativo de atividade antioxidante e de atividade antibacteriana seletiva frente a Salmonella Enteritidis (ATCC 11076), Enterococcus faecalis (ATCC 19433), Escherichia coli (ATCC 11229) e Staphylococcus aureus (ATCC 25923). Figuram-se, portanto, como uma excelente fonte de proteína, carboidratos e de compostos fenólicos relativamente estável por período de 270 dias de estocagem a temperatura ambiente, tornando-se importante na segurança alimentar e nutricional, saúde e bem-estar social. / Experiments for the evaluation of the physic-chemical, phytochemical and biological properties, and their stability in mature tuber roots, young stems and leaves of two cultivars, RBS Amelia and RBS Cuia and of two accessions of Ipomoea batatas, one having purple fleshed tuber roots with white spots (C) and another purple fleshed tuber (D), grown ecologically in “ Horta Comunitária da Lomba do Pinheiro (30º 06' 46'' S, 51° 06' 34'' O), Porto alegre, RS from the period of 2013 to 2015 were perfomed in the Hygiene, Quality and Microbiology and Food Chemistry laboratories of the Institute of Science and food Technology (ICTA), Federal University of Rio Grande do Sul (UFRGS). The quantitative assessment of physicochemical and phytochemical properties followed the described methods in the Manual of Technical Standards for Determination of Physical and Chemical Properties, Institute Adolfo Lutz. The total polyphenol content was determined by the Folin & Ciocalteu method, the content of anthocyanins at pH differential method, flavonoids, tan-nins, and vitamin C evaluated by spectrophotometric methods. Biological activity was analysed evaluated by determinating antioxidant properties of materials using fotocolorimétrico in vitro method, using the stable free radical 2,2-diphenyl-1-picrilhidrazila (DPPH) and also by evaluating antimicrobial activity, expressed as intensity of bacterial inhibition activity (IINIB) and intensity of bacterial inactivation activity (IINAB) using multiple tube system against different concentra-tions of Salmonella enteritidis (ATCC 11076), Enterococcus faecalis (ATCC 19433), Escherichia coli (ATCC 11229) and Staphylococcus aureus (ATCC 25923). It was also analyzed the physical and chemical, phytochemical and biological stability of tuber roots, stems and leaves stored at room temperature for periods of 90, 180 and 270 days, through the measurement and assessment of con-centrations of physico-chemical components, phytochemicals and biological activity on those points of storage. Protein levels were found ranging from 10.5 to 26.3%, total carbohydrates rang-ing from 15.7 to 61.1% with higher values observed in the RBS Amelia cultivar. Higher total phe-nolic content and anthocyanins were found in the purple fleshed roos, with values of up to 709.3 and 78,7mg / 100g, respectively. High anti-oxidant activity was also observed in most of the tested extracts which also showed antibacterial activity against all inocula tested, although its intensity was different. However, no antibacterial activity was detected in extracts prepared by heat, namely infusion and decoction. The stability of the properties of all materials was attended although some features have reduced its contents to 270 days of storage. The paper concludes that the tuber roots, stems and leaves of the RBS Amélia and RBS Cuia cultivars and sweet potato accessions studied constitute vegetables with high physical-chemical, phytochemical and biological potential, accord-ing to the high levels of total carbohidrates, considerable protein concentration, high total polyphe-nol values, or anthocyanins, mainly in pupuple fleshed roots accesses. The same materials have an indicative of antioxidant activity and selective antibacterial activity against Salmonella enteritidis (ATCC 11076), Enterococcus faecalis (ATCC 19433), Escherichia coli (ATCC 11229) and Sta-Staphylococcus aureus (ATCC 25923). Therefore, this specie can be seen as an excellent source of protein, carbohydrates and relatively stable phenolic compounds for a period of 270 days of storage at room temperature, making it important for food security and nutrition, health and social welfare.

Chez les mammifères femelles, le processus d’inactivation du chromosome X (XCI) assure la compensation de dose entre les deux sexes. Chez la souris, l’inactivation du X est établie de manière aléatoire dans l’épiblaste au stade blastocyste et peut être récapitulée in vitro dans les cellules souches embryonnaires. L’ARN non-codant Xist, exprimé à partir du centre d’inactivation du X (Xic), est le régulateur principal de ce processus. Il décore en cis le chromosome choisi pour être inactivé et initie la répression de ses gènes. Ainsi, de manière remarquable, les deux chromosomes X sont traités différemment pendant l’initiation de la XCI malgré leur homologie de séquence et leur localisation au sein d’un même noyau. De manière remarquable, ce processus implique le traitement différentiel de deux chromosomes homologues au sein d’un même noyau, avec des changements d’environnements nucléaires et chromatiniens entre le X actif et le X inactif. Il a été proposé que la localisation nucléaire pourrait jouer un rôle important dans l’initiation de l’expression monoallélique des gènes, non seulement pour l’initiation de la XCI mais aussi pour des processus tels que l’exclusion allélique dans les cellules lymphoides. Par exemple, l’association de loci avec des compartiments hétérochromatiques nucléaires et l’association en trans de loci homologues pourraient être impliquées dans la régulation des gènes monoalléliques. Ainsi, j’ai utilisé le système bactérien TetR/TetO afin d’analyser le rôle de l’organisation nucléaire du chromosome X et du Xic dans l’initiation de la XCI. J’ai pu montrer que si le recrutement des protéines de fusion TetR-LaminB1 ou -Cbx5 au niveau de la cassette TetO insérée dans le Xic permet la répression des gènes à proximité, cet évènement n’est pas toujours accompagné d’une relocalisation nucléaire. De plus, l’association forcée du Xic avec la périphérie nucléaire (TetR-LaminB1) n’a pas d’influence sur le choix du chromosome X à inactiver. Enfin, si la relocalisation des deux Xic à la périphérie nucléaire induit une réduction des évènements d’association en trans entre les deux loci, elle n’a pas d’effet sur l’initiation de l’inactivation. En résumé, ces résultats suggèrent que l’organisation nucléaire du chromosome X et du Xic et l’association des Xic en trans ne sont pas des facteurs déterministes pour le choix et l’initiation de la XCI, mais pourraient être le résultat des changements d’expression des gènes liés à l’X au cours de la différenciation des cellules souches ainsi que de l’augmentation de l’expression de Xist.Enfin, j’ai également analysé le rôle de la protéine CTCF, qui a été proposée pour être importante dans l’organisation structurale du génome, dans le contexte du Xic et de l’initiation de l’inactivation. Ainsi, le recrutement de CTCF au niveau de cassette TetO insérée dans le Xic induit localement une réduction mineure des interactions en cis et la répression des gènes du Xic, à l’exception de Xist dont l’expression est augmentée. Pour autant, la présence ectopique de CTCF n’a pas d’incidence majeure sur l’organisation générale du Xic. / X-chromosome inactivation (XCI) ensures dosage compensation in female mammals. Random XCI is established in the epiblast of female mouse embryos and can be recapitulated in vitro in differentiating embryonic stem cells (ESCs). The major regulator of XCI is the long non-coding RNA Xist, which is expressed from the X-inactivation center (Xic), covers the chromosome in cis and initiates gene silencing. During XCI, the two X chromosomes are treated very differently, despite their homology and the fact that they reside in the same nucleus. Nuclear localization has been hypothesized to play a role in monoallelic gene regulation, not only during XCI but also in other contexts. For example, association with heterochromatin and homologous trans interactions (“pairing”) have been implicated in the establishment of monoallelic gene expression in lymphoid cells and transient pairing has been suggested to participate in symmetry breaking during random XCI. Using the bacterial tetO/tetR system to alter the subnuclear localization and environment of one or both Xics, we have tested the function of subnuclear localization and trans interactions between the Xic loci during initiation of XCI. Using stable expression and reversible binding of TetR fusion proteins (e.g. LaminB1, Cbx5) we show that binding of these proteins can induce local gene repression and chromatin changes, although this is not always associated with subnuclear relocalization. We further show that the forced association of the Xic with the nuclear envelope, does not impact on the choice-making process during XCI. In particular, tethering both Xics to the nuclear lamina during early ESC differentiation resulted in a substantial reduction of homologous pairing events, but had no obvious impact on the onset of random, monoallelic Xist expression. Taken together, our results suggest that nuclear localization and trans interactions of the Xic may be downstream events rather than causal in the regulation of the XCI process.Furthermore, we recruited CTCF, a protein suggested to be involved in structural organization of the genome, to the Xic using the tetO/tetR system. Upon binding of CTCF the overall structure of the Xic remained unaltered though few cis interactions appeared to be weakened, which was accompanied by gene repression in the Xic. Surprisingly, the only upregulated gene in the Xic was Xist in ESCs and during differentiation, which demonstrates that the induced minor changes of cis interactions might impact on gene regulation in the Xic.

Este trabalho teve o objetivo de identificar genes candidatos para deficiência mental de herança ligada ao cromossomo X (DMLX). Foram investigadas quatro famílias, nas quais a deficiência mental de causa desconhecida segregava num padrão típico de herança ligada ao X, e 24 irmandades que incluíam pelo menos dois afetados do sexo masculino. O padrão de inativação do cromossomo X foi determinado nas mães dos afetados, pois desvios extremos no padrão de inativação são frequentes em mulheres portadoras de mutações no cromossomo X que causam DMLX. Nas famílias com padrão de herança ligada ao X, a deficiência mental foi mapeada, usando marcadores moleculares do tipo microssatélite, localizados ao longo do cromossomo X. Desequilíbrios cromossômicos submicroscópicos foram investigados por hibridação genômica comparativa baseada em array (array-CGH). Genes candidatos posicionais que já haviam sido associados a deficiência mental foram sequenciados pelo método de Sanger, em busca de mutações patogênicas. Em três famílias com DMLX, o probando teve seu exoma sequenciado. Nas três famílias com DMLX em que o sequenciamento do exoma foi realizado, foram detectadas mutações missense, levando à substituição de resíduos de aminoácidos conservados, que segregavam com a deficiência mental. Essas mutações foram consideradas como prováveis causas dos fenótipos nessas famílias: c.12378C>G no gene HUWE1 (HECT, UBA and WWE domain containing 1, E3 ubiquitin protein ligase); c.1413C>A no gene SHROOM4 (shroom family member 4) e c.262G>A no gene KIAA2022. As mulheres heterozigóticas quanto a essas mutações apresentaram desvio completo de inativação do cromossomo X (100:0). Mutações de ponto ou interrupção desses genes por rearranjos cromossômicos foram previamente descritas em alguns pacientes com deficiência mental. Em uma família, em que a DMLX estava associada a microcefalia e malformação de Dandy-Walker, uma microduplicação de aproximadamente 300 kb em Xq28 (ChrX :153,578,110-153,880,794;Hg19) foi detectada segregando com a doença. As mulheres heterozigóticas quanto a essa duplicação apresentaram desvios de inativação do X (80:20 e 90:10). Duplicações semelhantes foram anteriormente descritas em três famílias europeias e em um caso esporádico; a malformação de Dandy-Walker foi documentada em alguns desses pacientes. No estudo das 24 irmandades com pelo menos dois indivíduos do sexo masculino apresentando deficiência mental, o padrão de inativação do cromossomo X de suas mães foi determinado. Quatro mulheres (16,7%) apresentaram desvio total de inativação do cromossomo X (100:0), frequência significativamente maior do que a descrita para população geral de mulheres adultas (cerca de 2 %). Considerando que desvios extremos de inativação do cromossomo X são observados em aproximadamente 30% das portadoras de mutações que causam DMLX, concluiu-se que as quatro mães de homens que apresentam deficiência mental eram provavelmente portadores de mutações no cromossomo X, causadoras da deficiência mental em seus filhos. Não foram encontradas microdeleções ou microduplicações no cromossomo X nos probandos das quatro irmandades. / This study aimed at identifying candidate genes for X-linked intellectual disability (ID). Four families in which ID of unknown cause segregated as an X-linked trait, and 24 sibships with at least two affected males were investigated. The pattern of X-inactivation was determined in the mothers of affected males, taking into account that extremely skewing of X-inactivation is frequently found in women carrying mutations causative of X-linked intellectual disability (XLID). In the XLID families, ID was mapped by the genotyping of microsatellite markers localized throughout the X chromosome. Cryptic X-chromosome imbalances were investigated by array-based comparative genomic hybridization (a-CGH). Positional candidate genes that had been associated with ID were directly sequenced in the search for causative mutations. In three XLID families, the propositus had their exome sequenced. In three XLID families missense mutations that led to substitutions of conservative aminoacid residues were found that segregated with ID, and were probably causative of the clinical phenotypes: c.12378C>G in HUWE1 (HECT, UBA and WWE domain containing 1, E3 ubiquitin protein ligase) gene; c.1413C>A in the SHROOM4 (shroom family member 4) gene; c.262G>A in the KIAA2022 gene. Heterozygotes for these mutations had completely skewed X-inactivation (100:0 inactivation ratio). Point mutations or disruption of these genes by rearrangement breakpoints have been previously described in a few patients with ID. In one family in which XLID was associated with microcephaly and Dandy-Walker malformation, a duplication of approximately 300 kb at Xq28 (ChrX:153,578,110-153,880,794 - Hg19) was found segregating with the disease. Heterozygotes for this duplication had skewed X-inactivation (80:20 and 90:10 inactivation ratios). Similar duplications have been described in three European families and one sporadic case, Dandy-Walker malformation being documented in some patients. In the study of the 24 sibships with at least two males presenting with ID, the maternal pattern of X-inactivation was determined. Four women (16.7%) showed completely skewing of X-inactivation (100:0 inactivation ratio), a frequency significantly higher than the reported frequency of skewing >= 95% in women from the general population (about 2%). Considering this finding and that extremely skewed X-inactivation have been reported in about 30% of carriers of mutations causing XLID, it was assumed that the four mothers of males presenting with ID were most probably carriers of the mutations causative of ID in their sons. Chromosome X microimbalances were not found in the propositus, in these four sibships.

This work studied the improvement of stability, physical properties and enzymatic inactivation in fruit beverages by applying the ultrasound technology (US). In the first part, the effect of the US application on peach juice processing was evaluated. The macroscopic changes on pulp sedimentation stability, turbidity, colour and rheological properties were evaluated. As a result, it was demonstrated that the improvement in each of the properties evidenced at the macroscopic level involves interaction of complex mechanisms which depend directly on changes at the microscopic level, such as the structure, size, composition and interaction between the continuous phase (serum) and dispersed phase (pulp) of the juice. These changes were assessed by microscopy and particle size distribution. In the second and third parts, the inactivation of the enzyme peroxidase (POD) was evaluated in coconut water. In the second part, the effect of the application of US was evaluated for first time for coconut water POD, using two types of ultrasonic equipment (US baht and US probe). It was demonstrated that the changes of enzyme activity during the US process depend on the many forms that the enzyme can adopt, mainly depending on the energy applied to the system. Subsequently, in the third part, the US was applied as a pre-treatment of subsequent thermal processing. The evaluation was carried out under non isothermal conditions, being the POD inactivation kinetics modelled using the Weibull distribution function. Finally, it was observed that the pre-treatment using ultrasound slightly decreased the enzyme activity. Furthermore, the ultrasound effects resulted in a more homogeneous population and heat-sensitive enzymes, significantly reducing the needed time of thermal processing. In conclusion, this work studied and demonstrated that the ultrasound technology is an interesting alternative to improve the physical properties and enzymatic stability in fruit beverages, reflecting the importance from both the academic and industrial point of view. / Neste trabalho estudou-se a melhoria na estabilidade, propriedades físicas e inativação enzimática em bebidas de frutas através da aplicação da tecnologia de ultrassom (US). Na primeira parte, foi avaliado o efeito do US no processamento de suco de pêssego. As alterações macroscópicas na estabilidade de sedimentação da polpa, turbidez, cor e propriedades reológicas foram analisadas. Foi demonstrado que a melhoria em cada uma das propriedades evidenciadas macroscopicamente envolve interação de mecanismos complexos que dependem diretamente de alterações microscópicas, tais como estrutura, tamanho, composição e interação entre as fases contínua (soro) e dispersa (polpa) do suco. Estas alterações foram avaliadas por microscopia e análise de distribuição de tamanho de partículas. Na segunda e terceira partes, a inativação da enzima peroxidase (POD) foi avaliada em água de coco. O efeito da aplicação do US na POD de água de coco foi estudado pela primeira vez, utilizando dois tipos de equipamentos (banho e sonda de US). Demonstrou-se que as alterações na atividade enzimática durante o processamento com US estão relacionadas às diferentes conformações que a enzima pode adotar, dependendo principalmente da energia aplicada ao sistema. Na terceira parte, o ultrassom foi então aplicado como pré-tratamento ao processamento térmico. A avaliação foi realizada sob condições não isotérmicas, sendo a cinética de inativação da POD modelada usando a função de distribuição de Weibull. Foi observado que o pré-tratamento com US diminuiu a atividade enzimática. Além disso, o efeito do US resultou em uma população de enzimas mais homogênea e termosensível, reduzindo significativamente o tempo necessário para o processamento térmico. Desta forma, este trabalho estudou e demonstrou que a tecnologia de ultrassom é uma alternativa interessante para melhorar as propriedades físicas e a estabilidade enzimática de bebidas à base de frutas, indicando sua importância tanto acadêmica quanto industrial.

<p>Multiple studies have demonstrated atmospheric cold plasma (ACP) as an effective non-thermal technology for microbial decontamination, surface modification, and functionality alteration in food processing and packaging. ACP constitutes charged particles, such as positive and negative ions, electrons, quanta of electromagnetic radiation, and excited and non-excited molecules, which corresponds to its predominant reactive properties. However, in many of these applications, the interactions between plasma and the components in food matrix are not well-understood. The <b>overall goals</b> of this dissertation were to 1) evaluate the interactions between high voltage atmospheric cold plasma (HVACP) and microbes in liquid and semi-solid food; 2) investigate plasma transfer into semi-solid foods and determine the relationship between microbial inactivation and plasma transfer; 3) explore the interactions between plasma and proteins. </p> <p>The first study explored the microbial (<i>Salmonella</i> <i>enterica</i> serovar Typhimurium, <i>S</i>. <i>enterica</i>) inactivation efficacy of HVACP. The physicochemical interactions between HVACP and biomolecules, including an enzyme (pectin methylesterase, PME), vitamin C and other components in orange juice (OJ) under different conditions was also evaluated. Both direct and indirect HVACP treatment of 25 mL OJ induced greater than a 5 log reduction in <i>S</i>. <i>enterica</i> following 30 s of treatment with air and MA65 gas with no storage. For 50 mL OJ, 120 s of direct HVACP treatment followed by 24 h storage achieved <i>S</i>. <i>enterica</i> reductions of 2.9 log in air and 4.7 log in MA65 gas. An indirect HVACP treatment of 120 s followed by 24 hours storage resulted in a 2.2 log reduction in air and a 3.8 log reduction in MA65. No significant (<i>P </i>< 0.05) Brix or pH change occurred following 120 s HVACP treatment. HVACP direct treatment reduced vitamin C content by 56% in air and PME activity by 74% in air and 82% in MA65. These results demonstrated that HVACP can significantly reduce <i>Salmonella</i> in OJ with minimal quality degradation.</p> <p>The second study in this dissertation examined the penetration process of plasma into semi-solid food and the resulting microbial inactivation efficacy. Agar gels of various densities (0.25, 0.5, 1.0, and 2%) with a pH indicator were inoculated with <i>S</i>. <i>enterica</i> (10⁷>CFU) and exposed directly (between the electrode) or indirectly (adjacent to the plasma field created between the two electrodes) to 90 kV at 60 Hz for up to 1.5 h. A long treatment time (1.5 h) caused sample temperature to increase 5~10 °C. The microbial analysis indicated a greater than 6 log₁₀ (CFU) reduction (both with air and MA65) in the zone with a pH change. Inactivation of bioluminescence cells in the plasma penetrated zone confirmed that the plasma, and its generated reactive species, inactivate microbial as it penetrates into the gel. A two-minute HVACP direct treatment with air at 90 kV induced greater than 5 log₁₀ (CFU)<i> S</i>. <i>enterica </i>reduction in applesauce. <em></em></p> <p>The third study investigated the interactions between HVACP and protein, using bovine serum albumin (BSA) as a model protein. The physicochemical and structural alteration of BSA and its reaction mechanism, when subjected to HVACP, were investigated. After treating 10 mL of BSA solution (50 mg/mL) at 90 kV for 20, 40, or 60 min, we characterized structural alteration and side-group modification. FTIR spectroscopy, Raman spectroscopy, and circular dichroism analysis indicated protein unfolding and decreased secondary structure (25 % loss of α-helix, 12% loss of β-sheet) in HVACP treated BSA. Average particle size in the protein solutions increased from 10 nm to 113 µm, with a broader distribution after 60 min HVACP treatment indicating protein aggregation. SDS-PAGE and mass spectrometer analysis observed a formation of new peptides of 1 to 10 kDa, indicating that the plasma triggered peptide bond cleavage. Chemical analysis and mass spectrometer results confirmed the plasma modifications on the side chains of amino acids. This study reveals that HVACP treatment may effectively introduce structural alteration, protein aggregation, peptide cleavage, and side-group modification to proteins in aqueous conditions, through several physicochemical interactions between plasma reactive species (reactive oxygen species and reactive nitrogen species) and the proteins. This finding can be readily applied to other plasma-protein studies or applications in the food system, such as enzyme inactivation or protein-based film modifications.</p>

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No. of bitstreams: 2 Dissertação - Taise Maria dos Anjos Oliveira - 2016.pdf: 1420716 bytes, checksum: 75628b01c19c9db89c0e4ed2e9469284 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-02-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Photodynamic inactivation is a technique based on the synergistic combination of a photosensitizer, light and oxygen, resulting in the generation of reactive oxygen species, which are cytotoxic and cause cell death by apoptosis or necrosis. Bovine herpesvirus 1 is responsible for diseases of the respiratory and reproductive systems, it is estimated to occur an economic loss of a billion dollars a year, due to the action of the virus in the herd. In order to develop new alternatives and /or therapeutic approaches that are effective in viral clearance, we evaluated two photosensitizers, zinc phthalocyanine and aluminum phthalocyanine, the photoinactivation bovine herpesvirus 1. As a control we used irradiated virus without the presence of photosensitizer (light control) viruses incubated with photosensitizers without radiating (photosensitizer control) and viruses without any treatment. The light control showed that the use of light without the presence of photosensitizer does not have phototoxic effect on the virus; control showed that the photosensitizer zinc and aluminum phthalocyanines have no cytotoxicity in the absence of light. Both controls were compared to the virus that received no treatment. To better evaluate the irradiation time and the optimal concentration of the two photosensitizers, viral suspension aliquots containing 105.75 TCID50/mL were used, incubated with photosensitizers at concentrations of 5 and 10 μM and irradiated at 0, 15, 30, 45, 60, 75, and 90 minutes. After irradiation, the sample was added in permissive cell cultures virus, which was analyzed for the presence of cytopathic effect and the results were expressed as viral titers. The zinc phthalocyanine showed better efficiency in photoinactivation bovine herpesvirus 1, wherein the concentration of 10 μM with 30 minutes of irradiation was the most effective. The phthalocyanine aluminum inactivated virus completely after 60 minutes of irradiation, but there was no significant difference between concentrations. These results indicate that both phthalocyanines have a good applicability in photodynamic viral inactivation, but the zinc phthalocyanine has a better efficiency. / A inativação fotodinâmica é uma técnica baseada na combinação sinérgica de um fotossensibilizador, oxigênio e luz, que resulta na geração de espécies reativas do oxigênio, que são citotóxicas e causam a morte celular por apoptose ou necrose. O herpesvírus bovino 1 é responsável por enfermidades no sistema respiratório e reprodutivo, estima-se que ocorra uma perda econômica de um bilhões de dólares por ano, devido à ação do vírus no rebanho. Visando novas alternativas e/ou abordagens terapêuticas que sejam eficazes na eliminação viral, avaliamos dois fotossensibilizadores, ftalocianina zinco e ftalocianina alumínio, na fotoinativação do herpesvírus bovino 1. Como controle foram utilizados vírus irradiado sem a presença do fotossensibilizador (controle da luz), vírus incubado com os fotossensibilizadores sem irradiar (controle do fotossensibilizador) e vírus sem nenhum tratamento. O controle da luz mostrou que o emprego da luz sem a presença do fotossensibilizador não possui efeito fototóxico sobre o vírus, o controle do fotossensibilizador mostrou que as ftalocianinas zinco e alumínio não possuem citotoxidade na ausência da luz. Ambos os controles foram comparados ao vírus que não recebeu nenhum tratamento. Para avaliar o melhor tempo de irradiação e a melhor concentração dos dois fotossensibilizadores, foram utilizadas alíquotas de suspensão viral contendo 105,75 TCID50/mL, incubadas com os fotossensibilizadores nas concentrações de 5 e 10 μM e irradiadas nos tempos 0, 15, 30, 45, 60, 75 e 90 minutos. Após a irradiação a amostra foi adicionada em culturas de células permissíveis ao vírus, onde foi analisada a presença de efeito citopático e os resultados foram expressos em títulos virais. A ftalocianina zinco apresentou melhor eficiência na fotoinativação do herpesvírus bovino 1, sendo a concentração de 10 μM com 30 minutos de irradiação a mais eficaz. A ftalocianina alumínio inativou o vírus totalmente após 60 minutos de irradiação, mas não apresentou diferença significativa entre as concentrações. Estes resultados indicam que ambas ftalocianinas possuem uma boa aplicabilidade na inativação fotodinâmica viral, porém a ftalocianina zinco possui uma melhor eficiência.

<p>One of the main obstacles to maintaining patients with short bowel syndrome on parenteral nutrition, or successfully transplanting these patients with a small bowel graft, is the many severe infections that occur. Evidence is accumulating that translocating bacteria from the patient’s bowel causes a significant part of these infections. In this thesis bacterial translocation is studied in a Thiry-Vella loop of defunctionalised small bowel in the rat. </p><p>Bacterial translocation to the mesenteric lymph nodes (MLNs) occurs in almost 100% of the rats after three days. No systemic spread of bacteria is observed unless there is additional immunosupression with depletion of Kupffer cells in the liver. However, blocking the function of α/β T cells does not increase the translocation. Removal of MLNs does not either aggravate bacterial translocation in the Thiry-Vella loop model. Conversely, after small bowel transplantation translocating bacteria spread systemically if the MLNs are removed. </p><p>The Thiry-Vella loop should also be a suitable model for the testing of potentially translocation-inhibiting substances. Reinforcement of the intestinal barrier with glutamine or phosphatidylcholine proved insufficient in decreasing bacterial translocation. Even selective bowel decontamination with tobramycin failed to abolish bacterial translocation. Thus, it seems that the driving force for translocation in this model is strong regardless of the relatively small trauma of intestinal defunctionalisation.</p><p>Flow cytometric studies of the immune cells in the spleen MLNs showed a decrease in MHC class II positive T cells in the MLNs of the Thiry-Vella loop. Concurrently the number of macrophages increased with time as observed by immunohistochemistry. The fraction of MHC class II negative macrophages increased in the spleens of rats treated with glutamine. </p><p>In conclusion, the Thiry-Vella loop model offers possibilities of immunological as well as mechanistic studies on bacterial translocation from small intestine.</p>