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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Regulation of Mammary Lactogenic Differentiation by Singleminded-2s

Wellberg, Elizabeth 2009 May 1900 (has links)
Sim2s is a basic helix-loop-helix Per-Arnt-Sim (bHLH-PAS) transcription factor. In Drosophila, the Sim2 homolog, sim, is necessary for cell fate determination during central nervous system (CNS) development. In mammals, both Sim2 isoforms are involved in development of various tissues, including muscle, cartilage, and mammary gland. Loss-of-function studies revealed a role for Sim2s in specifying epithelial cell fate during mammary development and inhibiting growth and invasion of aggressive breast cancer cells. This study determined the role of Sim2s in mammary epithelial cell differentiation. Our hypothesis is that Sim2s is sufficient to promote lactogenic differentiation in vivo, characterized by expression of lactation-specific genes. Two models were used to test this hypothesis: (1) a transgenic mouse, expressing Sim2s under control of the MMTV-LTR, and (2) the mouse mammary epithelial cell line HC11. Together, these models allow analysis of the effect of Sim2s on global mammary gland differentiation and the mechanism through which it accomplishes this in a relatively homogenous population of cells. We determined that precocious expression of Sim2s in vivo is associated with upregulation of a subset of milk protein genes in nulliparous females. During early pregnancy, Sim2s regulation of lactogenic differentiation extended to a larger group of genes. Following pup removal, Sim2s appears to promote survival of alveolar epithelial cells. In vitro, Sim2s expression is necessary for maximal Csn2 expression, as determined by loss-of-function studies. Overexpression of Sim2s is sufficient to enhance prolactin-mediated Csn2 expression. Chromatin immunoprecipitation assays performed in HC11 cells revealed enhanced recruitment of Stat5a and RNA Polymerase II (RNAPII) to the regulatory region of Csn2 in the presence of Sim2s. In addition, Sim2s and RNAPII were found in a complex that was localized to both the promoter and coding region of the Csn2 gene. These studies support the idea that Sim2s is upregulated in a developmental stage-specific manner in the mouse mammary gland to promote the survival and differentiation of alveolar epithelial cells expressing high levels of milk protein genes. Further, Sim2s may regulate the function of a specific subset of alveolar cells by targeting the RNAPII holoenzyme complex to genes expressed during lactogenic differentiation.
2

Regulation of Mammary Lactogenic Differentiation by Singleminded-2s

Wellberg, Elizabeth 2009 May 1900 (has links)
Sim2s is a basic helix-loop-helix Per-Arnt-Sim (bHLH-PAS) transcription factor. In Drosophila, the Sim2 homolog, sim, is necessary for cell fate determination during central nervous system (CNS) development. In mammals, both Sim2 isoforms are involved in development of various tissues, including muscle, cartilage, and mammary gland. Loss-of-function studies revealed a role for Sim2s in specifying epithelial cell fate during mammary development and inhibiting growth and invasion of aggressive breast cancer cells. This study determined the role of Sim2s in mammary epithelial cell differentiation. Our hypothesis is that Sim2s is sufficient to promote lactogenic differentiation in vivo, characterized by expression of lactation-specific genes. Two models were used to test this hypothesis: (1) a transgenic mouse, expressing Sim2s under control of the MMTV-LTR, and (2) the mouse mammary epithelial cell line HC11. Together, these models allow analysis of the effect of Sim2s on global mammary gland differentiation and the mechanism through which it accomplishes this in a relatively homogenous population of cells. We determined that precocious expression of Sim2s in vivo is associated with upregulation of a subset of milk protein genes in nulliparous females. During early pregnancy, Sim2s regulation of lactogenic differentiation extended to a larger group of genes. Following pup removal, Sim2s appears to promote survival of alveolar epithelial cells. In vitro, Sim2s expression is necessary for maximal Csn2 expression, as determined by loss-of-function studies. Overexpression of Sim2s is sufficient to enhance prolactin-mediated Csn2 expression. Chromatin immunoprecipitation assays performed in HC11 cells revealed enhanced recruitment of Stat5a and RNA Polymerase II (RNAPII) to the regulatory region of Csn2 in the presence of Sim2s. In addition, Sim2s and RNAPII were found in a complex that was localized to both the promoter and coding region of the Csn2 gene. These studies support the idea that Sim2s is upregulated in a developmental stage-specific manner in the mouse mammary gland to promote the survival and differentiation of alveolar epithelial cells expressing high levels of milk protein genes. Further, Sim2s may regulate the function of a specific subset of alveolar cells by targeting the RNAPII holoenzyme complex to genes expressed during lactogenic differentiation.
3

Efeitos de glicocorticoide sobre a expressão de genes de relógio nas células ZEM-2S de Danio rerio / Glucocorticoid effects on clock gene expression in Danio rerio ZEM-2S cells

Sousa, Jennifer Caroline de 23 February 2015 (has links)
O estudo da expressão circadiana de genes de relógio tem sugerido hormônios como potenciais agentes sincronizadores. A regulação da ritmicidade de relógios periféricos é, aparentemente, mais um dentre os diferentes efeitos fisiológicos atribuídos aos glicocorticoides (GCs), que se constituem como candidatos a zeitgeber dado ao fato de que seus níveis circulantes apresentam padrões diários robustos e são uma das principais eferências do relógio central em mamíferos. Entretanto, em outros vertebrados, o papel dessa classe hormonal em relação a este aspecto ainda é pouco explorado e se a regulação é direta ou indireta, quais suas consequências e de que maneira ela ocorre são indagações a serem respondidas. Empregando a técnica do PCR quantitativo, avaliamos o perfil de expressão dos genes da alça negativa do núcleo da maquinaria molecular do relógio, per1b e cry1b, em células ZEM-2S do teleósteo Danio rerio expostas ao regime fotoperiódico 12:12 CE ou mantidas em escuro constante (EE) e mantidas em EE, mas condicionadas a trocas diárias de meio de cultura ou a pulsos diários de dexametasona a 10-7 M (DEXA), agonista sintético de glicocorticoide. Em 12:12 CE, ambos os genes apresentaram variação temporal, com picos de expressão observados na fase clara do ciclo. Em EE, per1b mostrou um perfil oscilatório de amplitude atenuada, enquanto para cry1b, não foi detectada oscilação ao longo das 24 h. Trocas de meio em EE alteraram significativamente o padrão de expressão de per1b e cry1b, no entanto, os pulsos diários de DEXA promoveram uma oscilação temporal muito mais pronunciada para per1b, modulando positiva ou negativamente sua expressão nos diferentes pontos temporais. O emprego do antagonista RU 486 na concentração de 10-5 M aboliu o pico de expressão detectado no ZT 16, porém, na concentração de 10-6 M, a expressão gênica foi aumentada em cerca de 3 vezes comparado ao tratamento apenas com DEXA. O gene cry1b, por sua vez, não se apresentou suscetível aos pulsos diários de DEXA. Estes resultados permitem confirmar as células ZEM-2S como modelo de relógios periféricos em cultura dada a fotossensibilidade intrínseca das mesmas, reafirmando o papel do ciclo CE como principal agente sincronizador. Os glicocorticoides certamente exercem modulação de per1b por meio da via genômica direta, sem, no entanto excluir uma possível ação via receptor de membrana, tendo em vista a atividade agonista de RU 486, podendo se constituir em um dos fatores que regulam o complexo funcionamento da maquinaria molecular do relógio biológico de zebrafish / Studies on the circadian expression of clock genes have suggested hormones as potential synchronizing agents. Regulation of rhythmicity of peripheral clocks is among the various physiological effects of glucocorticoids (GCs), which are candidate to zeitgeber, since their circulating levels present robust daily patterns and are one of the major outputs of the mammalian central pacemaker. However, in other vertebrates, such a feature has yet to be clarified as well as if the hormonal regulation is direct or indirect, what are its mechanisms and consequences. Applying qPCR technique, we evaluated the gene expression profile of per1b and cry1b, negative feedback loop members of the molecular machinery of biological clock, in ZEM-2S cells of the teleost Danio rerio. The cells were exposed to photoperiod regimen of 12:12 LD; kept in constant darkness (DD); kept in DD but subject to medium changes or treated with daily pulses of 10-7 M dexamethasone (10-7 M DEXA), a glucocorticoid synthetic analogue. In 12:12 LD, both genes presented temporal variation, with peaks of expression in the light phase. In DD, per1b showed an oscillatory profile with attenuated amplitude, whereas cry1b did not oscillate throughout 24 h. DEXA-free medium changes in constant DD conditions altered significantly per1b and cry1b expression profiles. Nevertheless, DEXA daily pulses promoted a temporal oscillation much more pronounced for per1b, modulating positively or negatively its expression at different time points, whereas cry1b was not susceptible to the hormone. RU 486 at 10-5 M abolished the peak of expression of per1b previously observed at ZT 16. On the other hand, RU 486 at 10-6 M increased the gene expression around 3-fold in comparison to just DEXA treatment. These results confirm ZEM-2S cells as a model of peripheral clocks in culture due to their intrinsec photosensitivity, emphasizing the light/dark cycle as a major synchronizing agent. The glucocorticoid modulates per1b expression, probably through a direct genomic pathway, without excluding however its possible action through membrane receptors, since RU 486 exerted agonistic activity. Glucocorticoids could, therefore, be one of the factors that regulate the complex molecular machinery of zebrafish biological clock
4

Efeitos de glicocorticoide sobre a expressão de genes de relógio nas células ZEM-2S de Danio rerio / Glucocorticoid effects on clock gene expression in Danio rerio ZEM-2S cells

Jennifer Caroline de Sousa 23 February 2015 (has links)
O estudo da expressão circadiana de genes de relógio tem sugerido hormônios como potenciais agentes sincronizadores. A regulação da ritmicidade de relógios periféricos é, aparentemente, mais um dentre os diferentes efeitos fisiológicos atribuídos aos glicocorticoides (GCs), que se constituem como candidatos a zeitgeber dado ao fato de que seus níveis circulantes apresentam padrões diários robustos e são uma das principais eferências do relógio central em mamíferos. Entretanto, em outros vertebrados, o papel dessa classe hormonal em relação a este aspecto ainda é pouco explorado e se a regulação é direta ou indireta, quais suas consequências e de que maneira ela ocorre são indagações a serem respondidas. Empregando a técnica do PCR quantitativo, avaliamos o perfil de expressão dos genes da alça negativa do núcleo da maquinaria molecular do relógio, per1b e cry1b, em células ZEM-2S do teleósteo Danio rerio expostas ao regime fotoperiódico 12:12 CE ou mantidas em escuro constante (EE) e mantidas em EE, mas condicionadas a trocas diárias de meio de cultura ou a pulsos diários de dexametasona a 10-7 M (DEXA), agonista sintético de glicocorticoide. Em 12:12 CE, ambos os genes apresentaram variação temporal, com picos de expressão observados na fase clara do ciclo. Em EE, per1b mostrou um perfil oscilatório de amplitude atenuada, enquanto para cry1b, não foi detectada oscilação ao longo das 24 h. Trocas de meio em EE alteraram significativamente o padrão de expressão de per1b e cry1b, no entanto, os pulsos diários de DEXA promoveram uma oscilação temporal muito mais pronunciada para per1b, modulando positiva ou negativamente sua expressão nos diferentes pontos temporais. O emprego do antagonista RU 486 na concentração de 10-5 M aboliu o pico de expressão detectado no ZT 16, porém, na concentração de 10-6 M, a expressão gênica foi aumentada em cerca de 3 vezes comparado ao tratamento apenas com DEXA. O gene cry1b, por sua vez, não se apresentou suscetível aos pulsos diários de DEXA. Estes resultados permitem confirmar as células ZEM-2S como modelo de relógios periféricos em cultura dada a fotossensibilidade intrínseca das mesmas, reafirmando o papel do ciclo CE como principal agente sincronizador. Os glicocorticoides certamente exercem modulação de per1b por meio da via genômica direta, sem, no entanto excluir uma possível ação via receptor de membrana, tendo em vista a atividade agonista de RU 486, podendo se constituir em um dos fatores que regulam o complexo funcionamento da maquinaria molecular do relógio biológico de zebrafish / Studies on the circadian expression of clock genes have suggested hormones as potential synchronizing agents. Regulation of rhythmicity of peripheral clocks is among the various physiological effects of glucocorticoids (GCs), which are candidate to zeitgeber, since their circulating levels present robust daily patterns and are one of the major outputs of the mammalian central pacemaker. However, in other vertebrates, such a feature has yet to be clarified as well as if the hormonal regulation is direct or indirect, what are its mechanisms and consequences. Applying qPCR technique, we evaluated the gene expression profile of per1b and cry1b, negative feedback loop members of the molecular machinery of biological clock, in ZEM-2S cells of the teleost Danio rerio. The cells were exposed to photoperiod regimen of 12:12 LD; kept in constant darkness (DD); kept in DD but subject to medium changes or treated with daily pulses of 10-7 M dexamethasone (10-7 M DEXA), a glucocorticoid synthetic analogue. In 12:12 LD, both genes presented temporal variation, with peaks of expression in the light phase. In DD, per1b showed an oscillatory profile with attenuated amplitude, whereas cry1b did not oscillate throughout 24 h. DEXA-free medium changes in constant DD conditions altered significantly per1b and cry1b expression profiles. Nevertheless, DEXA daily pulses promoted a temporal oscillation much more pronounced for per1b, modulating positively or negatively its expression at different time points, whereas cry1b was not susceptible to the hormone. RU 486 at 10-5 M abolished the peak of expression of per1b previously observed at ZT 16. On the other hand, RU 486 at 10-6 M increased the gene expression around 3-fold in comparison to just DEXA treatment. These results confirm ZEM-2S cells as a model of peripheral clocks in culture due to their intrinsec photosensitivity, emphasizing the light/dark cycle as a major synchronizing agent. The glucocorticoid modulates per1b expression, probably through a direct genomic pathway, without excluding however its possible action through membrane receptors, since RU 486 exerted agonistic activity. Glucocorticoids could, therefore, be one of the factors that regulate the complex molecular machinery of zebrafish biological clock
5

Regulation of Mammary cell Differentiation and Metabolism by Singleminded-2s

Scribner, Kelly C 16 December 2013 (has links)
Ductal carcinoma in situ (DCIS) has been shown to be a precursor to invasive ductal cancer (IDC). Though the progression of DCIS to IDC is believed to be an important aspect of tumor aggressiveness, prognosis and molecular markers that predict progression are poorly understood. Therefore, determining the mechanisms by which some DCIS progress is critical for future breast cancer diagnostics and treatment. Singleminded-2s (SIM2s) is a member of the bHLH/PAS family of transcription factors and a key regulator of differentiation. SIM2s is highly expressed in mammary epithelial cells and lost in breast cancer. Loss of Sim2s causes aberrant mouse mammary development with features suggestive of malignant transformation, whereas over-expression of Sim2s promotes precocious alveolar differentiation, suggesting that Sim2s is required for establishing and enhancing mammary gland differentiation. We hypothesize that SIM2s expression must be lost in premalignant lesions for breast cancer to develop. We first analyzed Sim2s in the involuting mammary gland, which is a highly tumorpromoting environment. Sim2s is down-regulated during involution, and forced expression delays involution. We then analyzed SIM2s expression in human breast cancer samples and found that SIM2s is lost with progression from DCIS to IDC, and this loss correlates with metastasis. SIM2s expression in DCIS promoted a differentiated phenotype and suppressed genes associated with de-differentiation. Furthermore, loss of SIM2s expression in DCIS xenografts increased metastasis likely due to an increase in hedgehog signaling and matrix metalloproteinase expression. Interestingly, we found metabolic shifts with gain and loss of SIM2s in not only DCIS cells, but also MCF7 and SUM159 cells. SIM2s expression decreased aerobic glycolysis and promoted oxidative phosphorylation through direct upregulation of CDKN1a and senescence. Loss of SIM2s, conversely, promotes mitochondrial dysfunction and induction of the Warburg effect. This is the first time CDKN1a and cellular senescence have been indicated as causative to metabolic shifts within cancer cells. These studies show a new role for SIM2s in metabolic homeostasis, and this regulation is lost during tumorigenesis. These data indicate SIM2s is at the apex where aging, metabolism, and disease meet – regulating the delicate relationship between the three.
6

Development of a Dynamic Event Tree Branching Methodology to Integrate Safety and Physical Security Analyses

Cohn, Brian E. January 2020 (has links)
No description available.
7

THE THEORETICAL STUDY OF TORSION –VIBRATIONAL DYNAMICS IN METHANOL AND THE IMPROVEMENT OF CW-CRDS EXPERIMENTAL APPARATUS

Clasp, Trocia N. January 2007 (has links)
No description available.
8

Measurement of the ψ(2S) production in presence of a Quark-Gluon Plasma / Mesure de la production de ψ(2S) en présence d’un Plasma de Quark et de Gluons

Feuillard, Victor 16 November 2017 (has links)
La matière nucléaire, constituant le noyau des atomes, est formée de quarks et de gluons, dont l’interaction est décrite par la théorie de la chromodynamique quantique (QCD). Dans des conditions normales, quarks et gluons ne peuvent être observés de façon isolée et sont confinés dans des hadrons tels que les protons et les neutrons. Le Plasma de Quarks et de Gluons (PQG) est un état de la matière nucléaire prédit par la QCD pour lequel ces quarks et gluons sont déconfinés. Expérimentalement, le PQG peut être créé dans des collisions d’ions lourds ultra-relativistes, telles que les collisions d’ions lourds effectuées au LHC, correspondant à des vitesses proche de celle de la lumière. Il est possible d’obtenir des informations sur le PQG en mesurant un large nombre d’observables. En particulier, la production de charmonium tels que le J/ψ et le ψ(2S), particules lourdes constituées d’une paire de quarks charme et anti-charme () est mesurée pour étudier le plasma. En effet, la présence d’un PQG est censée modifier les taux de production des charmonia, à cause d’un équilibre entre un mécanisme d’écrantage de couleur du potentiel des quarks charme et un mécanisme dit de recombinaison. La position de cet équilibre dépend de l’énergie de collision, la température du plasma, et la nature de la particule considérée, et plus spécifiquement, il est attendu que le ψ(2S) soit plus supprimé que le J/ψ. Dans cette thèse, la production inclusive de ψ(2S) en collisions Pb − Pb à une énergie par collision nucléon-nucléon dans le référentiel du centre de masse de TeV est mesurée dans le canal de décroissance de dimuon avec le Spectromètre à Muons d’ALICE. L’analyse est basée sur les données collectées dans ALICE (A Large Ion Coliider Experiment) au LHC en 2015 correspondant à une luminosité intégrée de 225 μb−1. Le facteur de modification nucléaire RAA est étudié en fonction de la centralité des collisions, correspondant à la distance transverse entre les centre des noyaux de plomb. Le rapport des RAA du ψ(2S) et du J/ψ est également mesuré et montre que le ψ(2S) est plus supprimé que le J/ψ pour des collisions mi-centrales et centrales. Comparées aux prédictions théoriques, les mesures sont compatibles avec les modèles dans la limite des incertitudes. L’amélioration du Muon Trigger, le MID, est également étudié, en particulier le débit de données attendu pour des fréquences de collision de 100 kHz. Basée sur les données en collisions Pb − Pb à une énergie de TeV, les estimations prédisent que la technologie qui sera implémentée sur le MID possède une bande passante suffisante. / The nuclear matter, which constitues the atomic nuclei, is composed of quarks and gluons and interactions between them are described by quantum chromo-dynamics (QCD). Under ordinary conditions, quarks and gluons cannot be observed isolated and are confined inside hadrons such as protons and neutrons. The Quark-Gluon Plasma (QGP) is a state of nuclear matter predicted by QCD where quarks and gluons are deconfined. Experimentally, a QGP can be created in ultra-relativistic heavy ion collisions such as the lead-lead collisions delivered at the LHC, corresponding to speeds close to the speed of light. It is possible to obtain information on the characteris- tics of the QGP by measuring a large number of observables. In particular, the production of charmonium states such as the J/ψ and the ψ(2S), heavy particles composed of a charm and anti-charm pair (), is studied to investigate the plasma. Indeed, the presence of QGP is expected to modify the charmonium production yields, due to a balance between the mechanism of color screening of the charm quark potential and a mechanism called recombination. This balance depends on the collision energy, the temperature of the plasma and nature on the considered particle, in particular one expects the ψ(2S) to be more suppressed than the J/ψ. In this thesis the inclusive production of ψ(2S) in Pb − Pb collisions at an energy per nucleon-nucleon collision in the center of mass frame of TeV is measured in the dimuon-decay channel, using the ALICE Muon Spectrometer. The analysis is based on the data collected in ALICE (A Large Ion Collider Experiment) at the LHC in 2015 with an integrated luminosity of 225 μb−1. The nuclear modification factor RAA is studied as a function of centrality. The ratio of the ψ(2S) and J/ψ RAA is also evaluated and shows that the ψ(2S) is more suppressed than the J/ψ for mid-central and central events. Compared with theoretical predictions, the measurements are, within uncertainty, in agreement with theoretical model. The upgrade of the Muon Trigger, the MID (Muon Identifier), is also studied, in particular the expected data flow at a collisions rate of 100 kHz. Based on the Pb − Pb data at a collision energy of TeV, the estimations predict that the technology that will be implemented in the MID provides a sufficient bandwidth to sustain the data flow.
9

Efeito do estresse térmico no relógio biológico de Danio rerio: um elo entre temperatura , luz, canais termoTRPs e genes de relógio / Thermal stress effects on Danio rerio biological clock: a link between temperature, light, thermo-TRP channels and clock genes

Costa, Marcos Rodrigo Jeronimo da 03 August 2016 (has links)
A adaptação temporal é fundamental para a sobrevivência de espécies que precisam coordenar sua fisiologia e comportamentos ajustando-se a sinais externos. Ritmos biológicos não são simplesmente uma resposta às mudanças de 24 horas no ambiente físico impostas pela rotação da Terra sobre o seu próprio eixo, ao contrário, surgem a partir de um sistema de cronometragem endógeno. No teleósteo Danio rerio, ainda não foi identificada a presença de uma região que atue como relógio central; alguns estudos têm evidenciado a existência de células e tecidos que contêm relógios circadianos autônomos, fotossensíveis, comprovando um outro tipo de regulação dos ritmos circadianos onde a percepção do ambiente e o ajuste do período circadiano são efetivados diretamente em nível celular. As consequências deletérias do aumento da temperatura são impedidas, em certa medida, por uma resposta adaptativa que assegura a sobrevivência celular na presença de calor. Esta via de sobrevivência ativada por calor, conhecida como resposta ao choque térmico, é composta por uma cascata de eventos que conduzem à indução de proteínas de choque térmico (HSPs) que minimizam a lesão celular aguda. Acredita-se que os sistemas de percepção dos ciclos diários de temperatura e luminosidade sofreram as mesmas pressões seletivas em sua co-evolução, resultando em sua associação. As bases da sensação térmica estão em um grupo de canais altamente conservados, presente em todos os metazoários estudados até o momento e envolvidos em uma série de modalidades sensoriais, os canais de potencial receptor transiente (TRP); os que respondem a estímulos térmicos foram agrupados em uma subfamília e denominados termoTRPs. O objetivo deste trabalho foi investigar a influência do pulso de temperatura (33 ºC) na expressão de genes de relógio e de proteínas de choque térmico, bem como o papel do canal TRPV1, em células embrionárias de blástula de Danio rerio, denominadas ZEM-2S, submetidas a escuro constante (DD) ou ciclos claro-escuro (LD 12:12). Através de PCR em tempo real (quantitativo) demonstrou-se que as células ZEM-2S expressam os genes dos seguintes canais TRP: trpA1a, trpA1b, trpV1/2, trpV4, trpC6, trpM2, trpM4a, trpM4b/c e trpM5. Após um pulso de temperatura, observou-se um aumento no transcrito de hsp90 aa1 em células mantidas tanto em DD como em LD, sendo a expressão de hsp90 aa1 em LD, no ponto uma hora, duas vezes menor quando comparada a sua expressão no mesmo ponto temporal em DD. O pulso de temperatura não promoveu efeito em nenhum dos genes do relógio estudados (bmal1a, bmal1, bmal2, cry1a, cry1b, per1, per2) quando as células foram mantidas em DD. Porém, o transcrito de per2 aumentou em resposta ao pulso de temperatura quando as células foram sincronizadas pelos ciclos claro-escuro. A inibição do canal TRPV1 não alterou o efeito induzido pelo pulso de temperatura na expressão do gene hsp90 aa1 em células ZEM-2S mantidas em DD. Por outro lado, nossos dados permitem afirmar que o mesmo participa parcialmente na indução do aumento da expressão do gene per2 pelo estímulo térmico em células mantidas em LD, tendo em vista um decaimento significativo na resposta deste gene. Os dados obtidos neste trabalho abrem uma nova perspectiva sobre a investigação da relação temperatura e genes de relógio, colocando um novo “ator” na regulação deste fenômeno: o canal TRPV1 / Temporal adaptation is essential for the survival of species which need to coordinately adjust their physiology and behavior to external signals. Biological rhythms are not just a response to the 24 hour changes in the physical environment imposed by the rotation of the Earth around its own axis, but they arise from an endogenous timing system. In the teleost Danio rerio, there has not been identified so far a region in the nervous system that could act as a central clock; some studies have reported the existence of cells and tissues which contain photosensitive, autonomous circadian clocks, demonstrating the existence of another type of circadian rhythm regulation in which environment perception and entrainment of the circadian period are directly effected at cell level. The deleterious consequences of temperature increase are prevented by an adaptive response which assures cell survival in the presence of heat. This survival pathway activated by heat, known as response to temperature shock, is signaled by a cascade of events leading to the induction of thermal shock proteins (HSPs) which attenuate the acute cell lesion. It is believed that the systems perceiving temperature and light daily cycles were subject to the same selective pressures during their co-evolution, resulting in their association. The base of thermal sensation is a family of highly conserved channels, present in all metazoans studied to date, and involved in a variety of sensorial modalities, the transient receptor potential channels (TRP); those responding to thermal stimuli were grouped in a sub-family named thermo-TRPs. The aim of this work was to investigate the influence of a temperature pulse (33 ºC) on the expression of clock and heat shock protein genes, as well as the role of TRPV1 channel, in blastula embryonic cells of Danio rerio, named ZEM-2S, subject to constant dark (DD) or light-dark cycles (LD). Using quantitative PCR, we demonstrated that ZEM-2S cells express genes for the following TRP channels: trpA1a, trpA1b, trpV1/2, trpV4, trpC6, trpM2, trpM4a, trpM4b/c and trpM5. After the pulse of temperature, we observed an increase of hsp90 aa1 transcripts in DD as well as in LD; hsp90 aa1 expression 1 hour after the stimulus was two-fold lower in LD than in DD. Temperature pulse did not affect the expression of any of the studied clock genes (bmal1a, bmal1, bmal2, cry1a, cry1b, per1, per2), when the cells were kept in DD. However, per2 transcript increased in response to the temperature pulse when the cells were synchronized by light-dark cycles. Inhibition of TRPV1 channel did not change the effect induced by the temperature pulse on hsp90 aa1 in ZEM-2S cells kept in DD. On the other hand, our data suggest that this channel participates, at least partially, in the temperature-induced increase of per2 in cells maintained in LD, as indicated by the significant decay observed in the gene response in the presence of the inhibitor. Our results open new investigative perspective about the relationship between temperature and clock genes, placing a new “actor” in the regulation of the phenomenon: the TRPV1 channel
10

Purificação, caracterização bioquímica e atividade antimicrobiana de uma nova albumina 2s da torta da mamoneira (Ricinus communis L.), com atividade inibitória contra tripsina / Purification, biochemical characterization and antimicrobial activity of a novel 2s albumin from castor bean(ricinus cmmunis L.) cake displaying trypsin inhibitory activity

Souza, Pedro Filho Noronha de January 2012 (has links)
SOUZA, Pedro Filho Noronha de.Purificação, caracterização bioquímica e atividade antimicrobiana de uma nova albumina 2s da torta da mamoneira (Ricinus communis L.), com atividade inibitória contra tripsina. 2012. 114 f. Dissertação (Mestrado em Bioquímica) - Universidade Federal do Ceará, Fortaleza-CE, 2012. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-06-27T14:14:02Z No. of bitstreams: 1 2012_dis_pfnsouza.pdf: 1847174 bytes, checksum: 6675a05ab248cfb741080071e366161c (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-08-02T20:11:34Z (GMT) No. of bitstreams: 1 2012_dis_pfnsouza.pdf: 1847174 bytes, checksum: 6675a05ab248cfb741080071e366161c (MD5) / Made available in DSpace on 2016-08-02T20:11:34Z (GMT). No. of bitstreams: 1 2012_dis_pfnsouza.pdf: 1847174 bytes, checksum: 6675a05ab248cfb741080071e366161c (MD5) Previous issue date: 2012 / Castor bean (Ricinus communis L.) is an important crop for the Northeast of Brazil, which recently has been used to produce biodiesel. Around 90% of the castor bean production in Brazil is concentrated at Northeast region and the state of Ceará is the second largest producer. During the oil extraction process from the castor bean seeds, a resulting residue, called castor cake, is underutilized. However, this byproduct is rich in protein and micronutrients such as nitrogen, phosphorus and potassium, an attribute that qualifies it as a stuff that could be used as organic fertilizer or as animal feed, for example, bringing added value to it. Unfortunately, its use as food has not been possible because of the presence of toxic elements and allergens (ricin, ricinine, complex allergens) in its composition, unless it were previously submitted to a detoxification process. To date, the existing technologies to this end are not economically viable on an industrial scale. Therefore, studies to add value to this abundant byproduct generated in the castor bean biodiesel productive chain is of paramount importance. Thus, in this context, this study was performed to identify, isolate, purify and characterize new bioactive molecules of de-oiled castor cake with biotechnological potential. Through extraction of soluble proteins with 50 mM Tris-HCl pH 7.5, fractionation with ammonium sulfate (50-75%), following by hydrophobic interaction chromatography in Phenyl-Sepharose column and ion exchange chromatography (DEAE-Sepharose), a protein, named Rc-2S-Alb, able to inhibit trypsin was purified. Rc-2S-Alb has a molecular mass of approximately 75.8 kDa, as determined by SDS-PAGE, and under reducing conditions showed a large and a small protein band of 15.8 kDa and 10.5 kDa, respectively. Its NH2-terminal sequence showed similarity with the following proteins: putative 2S albumin precursor (89%), chain A of RicC3 (89%), and chain A of mabilin-1 (89%), all from R. communis seeds; and with the short chain of a "napin-like" protein from Brassica napus (89%). In addition, these similar proteins have two highly conserved domains: QEVQRKDLS and YLRQS. Comparison of the partial primary structure of Rc-2S-Alb generated by the ESI-Q-TOF MS/MS analysis of 17 tryptic peptides, showed 43% similarity to Mabinlin-1 (pI/Mr 6.7 and 29.3 kDa) from R. communis. There were also high similarities among the three-dimensional structures of Rc-2S-Alb, RicC3 and Mabinlin-1. Rc-2S-Alb did not inhibit the spore germination of the phytopathogenic fungi Fusarium oxysporum and Rizoctonia solani, but promoted aggregation of their respective spores. Moreover, Rc-2S-Alb did not inhibit the mycelial growth of Fusarium oxysporum, Fusarium solani, Rizoctonia solani and Collethotricum gloeosporioides. Contrary, Rc-2S-Alb was effective in inhibiting the growth of the human pathogenic bacteria Pseudomonas aeruginosae, Klebsiella pneumoniae and Bacillus subtilis, at low concentrations. In conclusion, it was established a protocol to purify a new 2S albumin from castor bean cake, which inhibits trypsin and has important antibacterial activity. Thus, the Rc-2S-Alb should be further studied in order to verify its effectiveness as new alternative therapeutic agent against resistant bacteria to commercial antibiotics, which will contribute to improve the human health. / A mamoneira (Ricinus communis L.) é uma cultura importante para a região Nordeste do Brasil, onde, recentemente, tem sido utilizada para produção de biodiesel. O Nordeste detém 90% da produção brasileira de mamona, sendo o Ceará, o segundo maior produtor. No processo de extração de óleo das sementes de mamoneira para produção de biodiesel, o resíduo resultante, denominado de torta da mamona, representa um subproduto pouco utilizado. Apesar disso, essa torta é rica em proteínas e micronutrientes, como nitrogênio, fósforo e potássio, atributo que a qualifica como um insumo que poderia ser utilizado como adubo orgânico, ou mesmo como ração animal, o que lhe agregaria valor comercial. Todavia, seu uso como alimento não tem sido possível por causa da presença de elementos tóxicos e alergênicos (Ricina, Ricinina, Complexos Alergênicos) na sua composição, a não ser que passasse por processamento para sua destoxificação. Infelizmente, tecnologia economicamente viável para esse fim, em escala industrial, ainda é inexistente. Portanto, há necessidade de pesquisas que venham a agregar valor a esse subproduto abundante da cadeia produtiva do biodiesel. Assim, nesse contexto, o presente trabalho está inserido num projeto cujo objetivo maior é identificar, isolar, purificar e caracterizar novas moléculas bioativas da torta delipidada de sementes de mamoeira com potencial biotecnológico. Através de extração de proteínas solúveis com tampão Tris-HCl, 50 mM, pH 7,5, e fracionamento do extrato obtido com sulfato de amônio (50-75%), cromatografia de interação hidrofóbica em coluna Phenyl-Sepharose e cromatografia de troca iônica (DEAE-Sepharose) foi possível purificar uma albumina 2S, denominada Rc-2S-Alb, capaz de inibir tripsina. A Rc-2S-Alb apresentou massa molecular de, aproximadamente, 75,8 kDa, determinada por SDS-PAGE, mas, em condições redutoras, apareceu como uma banda maior de 15,8 kDa e outra menor com 10,5 kDa. Sua sequência NH2-terminal revelou haver similaridade (89%) com o precursor putativo da albumina 2S de R. communis já descrita, com a cadeia A da estrutura da RicC3 (89%), com a cadeia A da mabilin-1, ambas, também, de R. communis (89%), com a cadeia pequena de uma proteina “napin-like”de Brassica napus (89%). Em todas essas proteínas similares, dois domínios, QEVQRKDLS e YLRQS, são altamente conservados. Comparação da estrutura primária gerada por ESI-Q-TOF MS/MS, a apartir de 17 peptídeos trípticos da Rc-2S-Alb, mostrou similaridade de 43% com a Mabinlin-1 (pI/Mr de 6,7 e 29.3 kDa) de R. communis. Em relação à estrutura tridimensional da Rc-2S-Alb, ela apresentou similaridade com a de Ric C3 e Mabinlin-1. A Rc-2S-Alb foi incapaz de inibir a germinação de esporos dos fungos fitopatogênicos Fusarium oxysporum e Rizoctonia solani, mas foi capaz de promover a aglomeração dos mesmos. A Rc-2S-Alb também não foi capaz de inibir o crescimento micelial dos fungos Fusarium oxysporum, Fusarium solani, Rizoctonia solani e Collethotricum gloeosporioides. Por outro lado, a Rc-2S-Alb foi eficiente em inibir o crescimento de Pseudomonas aeruginosae, Klebsiella pneumoniae e Bacillus subtilis, todas as bactérias patogênicas a seres humanos, quando em baixas concentrações. Como conclusão, foi possível a purificação de uma nova albumina 2S da torta da mamona, capaz de inibir tripsina e com atividade antibacteriana importante. Assim, a Rc-2S-Alb deve ser explorada no sentido de verificar sua eficácia como um novo agente terapêutico alternativo no combate a bactérias resistentes aos produtos disponíveis, hoje, no mercado, o que, se confirmado, poderá contribuir para a melhoria da saúde humana.

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