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Potencialização da ação de produtos lipofílicos provenientes de espécies de Hypericum nativas do sul do Brasil / Potentiation of action of lipophilic products from Hypericum species native to south BrazilMeirelles, Gabriela de Carvalho January 2016 (has links)
Plantas do gênero Hypericum (Hypericaceae) são reconhecidas fontes de moléculas com fins terapêuticos. Para espécies nativas do sul do Brasil, atividades como antifúngica e antinociceptiva já foram relatadas, atribuídas principalmente a compostos extraídos em suas frações lipofílicas como derivados de floroglucinol, benzopiranos e benzofenonas. Neste estudo, o potencial sinérgico entre frações lipofílicas de H. carinatum e o fármaco fluconazol, frente a fungos leveduriformes emergentes, foi avaliado por duas metodologias distintas: checkerboard e isobolograma. Para isolados de Candida krusei e C. famata o efeito da associação foi superior ao do fármaco isolado. Dessa forma, o perfil de suscetibilidade observado sugere que a fração esteja auxiliando a ação do fármaco. Ainda abordando o potencial terapêutico de espécies de Hypericum, a investigação da atividade antinociceptiva (via oral) do benzopirano HP1 de H. polyanthemum, quando incorporado em nanoemulsões, foi avaliada. Os resultados demonstraram que HP1 pode ser adequadamente incorporado em nanoemulsões, dada sua solubilidade no núcleo oleoso. Em relação ao efeito antinociceptivo, nanoemulsões contendo HP1 demonstraram o mesmo efeito do composto livre, em magnitude, porém em dose inferior. A redução da dose ativa sugere que uma melhor solubilização do composto possa ter ocorrido quando o mesmo está inserido em nanoemulsões. Nesse contexto, estudos de permeabilidade intestinal ex vivo (Ussing chambers) de HP1, na sua forma livre e incorporado em nanoemulsões, foram realizados. Os resultados demonstraram que a permeabilidade intestinal do benzopirano HP1, quando incorporado em nanoemulsões, foi cerca de 4 vezes maior em relação a forma livre. Além disso, experimentos de lipólise in vitro mostraram que enzimas presentes no trato gastrointestinal são hábeis em hidrolisar nanoemulsões a espécies coloidais, mais solúveis e facilmente absorvíveis pelas células intestinais. Ainda, a permeabilidade intestinal do benzopirano HP1, na sua forma livre, no sentido absortivo foi maior que no sentido secretório indicando que transportadores ativos estão, ao menos em parte, auxiliando a absorção deste composto pelas células intestinais. Dessa forma, com vistas a elucidar o provável transportador ativo de HP1, dada a semelhança estrutural deste benzopirano com moléculas canabinoides e a relação existente entre os sistemas opioide e canabinoide, a influência deste último na absorção de HP1 foi investigada. Os resultados demonstraram que o benzopirano HP1 pode estar relacionado ao sistema canabinoide, mas a natureza dessa ligação, seja de transporte, agonismo/antagonismo ou físico-química, não foi possível de ser elucidada. Os resultados obtidos nesta tese são relevantes à medida que espécies de fungos leveduriformes emergentes se mostram cada vez mais resistentes aos fármacos comumente utilizados. Além disso, a importância destes resultados se dá pela viabilidade de incorporação do benzopirano HP1 em nanoemulsões e a capacidade desses sistemas em reduzir a dose ativa no benzopirano HP1 por uma maior solubilização do composto e assim, melhor absorção. Dessa maneira, os resultados deste trabalho representam o alto potencial biológico de espécies de Hypericum e abrem possibilidade para mais estudos utilizando estas plantas. / Plants from genus Hypericum (Hypericaceae) are recognized as a source of therapeutical agents. To south Brazil species, acitivities like antifungal and antinociceptive had already been demonstrated, attributed mainly to compounds from lipophilic fractions as phloroglucinol derivatives, benzophenones and benzopyrans. In this study, antifungal potential of lipophilic fractions of H. carinatum and fluconazole against emerging yeasts was evaluated by two methodologies for multiple dose-response analyzes: checkerboard and isobologram. To Candida krusei and C. famata isolates the effect of association was higher than the effect of fluconazole alone. Thus, the susceptibility profile observed for these species suggests that, somehow, the fractions are facilitating the action of drug. Still on therapeutical potential of Hypericum species, the antinociceptive study of a benzopyran (HP1) isolated from H. polyanthemum, incorporate in nanoemulsions, was evaluated. The results demonstrated that HP1 could be incorporated in a nanoemulsion system, given the high solubility in the oil core. Regarding the antinociceptive effect, HP1 loaded in nanoemulsions showed the same effect of free form, in magnitude, at lower doses. These results suggest a better solubilization of HP1 when loaded in nanoemulsions, and, thus, better absorption by organism. In this context, ex vivo intestinal permeability studies (Ussing chambers) of HP1 free form and loaded in nanoemulsions were performed. The results showed that the intestinal permeability of HP1 loaded in nanoemulsions were about 4 times higher than HP1 free form. Besides, the intestinal permeability of HP1 free form in absorptive direction was higher than secretory direction indicating that active transporters are, at least in part, involved in HP1 intestinal absorption. Thus, in order to elucidate the probable active transporter of HP1 and since its structure looks like a cannabinoid molecule and there is a relation between the opioid and cannabinoid pathways, the influence of intestinal cannabinoid system in HP1 absorption was investigated. The results indicated that the benzopyran HP1 may be related to cannabinoid system, but the nature of this interaction: transport, agonism/antagonism or physico-chemical is still unknown. The outcomes obtained are relevant since the resistance of emerging yeast species to available drugs, used for a variety of fungal infections, is increasing. The importance of these findings lies also in the feasibility of incorporating HP1 into nanoemulsions, and the capacity of these systems in reduce the antinociceptive active doses, by higher solubilization, and thus, absorption. Then, together the results represent the high biological potential of Hypericum species and open new possibilities to further studies with these plants.
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Characterisation of HP1γ in mammalian cellsWiese, Meike January 2018 (has links)
The degree of chromatin compaction plays a fundamental role in controlling the accessibility of DNA to the transcription machinery as well as other DNA-dependent biological pathways. The mammalian HP1 (Heterochromatin protein 1) protein family consists of three members: HP1α, β and γ. Each paralogue regulates formation and maintenance of heterochromatin by binding to the repressive chromatin marks H3K9me2/3 with their chromodomains (CDs). Despite high sequence conservation, each HP1 paralogue possesses specific functions, which are likely to be cell type specific. The aim of my thesis was to find novel functions for HP1γ in mouse embryonic stem cells (mESCs) and breast cancer cells. Mass spectrometry analysis identified citrullination of residues R38 and R39 within the CD of HP1γ. I show that these residues are citrullinated by peptidyl arginine deiminase 4 (PADI4) in vitro and in vivo. Mutations in HP1γ (R38/9A), designed to mimic the loss of charge accompanied with citrullination, affect HP1γ’s binding to H3K9me3 peptides and reduce its residence time on chromatin in differentiated mESCs, indicating a role for citrullination in regulating HP1γ binding to chromatin during differentiation. Furthermore, I studied the phenotype of HP1γ depletion in two human breast cancer models and found that HP1γ is essential for cell proliferation and viability of cancer, but not of normal epithelial cells. I performed whole transcriptome analysis in breast cancer cells depleted of HP1γ and cross-referenced it with its genomic localisation, which identified increased expression of interferon/antiviral defense genes and activation of pro-apoptotic pathways. Whilst genes involved in these pathways were not directly bound by HP1γ, this analysis also identified HP1γ as a novel regulator of zinc finger (ZNF) genes. In summary, I identified novel post-translational modifications in HP1γ and characterised them in mESCs. I further demonstrated a role for HP1γ regulating breast cancer cell viability and identified HP1γ as a novel regulator of ZNF genes. My findings highlight HP1γ as a potential target for breast cancer therapy.
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Potencialização da ação de produtos lipofílicos provenientes de espécies de Hypericum nativas do sul do Brasil / Potentiation of action of lipophilic products from Hypericum species native to south BrazilMeirelles, Gabriela de Carvalho January 2016 (has links)
Plantas do gênero Hypericum (Hypericaceae) são reconhecidas fontes de moléculas com fins terapêuticos. Para espécies nativas do sul do Brasil, atividades como antifúngica e antinociceptiva já foram relatadas, atribuídas principalmente a compostos extraídos em suas frações lipofílicas como derivados de floroglucinol, benzopiranos e benzofenonas. Neste estudo, o potencial sinérgico entre frações lipofílicas de H. carinatum e o fármaco fluconazol, frente a fungos leveduriformes emergentes, foi avaliado por duas metodologias distintas: checkerboard e isobolograma. Para isolados de Candida krusei e C. famata o efeito da associação foi superior ao do fármaco isolado. Dessa forma, o perfil de suscetibilidade observado sugere que a fração esteja auxiliando a ação do fármaco. Ainda abordando o potencial terapêutico de espécies de Hypericum, a investigação da atividade antinociceptiva (via oral) do benzopirano HP1 de H. polyanthemum, quando incorporado em nanoemulsões, foi avaliada. Os resultados demonstraram que HP1 pode ser adequadamente incorporado em nanoemulsões, dada sua solubilidade no núcleo oleoso. Em relação ao efeito antinociceptivo, nanoemulsões contendo HP1 demonstraram o mesmo efeito do composto livre, em magnitude, porém em dose inferior. A redução da dose ativa sugere que uma melhor solubilização do composto possa ter ocorrido quando o mesmo está inserido em nanoemulsões. Nesse contexto, estudos de permeabilidade intestinal ex vivo (Ussing chambers) de HP1, na sua forma livre e incorporado em nanoemulsões, foram realizados. Os resultados demonstraram que a permeabilidade intestinal do benzopirano HP1, quando incorporado em nanoemulsões, foi cerca de 4 vezes maior em relação a forma livre. Além disso, experimentos de lipólise in vitro mostraram que enzimas presentes no trato gastrointestinal são hábeis em hidrolisar nanoemulsões a espécies coloidais, mais solúveis e facilmente absorvíveis pelas células intestinais. Ainda, a permeabilidade intestinal do benzopirano HP1, na sua forma livre, no sentido absortivo foi maior que no sentido secretório indicando que transportadores ativos estão, ao menos em parte, auxiliando a absorção deste composto pelas células intestinais. Dessa forma, com vistas a elucidar o provável transportador ativo de HP1, dada a semelhança estrutural deste benzopirano com moléculas canabinoides e a relação existente entre os sistemas opioide e canabinoide, a influência deste último na absorção de HP1 foi investigada. Os resultados demonstraram que o benzopirano HP1 pode estar relacionado ao sistema canabinoide, mas a natureza dessa ligação, seja de transporte, agonismo/antagonismo ou físico-química, não foi possível de ser elucidada. Os resultados obtidos nesta tese são relevantes à medida que espécies de fungos leveduriformes emergentes se mostram cada vez mais resistentes aos fármacos comumente utilizados. Além disso, a importância destes resultados se dá pela viabilidade de incorporação do benzopirano HP1 em nanoemulsões e a capacidade desses sistemas em reduzir a dose ativa no benzopirano HP1 por uma maior solubilização do composto e assim, melhor absorção. Dessa maneira, os resultados deste trabalho representam o alto potencial biológico de espécies de Hypericum e abrem possibilidade para mais estudos utilizando estas plantas. / Plants from genus Hypericum (Hypericaceae) are recognized as a source of therapeutical agents. To south Brazil species, acitivities like antifungal and antinociceptive had already been demonstrated, attributed mainly to compounds from lipophilic fractions as phloroglucinol derivatives, benzophenones and benzopyrans. In this study, antifungal potential of lipophilic fractions of H. carinatum and fluconazole against emerging yeasts was evaluated by two methodologies for multiple dose-response analyzes: checkerboard and isobologram. To Candida krusei and C. famata isolates the effect of association was higher than the effect of fluconazole alone. Thus, the susceptibility profile observed for these species suggests that, somehow, the fractions are facilitating the action of drug. Still on therapeutical potential of Hypericum species, the antinociceptive study of a benzopyran (HP1) isolated from H. polyanthemum, incorporate in nanoemulsions, was evaluated. The results demonstrated that HP1 could be incorporated in a nanoemulsion system, given the high solubility in the oil core. Regarding the antinociceptive effect, HP1 loaded in nanoemulsions showed the same effect of free form, in magnitude, at lower doses. These results suggest a better solubilization of HP1 when loaded in nanoemulsions, and, thus, better absorption by organism. In this context, ex vivo intestinal permeability studies (Ussing chambers) of HP1 free form and loaded in nanoemulsions were performed. The results showed that the intestinal permeability of HP1 loaded in nanoemulsions were about 4 times higher than HP1 free form. Besides, the intestinal permeability of HP1 free form in absorptive direction was higher than secretory direction indicating that active transporters are, at least in part, involved in HP1 intestinal absorption. Thus, in order to elucidate the probable active transporter of HP1 and since its structure looks like a cannabinoid molecule and there is a relation between the opioid and cannabinoid pathways, the influence of intestinal cannabinoid system in HP1 absorption was investigated. The results indicated that the benzopyran HP1 may be related to cannabinoid system, but the nature of this interaction: transport, agonism/antagonism or physico-chemical is still unknown. The outcomes obtained are relevant since the resistance of emerging yeast species to available drugs, used for a variety of fungal infections, is increasing. The importance of these findings lies also in the feasibility of incorporating HP1 into nanoemulsions, and the capacity of these systems in reduce the antinociceptive active doses, by higher solubilization, and thus, absorption. Then, together the results represent the high biological potential of Hypericum species and open new possibilities to further studies with these plants.
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Composante épigénétique dans le déclenchement de l'inflammation chez les patients atteints de la sclérose en plaques / Epigenetic component in the onset of inflammation in the context of multiple sclerosisAzebi, Saliha 30 September 2015 (has links)
La sclérose en plaques est une maladie auto-immune dirigée contre les protéines de la myéline du cerveau. Plusieurs mécanismes physiopathologiques sont impliqués dans la SEP tels que l'inflammation, la démyélinisation et l’atteinte axonale. La SEP est associée à une expression accrue de cytokines et à une activation des rétrovirus endogènes humains (HERVs). Dans les conditions physiologiques, ces unités transcriptionnelles sont maintenues dans un état réprimé par un même mécanisme répresseur dépendant de la chromatine : la tri-méthylation de la lysine 9 de l’histone H3 (H3K9me3), qui crée un site de liaison aux protéines de la famille HP1. Nous avons trouvé qu’à la fois, les gènes de l’immunité et les HERVs nécessitent les protéines hétérochromatiniennes HP1α pour leur répression transcriptionnelle. Nous avons montré que la peptidylarginine déiminase 4, une enzyme qui joue un rôle dans la SEP, affaiblit la liaison de HP1α à la lysine 9 tri-méthylée de l'histone H3 en citrullinant l’arginine 8. Nous avons apporté la preuve que de multiples événements de la réactivation de la transcription chez les patients atteints de la SEP peuvent être expliqués par un défaut du mécanisme unique de répression génique. Nous avons également montré qu'il est possible de renforcer la répression de HP1 à l'aide de petites molécules. Par exemple, l’EGCG, un composé de thé vert, est en mesure de réduire à la fois l’expression des HERVs et des cytokines en augmentant l'activité de l’histone méthyltransférase SUV39H1. Cela conduit à une accumulation de la marque répressive H3K9me3, qui va favoriser la liaison de HP1.Ensemble, ces résultats suggèrent que HP1 est une composante importante de la SEP au niveau de la régulation des cytokines et des HERVs. / Multiple Sclerosis is an autoimmune disease resulting in damage to myelin structures of the brain. Several physiopathological mechanisms are involved in MS including inflammation, demyelination, and axonal damage. MS is associated with increased cytokine expression and activation of human endogenous retroviruses (HERVs). These two types of transcriptional units are kept in check by chromatin-dependent silencing associated with lysine 9 trimethylation of histone H3, and subsequent of HP1 proteins. We find that both the cytokine genes and the HERVs require the heterochromatin protein HP1 for their transcriptional repression. Furthermore, we have shown that the peptidylarginine deiminase 4, an enzyme with a suspected role in MS, weakens the binding of HP1 to tri-methylated histone H3 lysine 9 by citrullinating histone H3 arginine 8. We thereby evidence that multiple events of transcriptional reactivation in MS patients can be explained by deficiency of a single mechanism of gene silencing. We have also shown that it is possible to reinforce HP1 repression by using small molecules. For example, EGCG, a green tea compound, is able to reduce both HERVs and cytokines expression by increasing histone methyltransferase activity SUV39H1. This leads to increased accumulation of H3K9me3 repressive marks and favors binding of HP1. All together, these results suggest that HP1 is an important component of the regulation of cytokine genes and HERVs in MS patients.
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Investigating the phase separation of recombinant Heterochromatin Proteins 1 (HP1) of Caenorhabditis elegansAlotaibi, Aljoharah 09 August 2023 (has links)
The proper packaging of the genome in eukaryotic nuclei is essential for proper gene expression and cell function. Chromatin at the large scale is divided into two major compartments heterochromatin and euchromatin. Heterochromatin compromises the transcriptionally inactive tightly packaged regions of chromatin, while euchromatin is the transcriptionally active region of chromatin.
The Heterochromatin Protein family (HP1) proteins are epigenetic hallmarks of constitutive heterochromatin. Recent evidence suggests human HP1α undergoes liquid-liquid phase separation suggesting a role for HP1 phase separation in the formation of compacted heterochromatin within HP1 droplets. Phase separation is a biophysical property of proteins with intrinsically disordered domains which are protein domains that lack a defined secondary structure and have the ability to undertake multiple conformations.
In this thesis, I investigated the ability of C. elegans HP1 homologs HPL-2A and HPL-1 to phase separate utilizing directed mutations to elucidate the intermolecular interactions that govern this phenomenon and different assays to assess their phase separation.
I concluded that HPL-2A is a bona fide phase separating protein that selectively condenses chromatin. HPL-2A’s phase separation depends on specific interactions, mainly dimerization and the presence of lysine and arginine residues in the hinge region. HPL-2A has a specific IDR that drives its phase separation which is the hinge region as the CTE and NTE are not essential for its phase separation.
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Molekulare und funktionelle Analyse von Windei (CG12340) als Bindungspartner der Histonmethyltransferase Eggless während der Oogenese von <i>Drosophila</i> / Molecular and functional analysis of Windei (Wde) as binding partner of the histone methyltransferase Eggless during the oogenesis of <i>Drosophila</i>Koch, Carmen 20 January 2009 (has links)
No description available.
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Interaction fonctionnelle de la Poly(ADP-Ribose) polymérase-1 (PARP1) avec des protéines de l'hétérochromatine : impact sur la fonction de l'hétérochromatine et la réparation de l'ADN / Functional interaction between Poly(ADP-ribose) polymerase-1 (PARPl) and heterochromatin proteins : impact on heterochromatin function and DNA repairDe Vos, Mike 14 March 2014 (has links)
Nous avons identifié une association poly(ADP-ribose) (PAR)-dépendante entre PARP1 et UHRF1. UHRF1 est PARylé par PARP1 et lie le PAR de façon non covalente. L’absence de PARP1 (i) perturbe l’association de UHRF1 et DNMT1, (ii) induit une ubiquitination excessive de DNMT1 par UHRF1 favorisant sa dégradation au cours du cycle, (iii) favorise la transcription des régions de l’hétérochromatine péricentrique (pHC) (iv) et perturbe la localisation de la marque répressive H4K20me3 au niveau des foyers de l’pHC. Dans un deuxième temps, nous avons étudié le rôle de l’association KAP1-HP1 dans la réponse cellulaire aux dommages. L’interaction entre ces deux partenaires est essentielle pour le recrutement de KAP1 sur les sites de cassures. Après induction de cassures, l’absence d’interaction induit un délai dans la réparation des cassures double-brins et une diminution de la survie cellulaire. Une analyse détaillée suggère une déficience du mécanisme de réparation par recombinaison homologue. / We identified a poly(ADP-ribose) (PAR)-dependent interaction between PARP1 and UHRF1. UHRF1 is PARylated by PARP1 and binds PAR in a non-covalent way. The absence of PARP1 (i) impairs the UHRF1/DNMT1 interaction, (ii) induces excessive UHRF1-mediated ubiquitination of DNMT1 promoting its degradation during the cell cycle, (iii) increases the transcription of pericentric heterochromatin (pHC) regions (iv) and impairs the localization of the repressive histone mark H4K20me3 on pHC. In a second project we studied the role of the KAP1/HP1 interaction in response to DNA damage. The interaction between the two partners is essential for KAP1 recruitment to DNA damage sites. The absence of the interaction, after damage, induces a delay of the double strand break repair kinetics and decreases the cell survival rate. A more detailed analysis suggests a deficiency of the homologous recombination repair pathway.
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Energy landscaping : on the relationship between functionality and sequence mutations for multifunctional biomoleculesRöder, Konstantin January 2018 (has links)
The process of protein and RNA folding has been understood in general terms through the principle of minimal frustration, and is usually thought of as being guided by a folding funnel on the energy landscape, which is based around the native structure. However, more recently, various biomolecules have been associated with multifunnel energy landscapes, where each funnel exhibits a distinct structural ensemble and function. This work explores how the principle of minimal frustration may be extended to multifunnel energy landscapes that are associated with multifunctional biomolecules. To achieve this aim, the computational potential energy landscape framework is employed to analyse four example systems. Additionally, this study analyses mutants for all four systems, where the mutations are chosen to change properties of the systems without destabilising the native sequence ensemble entirely. The first system considered is a two-state coiled-coil. It is shown how mutations fundamentally change the energy landscape from the minimal frustrated organisation necessary to fulfil biological function. These changes can introduce alternative pathways for folding, as well as new structural ensembles. Similar effects are observed for ubiquitin. In addition, the landscape exploration allows us to calculate a number of experimentally determined properties for this protein, which exhibit excellent agreement, and we characterise folding at an atomistic level of detail. Next we consider the hormones oxytocin and vasopressin, which are themselves mutants of each other, along with a number of other mutants for both molecules. Again, the frustration in the landscape increases due to mutations, and a greater variety in the resulting structural ensembles is observed, leading to changes in binding affinities. Finally, the HP1 loop of RNA 7SK is analysed, revealing that the principles established for the energy landscapes of proteins extend to nucleic acids. Overall, the results indicate that sequences have evolved to exhibit the minimum number of funnels on the energy landscape to support multiple functions, extending the principle of minimal frustration to multifunnel energy landscapes.
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Reading the Epigenetic State of Chromatin Alters its AccessibilityGibson, Matthew D. January 2016 (has links)
No description available.
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