The heterochromatin 1 (HP1) family of non-histone chromosomal proteins is evolutionarily conserved and is involved in numerous biological processes, including the stabilization of heterochromatin, a state of compacted DNA along a protein scaffold. HP1 proteins and trimethylated histone H3 on lysine 9 (H3K9me3) are major constituents of heterochromatin and have been characterized extensively in vitro. The binding of HP1 proteins to H3K9 methylation marks plays an essential role in mammalian development and chromatin organization. However, due to their critical function, dissecting the molecular mechanism by which HP1 proteins exert their function in vivo is difficult. C. elegans is a unique model because not only are deletion mutants of the two HP1 homologs, HPL-1 and HPL-2, viable, but also H3K9 methylation is not essential to worm development. Interestingly, HPL-2 is alternatively spliced to generate two HP1 proteins, but in vivo experimentation has vastly ignored the potential contributions of the alternative transcripts to hpl-2 function, thus obfuscating which phenotypes associated with hpl-2 knockdown are due to the loss of one or more of the splicing variants. In this dissertation, I characterized the HPL-2 splicing variants (A and B) on a biochemical level in relation to the canonical human HP1b protein and on a physiological level in splicing variant-specific knockout worms. I show that both recombinant HPL-2A and HPL-2B bind H3K9me3 through their chromodomain (CD). But while HPL-2A acts as a canonical HP1 protein, namely it dimerizes and phase-separates like hHP1b, HPL-2B does not. In contrast to recombinant protein, in extracts both proteins rely on other factors, such as the MBT domain-containing protein LIN-61, for their recruitment to H3K9me3. Although HPL-2A and HPL-2B display distinct characteristics in vitro, both hpl-2a and hpl-2b worms are phenotypically wildtype. In agreement, knockout of either splicing variant leads to upregulated expression of the other one, suggesting a certain level of functional redundancy. Nevertheless, I show that the C-terminal extension of HPL-2B, which is absent in HPL-2A, resembles that of the CEC-4 heterochromatin anchor. I therefore hypothesize that the main functions of HPL-2 are distinct: HPL-2A mediates chromatin compaction and HPL-2B facilitates heterochromatin anchoring to the nuclear periphery.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第17930号 / 生博第293号 / 新制||生||38(附属図書館) / 30750 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 藤田 尚志, 教授 石川 冬木, 教授 松本 智裕 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

The ultimate goal of mitosis is the equal distribution of chromosomes between the two daughter cells. One of the key players that ensures faithful chromosome segregation is the chromosomal passenger complex (CPC). CPC localisation to mitotic centromeres is complex, involving interactions with Shugoshin and binding to phosphorylated histone H3T3. It was recently reported that Heterochromatin Protein 1 (HP1) has a positive impact on CPC function during mitosis. The interaction between HP1 and the CPC appears to be perturbed in cancer-­‐derived cell lines, resulting in decreased HP1 levels at mitotic centromeres and may be a potential cause for increased chromosome mis-­‐segregation rates. In this study, I tethered HP1α to centromeres via the DNA-­‐binding domain CENP-­‐B. However, instead of improving the rate of chromosome mis-­‐segregation, HP1α tethering resulted in activity of the spindle assembly checkpoint and destabilisation of kinetochore-­‐microtubule attachments, most likely caused by the robust recruitment of the CPC. Tethered HP1α even traps the CPC at centromeres during mitotic exit, resulting in a catalytically active CPC throughout interphase. However, it was not clear whether endogenous HP1 contributes to CPC localisation and function prior to mitosis. Here I also describe a substantial interaction between endogenous HP1 and the CPC during the G2 stage of the cell cycle. The two isoforms HP1α and HP1γ contribute to the clustering of the CPC into active foci in G2 cells, a process that is independent of CDK1 kinase activity. Furthermore, the H3S10ph focus formation in the G2 phase appears to be independent of H3T3ph and H2AT120ph, the two histone marks that determine the CPC localisation in early mitosis. Together, my results indicate that HP1 contributes to CPC concentration and activation at pericentromeric heterochromatin in G2. This novel mode of CPC localisation occurs before the Aurora B-­‐driven methyl/phos switch releases HP1 from chromatin, which possibly enables the H3T3ph and H2AT120ph driven localisation of the CPC during mitosis.

Within the cell nucleus, there are regions of highly condensed, transcriptionally silent chromatin called heterochromatin. Heterochromatin plays an important role in both chromosomal stability and gene regulation within the cell. Heterochromatin assembly is mediated by Heterochromatin Protein 1 (HP1) binding to epigenetically marked histone tails, most notably methylated H3K9. HP1 is post-translationally phosphorylated at serine and threonine residues, and this phosphorylation has been shown to increase HP1’s binding affinity for methylated H3K9 and heterochromatin formation. To study the effect of phosphorylation on heterochromatin assembly and HP1 localization within the nucleus, the unicellular protozoan Tetrahymena thermophila was used. Tetrahymena is an ideal model for this work because cells have a dynamic chromatin environment. Tetrahymena have an HP1-like protein, tHP1, which localizes to transcriptionally silent chromatin bodies within the otherwise transcriptionally active macronucleus. tHP1 is known to be phosphorylated at threonine-64 (site one) and at either serine-102 or threonine-103 (site two). Previous work shows that when phosphorylation at both sites is prevented, tHP1 exhibits decreased localization to chromatin bodies. In order to determine which site of phosphorylation accounts for tHP1’s localization to regions of heterochromatin, mutant proteins that allow phosphorylation at only one of the two sites were generated. The efforts to engineer a mutant protein that cannot be phosphorylated at site two and to visualize the protein’s localization throughout cell development are discussed. When phosphorylation is prevented at site two, tHP1 localization to regions of heterochromatin remains intact. These results suggest that phosphorylation at site one, not site two, may be responsible for tHP1 localization to macronuclear chromatin bodies. A mechanism by which site one phosphorylation influences tHP1 targeting to regions of heterochromatin is proposed. Furthermore, bioinformatics techniques are employed to identify other tHP1-like proteins within Tetrahymena. Characterization of these proteins will likely contribute to a more complete model of how heterochromatin is assembled in Tetrahymena.

Trimethylation of histone H3 lysine 9 (H3K9me3) and DNA methylation mark heterochromatin, contributing to gene silencing and normal cellular functions. My research investigated the control of H3K9me3 and DNA methylation in the filamentous fungus Neurospora crassa. The H3K9 methyltransferase complex, DCDC, consists of DIM-5, DIM-7, DIM-9, DDB1, and CUL4. Each component of DCDC is required for H3K9me3. The DIM-9/DDB1/CUL4 subunits are reminiscent of known cullin E3 ubiquitin ligases. I showed that core features of CUL4-based E3 ubiquitin ligases are not required for H3K9me3 and DNA methylation in Neurospora. H3K9me3 is bound by heterochromatin protein 1 (HP1) to recruit the DIM-2 DNA methyltransferase and the HCHC histone deacetylase complex. HCHC consists of HP1, CDP-2, HDA-1, and CHAP. Both HP1 and CDP-2 harbor conserved chromodomains that bind H3K9me3, and CHAP contains two putative AT-hook domains that bind A:T-rich DNA. To test the contributions of these domains to HCHC function, I deleted the chromodomains of HP1 and CDP-2. Deletion of the HP1 chromodomain resulted in a reduction of DNA methylation, which was not exacerbated by deletion of the CDP-2 chromodomain. A strain with deletions of chap and the HP1 chromodomain showed a DNA methylation phenotype comparable to the loss of the HDA-1 catalytic subunit. These findings support a model in which recognition of H3K9me3 and A:T-rich DNA by HP1 and CHAP, respectively, are required for proper HCHC function. To examine the relationships between H3K9me3, DNA methylation, and histone acetylation, I utilized in vivo protein tethering of core heterochromatin components. The requirement of DIM-7 for native heterochromatin, previously implicated in localizing the H3K9 methyltransferase DIM-5, was not bypassed by DIM-5 tethering, indicating that DIM-7 has additional roles within the DCDC. Artificial localization of the HCHC histone deacetylase, by tethering HP1 or HDA-1, resulted in induction of H3K9me3, DNA methylation, and gene silencing, but silencing did not require H3K9me3 or DNA methylation. HCHC-mediated establishment of H3K9me3 was not required for de novo heterochromatin formation at native heterochromatic loci suggesting a role in heterochromatin spreading. Together, this work implicates HDA-1 activity as a key driver of heterochromatin spreading and silencing. This dissertation includes previously published co-authored material.

Une réponse rapide et adaptée est nécessaire à la survie des cellules soumises à un stress. La réponse cellulaire au stress (HSR pour Heat-Shock response) médié par le facteur de transcription HSF1 est induite par les contextes environnementaux (chaleur, hypoxie, …) et les processus biologiques normaux et pathologiques (vieillissement, inflammation, …) associés à une accumulation de protéines endommagées (Morimoto, 1998). Ces protéines endommagées forment des agrégats toxiques aux conséquences létales pour les cellules.Conservé chez tous les eucaryotes, HSF1 orchestre les actions nécessaires à la survie et à la croissance des cellules malgré le stress. Ses cibles les mieux connues sont les gènes codants pour les Heat Shock Protein (HSP) qui font office de chaperon moléculaire. Une caractéristique de la HSR chez l’Homme est l’accumulation massive du facteur HSF1 en foyers nucléaires nommés Nuclear Stress Bodies (nSBs). Curieusement, ces foyers ciblent l’hétérochromatine péricentrique composée de séquences répétées en tandem de type Satellite III (SATIII), particulièrement au niveau du locus 9q12. HSF1 induit une forte transcription en ARN SATIII Sens (Jolly et al., 2004). Le rôle des nSBs est une des problématiques majeures de notre équipe cependant jusqu’à présent aucune fonction n’a été confirmée pour ces structures.Les nSBs, spécifiques aux cellules humaines, n’ont été décrits que dans des cellules en culture. Mon projet de thèse a consisté dans un premier temps à montrer la présence des nSBs in vivo chez l’Homme. Cette étude, réalisée sur du tissu testiculaire nous a également permis d’identifier une nouvelle cible SATIII majeure pour HSF1, la région Yq12. Dans les testicules, les nSBs sont associés à des processus méiotiques et post-méiotiques, suggérant un rôle dans le remodelage de l’hétérochromatine. Dans un deuxième temps, nous avons cherché à mieux comprendre le rôle des nSBs lors de la HSR. Nous avons pu montrer que l’étape de transcription des SATIII induit une déstabilisation de l’hétérochromatine péricentrique caractérisée par une dissociation des facteurs HP1 (Heterochromatin Protein 1) alpha et beta et une perte de la marque répressive H3K9me3. Au cours de la période de récupération qui accompagne la reformation de l’hétérochromatine, une transcription séquentielle d’ARN SATIII Sens puis Anti-sens précède la restructuration des loci 9q12. Nous avons également pu montrer que la transcription des SATIII est associée à un blocage de la mitose. Nous montrons que dans les cellules stressées, une altération de ce point de contrôle par un Knock down des ARN sat III par des approches LNA conduisent à une l’instabilité génomique des cellules tumorales avec apparition de cellules polynucléées. / A rapid and well-adapted response is required for cell survival upon stress. The cellular stress response (HSR) is mediated by the transcription factor Heat Shock Factor 1 (HSF1) (Morimoto, 1998). It is activated by environmental stress (heat, hypoxia, ...) and by a series of patho-physiological contexts (aging, inflammation, ...) involving protein damages.The best-characterized targets of HSF1 are genes encoding for Heat Shock Protein (HSP) acting as molecular chaperone. A specific feature of the HSR in human cells is the presence of HSF1 nuclear foci named Nuclear Stress Bodies (NSBs). Surprisingly, nSBs target pericentric heterochromatin consisting in tandem repeats of type III Satellite (SATIII) sequences, primarily at the 9q12 locus. HSF1 triggers a strong transcriptional activation of this locus (Jolly et al., 2004). The role of nSBS is a major issue since no function related to these structures has been reported so far.So far, nSBs have been only identified in cells in culture. My thesis project has been to further explore whether these structures also existed in normal tissues. Indeed, we have been able to identify the presence of nSBs in testis where they were found to be associated to meiotic and post-meiotic stages, suggesting a role related to heterochromatin remodeling. Moreover, we have identified the Yq12 locus as a new target of nSBs in these tissues. Secondly, we have brought new evidence that sat III sequences triggers a transient dissociation of HP1 (heterochromatin Protein 1) α and β as well as a loss of the repressive epigenetic H3K9me3 histone mark at pericentric heterochromatin. Interestingly we have also found that, following stress, a sequential accumulation of SATIII RNA in a Sense and Antisense orientation occurs, suggesting that this specific pattern of expression plays an important role in heterochromatin reformation. Finally, we have found that the accumulation of SATIII RNA is associated with a slowdown of mitosis. Indeed we have found that in stressed cells, accumulation of sat III impcats the progression of mitosis and that a knock down of sat III RNA using LNA approaches releases this blockade, leading to genomic instability of tumor cells and to the appearance of poly nucleated cells.

La chromatine est connue pour son rôle dans le maintien de l'identité cellulaire. Des perturbations dans la dynamique de la chromatine sont des événements courants dans les cancers. La structure de la chromatine et sa dynamique sont fortement dépendante des protéines HP1, connues pour être impliquées dans l’extinction de l’hétérochromatine, mais également dans la régulation de l'expression des gènes, la réplication et la réparation des dommages de l'ADN. Afin de mieux caractériser les fonctions d’HP1 chez les mammifères, nous avons étudié les conséquences de l'inactivation de leurs gènes chez la souris. De façon inattendue, nous démontrons que l'inactivation d’HP1a ou d’HP1g conduit à une prédisposition élevée des souris à développer des tumeurs spécifiquement dans le foie. Par conséquent, nous avons établi des modèles murins permettant l'inactivation simultanée d’HP1a/HP1b et HP1a/HP1g spécifiquement dans les hépatocytes. Ces modèles ont montré une augmentation significative de l'incidence du développement des tumeurs dans le foie, ce qui montre que les protéines HP1 sont des suppresseurs spécifiques de tumeurs hépatiques. L'analyse histologique de foies HP1abliverKO a montré des défauts qui ressemblent à ceux observés dans une pathologie connue du foie humain, la stéatohépatite non alcoolique. Afin de caractériser les mécanismes moléculaires sous-jacents ces fonctions des HP1, nous avons analysé le transcriptome de foies de souris âgées de 5 semaines. Ces analyses ont révélé que les gènes sur-exprimés en réponse à l’absence d’HP1ag ou HP1ab sont fortement enrichis en gènes codant pour des membres de la famille de répresseurs de transcription KRAB-ZFP. Ce résultat est intéressant car il est connu que ces répresseurs sont régulés par le corépresseur TRIM28 qui a besoin d’interagir avec HP1 pour remplir ses fonctions. Cela suggère donc une boucle d'autorégulation entre HP1, TRIM28 et KRAB-ZFP. En utilisant des souris exprimant une protéine TRIM28 qui est incapable d'interagir avec HP1 spécifiquement dans les hépatocytes, nous avons démontré que la perturbation de l'interaction entre TRIM28 et HP1 conduit au développement spontané de tumeurs dans le foie et conduit également à une surexpression des mêmes KRAB-ZFP que ceux dérégulée chez les souris HP1abliverKO et HP1agliverKO. L’immunoprécipitation de la chromatine (ChIP) a mis en évidence que TRIM28 et HP1 sont recrutés de façon interdépendante dans les régions 5 'et/ou 3' des gènes de KRAB-ZFP afin de réguler leur expression. Nous avons également observé la dérégulation de certains gènes liés au cancer, comme Tert, Nox4, AR, GPC3 et Arid1a. Ces modifications sont dépendantes de l’isotope d’HP1 inactivé, ce qui reflète les différents mécanismes moléculaires de l’oncogenèse. Afin d'élucider l'impact possible d’HP1 sur l'organisation générale du noyau, j'ai effectué une analyse par immunofluorescence sur cryosections du foie. Nos données suggèrent que les caractéristiques hétérochromatiques constitutives (H3K9me3) sont remplacées par des caractéristiques hétérochromatiques facultatives (H3K27me3) en l'absence de HP1ag et que les foyers péricentriques hétérochromatiques ont une légère tendance à être délocalisés. Enfin, pour mieux comprendre les profils chromosomiques dans la tumeur du foie HP1-dépendante, nous avons effectué une hybridation génomique comparative dans les foies tumoraux. Comme prévu, plusieurs événements de gain et de perte dans les variations du nombre de copies (CNV) dans certaines régions subchromosomales ont été observés, en particulier pour les chromosomes 4, où certains membres de KRAB-ZFP sont touchés. En résumé, nos résultats montrent que les protéines HP1 sont des suppresseurs de tumeur spécifique du foie. Ces données suggèrent également que la fonction principale d’HP1 au sein du foie est de réguler l'activité de TRIM28 et ainsi réguler l'expression et l'activité de répression des KRAB-ZFP et, finalement, l'homéostasie du foie. / Chromatin is known for its essential role in establishment and maintenance of cellular identity. Accordingly, disturbances in chromatin’s dynamics are common events in cancers. Chromatin structure and dynamics is highly dependent upon HP1, small non-histone chromosomal proteins that are known to be involved in heterochromatin silencing but also in gene expression regulation, DNA replication and DNA damage repair. To better characterize HP1 functions in mammals, we have studied the consequences of the inactivation of the corresponding genes in mice. Unexpectedly, we demonstrated that inactivation of either HP1a or HP1g lead to a high predisposition of mice to develop tumors specifically within liver. Hence, we established mice models allowing simultaneous inactivation of HP1a/HP1b and HP1a/HP1g specifically within hepatocytes. These models (HP1abliverKO and HP1agliverKO) displayed a significant increased incidence of tumor development within liver, demonstrating that HP1 are liver specific tumor suppressors. Histological analysis of HP1abliverKO livers showed defects that resembled those observed in a human liver pathology known as nonalcoholic steatohepatitis (NASH) characterized by an increase of steatosis, followed by an increased inflammation and the development of fibrosis that finally leads to tumors in old animals. In the case of HP1agliverKO mice, even though inflammation and tumor development were observed, this was not linked with steatosis, strongly suggesting that the underlying mechanisms are specific of each HP1 isoform. In order to reveal molecular mechanisms, we did expression analysis in the liver of 5 weeks old mice, which revealed a strong enrichment of genes encoding for members of the KRAB-ZFP of transcriptional repressors family within genes regulated by HP1ag or HP1ab. This result is of particular interest since it is known that these repressors are regulated by the corepressor TRIM28 which has been shown to require its interaction with HP1 to fulfill its functions suggesting a loop of auto-regulation between HP1, TRIM28 and KRAB-ZFP. Using mice expressing a TRIM28 protein unable to interact with HP1 specifically within hepatocytes, we demonstrated here that the disruption of the interaction between TRIM28 and HP1 lead to spontaneous development of tumors within liver and to over-expression of the same KRAB-ZFP as those deregulated in HP1abliverKO and HP1agliverKO mice. Chromatin immunoprecipitation (ChIP) pinpointed that TRIM28 and HP1 are inter-dependently recruited to the 5’ and/or 3’ ends of KRAB-ZFP genes to regulate their expression. We also observed deregulation of some cancer related genes, such as Tert (Telomerase reverse transcriptase), Nox4 (NADPH oxidase 4), AR (Androgen receptor), GPC3 (Glypican3), Arid1a (AT-Rich Interaction Domain 1A), and interestingly these alterations are depended upon the inactivated HP1 isotype, reflecting distinct molecular oncogenesis. In order to elucidate the possible impact of HP1 on global organization of the nucleus, I performed immunofluorescence analysis in the liver cryosections of 5 weeks old mice. Our data suggest that constitutive heterochromatic features (H3K9me3) are replaced by facultative heterochromatic features (H3K27me3) in absence of HP1ag and that heterochromatic pericentric foci tend to slightly be delocalized. Finally, to better understand the chromosomal rearrangements profile in HP1-dependent liver tumor, we performed Comparative genomic hybridization (CGH) in old tumoral liver. As anticipated, multiple events of gain and loss in copy number variations (CNV) in subchromosomal regions were observed, especially for chromosomes 4, where some KRAB-ZFP members are affected. Altogether, our data demonstrated that HP1 are liver-specific tumor suppressor. They also suggest that HP1 main function within liver is to regulate TRIM28 activity and thereby regulate the expression and repression activity of KRAB-ZFP and ultimately liver homeostasis.

No description available.

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.

HOAP was originally identified as a component of an ORC-containing multi-protein complex of Heterochromatin Protein 1 (HP1) from early Drosophila embryos. HOAP immunostaining showed prominent association of it with telomeres, and mutants for HOAP (cav1) showed it functions along with HP1 in forming a telomere capping complex that prevents telomeric fusions. Weaker HOAP immunostaining is also observed in regions of pericentric heterochromatin and euchromatin. To examine the role of HOAP at these non-telomeric sites, we applied Affymetric Drosophila Genome Arrays to undertake a microarray expression profiling study of genes that are mis-expressed in cav1 mutant larvae. The data from four publicly available databases were used to assess the normal expression patterns of the affected genes. We found that the majority (67%) of genes with decreased expression levels in cav1 mutants (log2R< -2.0, pvalue≤ 0.01) have normally testis-specific expression. These results could indicate a role of HOAP in testis-specific gene expression. Alternatively they could reflect reduced male viability due to the loss of HOAP, which resulted in the under-representation of males in the cav1 larval sample. The latter hypothesis is supported by the observation of 2.8-fold under-representation of males in cav1 larvae when I used a yellow+-marked X chromosome to differentially mark male and female cav1 larvae. Thus, this project is focused on determining and characterizing the cause of the reduced male viability. Here I report a role for both HOAP and HP1 in regulating the establishment promoter, SxlPe, of the sex determination masterswitch, Sex lethal (Sxl). Female-specific activation of SxlPe is essential to females as it provides SXL protein to initiate productive female-specific splicing of the late Sxl transcripts which are transcribed in both sexes. We find inappropriate firing of SxlPe and splicing of Sxl transcripts in male cav mutants, whereas mutants for HP1 display Sxl splicing defects in both sexes. Both proteins are associated with SxlPe sequences. In embryos from HP1 mothers and Sxl mutant fathers, female viability and RNA polymerase II recruitment to SxlPe is severely compromised. Our genetic and biochemical assays suggest a repressing activity for HOAP and both activating and repressing roles for HP1 at SxlPe.