Submitted by Luiz Felipe Barbosa (luiz.fbabreu2@ufpe.br) on 2015-03-13T13:11:59Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Nicole Leoni numerada.pdf: 2611677 bytes, checksum: e5bacea1c2ee6c31edf71b1c3cf3df25 (MD5) / Made available in DSpace on 2015-03-13T13:11:59Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Nicole Leoni numerada.pdf: 2611677 bytes, checksum: e5bacea1c2ee6c31edf71b1c3cf3df25 (MD5) Previous issue date: 2012 / Os efeitos do resveratrol e da utilização de lipídios em ensaio de restrição dietética (RD) sobre a senescência demográfica, a resposta neuromotora e a expressão do gene Hsp22 foram analisados em cinco linhagens de Drosophila melanogaster. Ao contrário do previsto para RD utilizando proporção entre proteínas e carboidratos como variável, a utilização de lipídio alterou tanto a taxa de mortalidade inicial como a taxa de envelhecimento, alem de surtir efeitos deletérios na fecundidade de fêmeas e no desempenho de machos no teste de geotaxia negativa. Fêmeas e machos atingiram maior longevidade em meio sem adição e com adição moderada de lipídios respectivamente. O resveratrol teve efeitos variados na longevidade, mas foi capaz de retardar a deterioração neuromotora em todas as linhagens, exceto na mutante Sir2-. Houve ocorrência de pufes Hsp22 maiores em larvas criadas em meio hipercalórico controle e hipocalórico tratado com resveratrol. Esses resultados indicam que: (1) nem o conteúdo energético, nem a proporção entre carboidratos e proteínas podem explicar totalmente a longevidade em D. melanogaster; (2) os efeitos dos lipídios dietéticos foram dependentes do sexo, aparentando ser levemente tóxicos com efeito hormético em machos; (3) os mecanismos que influenciam longevidade e senescência funcional são, pelo menos parcialmente, distintos; (4) o resveratrol é capaz de retardar o declínio funcional locomotor e apresenta efeitos na longevidade dependentes do genótipo, sexo, diluição do meio e quantidade administrada, aparentando estar envolvido com Sir2; (5) ausência do alelo funcional Sir2 pode regular positivamente Hsp22, o que explica maior longevidade dos mutantes Sir2- apesar da inatividade desta sirtuína; (6) aumento do nível de Sir2 induzido por RD pode inibir a expressão de Hsp22, corroborando a hipótese do envolvimento da acetilação de histona na regulação dessa proteína; (7) resveratrol, entretanto, pode reverter esse processo por alterar a afinidade química de Sir2 pelo lócus de Hsp22

Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-04-10T16:45:24Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Dissertação-NicoleLeoni.pdf: 2611853 bytes, checksum: 64bb3c5f2ca3ff1fd9b70a9e02babd6d (MD5) / Made available in DSpace on 2017-04-10T16:45:24Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Dissertação-NicoleLeoni.pdf: 2611853 bytes, checksum: 64bb3c5f2ca3ff1fd9b70a9e02babd6d (MD5) Previous issue date: 2012 / Os efeitos do resveratrol e da utilização de lipídios em ensaio de restrição dietética (RD) sobre a senescência demográfica, a resposta neuromotora e a expressão do gene Hsp22foram analisados em cinco linhagens de Drosophila melanogaster.Ao contrário do previsto para RD utilizando proporção entre proteínas e carboidratos como variável, a utilização de lipídioalterou tanto a taxa de mortalidade inicial como a taxa de envelhecimento, alem de surtir efeitos deletérios na fecundidade de fêmeas e no desempenho de machos no teste de geotaxia negativa.Fêmeas e machos atingiram maior longevidade em meio sem adição e com adição moderada de lipídios respectivamente. O resveratrol teveefeitos variados na longevidade, mas foi capaz de retardar a deterioração neuromotora em todas as linhagens, excetona mutante Sir2-. Houveocorrência de pufes Hsp22 maiores em larvas criadas em meio hipercalórico controle e hipocalórico tratado com resveratrol. Esses resultados indicam que:(1) nem o conteúdo energético, nem a proporção entre carboidratos e proteínas podem explicar totalmente a longevidade em D. melanogaster; (2)os efeitos dos lipídios dietéticos foram dependentes do sexo, aparentando ser levemente tóxicos com efeito hormético em machos; (3) os mecanismos que influenciam longevidade e senescência funcional são, pelo menos parcialmente,distintos; (4) o resveratrol é capaz de retardaro declínio funcional locomotor e apresenta efeitos na longevidade dependentes do genótipo, sexo, diluição do meioe quantidade administrada, aparentando estar envolvido com Sir2; (5)ausência do alelo funcional Sir2 pode regular positivamente Hsp22, o que explica maior longevidade dos mutantes Sir2-apesar da inatividade desta sirtuína; (6)aumento do nível de Sir2 induzido por RD pode inibir a expressão de Hsp22, corroborando a hipótese do envolvimento daacetilação de histona na regulação dessa proteína; (7) resveratrol, entretanto, pode reverter esse processo por alterar a afinidade química de Sir2 pelo lócus de Hsp22. / The effects of resveratrol and dietary restriction (DR) using lipids on demographic senescence, neuromotor response and Hsp22gene expression were analyzed in five strains of Drosophila melanogaster.In contrasttoDR employing the ratio of protein to carbohydrates as variable, the use of lipids modified both initial mortality rate and rate of aging.Females and males reached greater longevity on fat-free medium and on medium containing moderate quantity of lipids respectively. DR did not improve the performance of males in negative geotaxis assay and addition of lipids caused a decrease in fertility in females.Effects of resveratrol on longevity and mortality varied, but it was able to delay neuromotor deterioration in all strains, except on Sir-2 mutant.In general, there was occurrence of larger Hsp22puffs in lavae reared onhypercaloric medium control and on hypocaloric medium treated with resveratrol. These results indicate that: (1) neither energy content nor ratio of proteins to carbohydrates can fully predict the longevity of D. melanogaster; (2) the effects of dietary lipids on this organism are sex dependent and appear to be slightly toxic, with hormetic effect in males; (3) the mechanisms that influence longevity and functional senescence are, atleast partially, distinct; (4) resveratrol can delay functional locomotive decline and its effects on longevity and mortality depend on its concentration, on the strain ́sgenotype, sex and dilution of the medium, appearing to be involved with Sir2; (5) absence of functional Sir2 allele can upregulate Hsp22, which can explain the greater longevity of Sir2-mutants, despite its sirtuin ́s inactivity; (6) increased level of Sir2 induced by DR may inhibit the expression of Hsp22, supporting the hypothesis thathistone acetylation is involved in regulating this protein; (7) resveratrol, however, can reverse this process by altering the chemical affinity of Sir2 to Hsp22 ́s locus.

La reperméabilisation par thrombolyse, angioplastie ou chirurgie cardiaque des artères coronaires au décours d’un épisode d’ischémie myocardique est à l’origine d’une réduction spectaculaire de la morbi-mortalité de l’insuffisance coronaire aiguë. Elle est cependant la cause de lésions supplémentaires. La recherche de nouvelles approches cardioprotectrices complémentaires des méthodes de reperfusion actuelles est donc indispensable pour réduire les conséquences de l’ischémie myocardique. La découverte de puissants mécanismes cardioprotecteurs endogènes qui consistent en de brefs épisodes d’ischémie-reperfusion (IR) réalisés avant l’ischémie (pré-conditionnement ischémique, PCI) ou lors de la reperfusion (post-conditionnement ischémique) a permis de déterminer les mécanismes intervenant dans l’établissement des lésions d’IR. Le rôle prépondérant de la mitochondrie et l’augmentation de la perméabilité de ses membranes notamment via l’ouverture du pore de transition de perméabilité mitochondriale (mPTP) a pu être mis en évidence.Depré et al. (2001) ont décrit une stratégie cardioprotectrice novatrice : la surexpression de la protéine Hsp22 chez la souris protège le myocarde de l’infarctus et induit l’hypertrophie de ce dernier. Cette protéine est en effet capable d’activer des acteurs clés de la survie cellulaire notamment la voie des NO synthases qui est fortement impliquée dans le processus de PCI.Dans un premier temps, nous avons étudié les effets d’Hsp22 sur les fonctions mitochondriales et analysé le rôle du NO dans ces effets à l’aide d’un modèle murin sur-exprimant Hsp22 dans le myocarde. Nous avons montré qu’à l’état basal, la sur-expression d’Hsp22 augmente la production mitochondriale de NO, ce qui stimule la phosphorylation oxydative et s’accompagne de l’augmentation de la production d’espèces réactives de l’oxygène (ERO) par le complexe I de la chaîne respiratoire mitochondriale. Cette sur-expression réduit également la capacité maximale des complexes I et III à produire des ERO et limite l’ouverture du mPTP. Après anoxie, la sur-expression d’ Hsp22 exacerbe la diminution de phosphorylation oxydative par un mécanisme dépendant du NO et réduit la surproduction d’ERO par la chaîne respiratoire. Ces caractéristiques sont semblables à celles conférées par le PCI. La protéine Hsp22 induit donc au niveau mitochondrial des modifications qui pourraient participer à son effet cardioprotecteur. Une étude sur un modèle d’IR in vivo a permis de confirmer qu’Hsp22 limite fortement la taille de l’infarctus et de montrer que cet effet est associé à une réduction de l’atteinte des fonctions mitochondriales après IR. Les ERO sont des médiateurs clés dans le PCI mais également dans le développement de l’hypertrophie myocardique et du vieillissement prématuré. Or, Hsp22 stimule la production d’ERO mitochondriales, induit une hypertrophie myocardique et un effet cardioprotecteur similaire au PCI. Nous avons donc étudié le rôle des ERO dans les effets induits par Hsp22. Nos résultats ont montré qu’Hsp22 active les principales sources d’ERO cellulaires dans le myocarde aboutissant à un stress oxydant. Cet effet est associé à une forte réduction de la durée de vie des animaux sur-exprimant Hsp22 et à l’apparition de marqueurs de vieillissement prématuré dans le myocarde. Un traitement antioxydant permet de réduire cette sur-production d’ERO ainsi que l’hypertrophie myocardique et de rétablir l’espérance de vie des animaux transgéniques. Enfin, l’effet cardioprotecteur induit par Hsp22 est maintenu avec l’âge et n’est pas dépendant des ERO.En conclusion, l’effet cardioprotecteur durable induit par Hsp22 est associé à une protection des fonctions mitochondriales NO dépendante mais s’accompagne d’un stress oxydant responsable de l’hypertrophie myocardique et de la réduction de la durée de vie. Un traitement antioxydant est capable d’inhiber les effets délétères induits pas Hsp22 sans affecter son effet cardioprotecteur. / Development of reperfusion strategies such as thrombolysis, angioplasty and cardiac surgery to restore blood flow after myocardial ischemia is responsible for a spectacular reduction in deleterious consequences resulting from acute coronary syndrome. However reperfusion itself causes supplementary lesions. Research for new complementary cardioprotective strategies is needed to reduce the impact of myocardial ischemia. The discovery of powerful intrinsic cardioprotective processes consisting in repeated short cycles of ischemia-reperfusion (IR) before the ischemic episode (ischemic preconditioning) or at the moment of the reperfusion (ischemic postconditioning) has allowed to analyze the mechanisms involved in IR lesions and highlighted a crucial role of mitochondria and more particularly of the increase in its membrane permeability via the opening of the mitochondrial permeability transition pore (mPTP). Dr C. Depré et al. (2001) demonstrated that the over-expression of Hsp22 protein coding gene which induced myocardial hypertrophy protected from myocardial infarction. The mechanism of this innovative cardioprotective strategy is not fully understood but Hsp22 promotes the activation of cellular survival pathways such as the NO synthase pathway which is also involved in ischemic preconditioning (Depré et al., 2006).The goal of the first part of our study was to evaluate the effects of Hsp22 on mitochondrial functions and the role of NO in these effects using a transgenic mouse model overexpressing Hsp22 in the heart. Our results showed that Hsp22 overexpression increases mitochondrial NO production which stimulated oxidative phosphorylation in basal state. This was accompanied by an increased in reactive oxygen species (ROS) production by mitochondrial respiratory chain complex I. This overexpression also reduced the maximal capability of complex I and III to produce ROS production and limited mPTP opening. After anoxia, Hsp22 overexpression increaseed oxidative phosphorylation inhibition by a NO-dependent mechanism and limited the burst of ROS production from the respiratory chain.. Thus, Hsp22 modulates mitochondrial functions and this could participate to its cardioprotective effect as these characteristics replicate those of ischemic preconditioning. In the next step, we confirmed that Hsp22 overexpression highly reduced infarct size in an in vivo model of IR and showed that this was associated with a better preservation of mitochondrial functions.As ROS are key mediators of preconditioning but also of myocardial hypertrophy and aging and Hsp22 stimulates mitochondrial ROS production, induces a myocardial hypertrophy and a cardioprotective effect replicating preconditioning, we explored the role of ROS in Hsp22-induced effects in the last part of the study. Our results showed that Hsp22 overexpression activated major cellular sources of ROS leading to myocardial oxidative stress. This was associated with an extensive reduction of lifespan and the appearance of aging markers in the myocardium of young transgenic mice. Antioxidant treatment reduced the overproduction of ROS induced by Hsp22, decreased myocardial hypertrophy and restored lifespan in Hsp22 overexpressing mice showing the role of ROS in these effects. Finally, the cardioprotective effect induced by Hsp22 was maintained in old mice and was not dependent of ROS production. In conclusion, long-lasting cardioprotective effect induced by Hsp22 is associated with a NO-dependent preservation of mitochondrial functions and an oxidative stress responsible for myocardial hypertrophy and reduced lifespan. Antioxidant treatment is able to inhibit deleterious consequences of Hsp22 overexpression without affecting its cardioprotective effect.

Submitted by ANA CLAUDIA SILVA BRAGA null (anabragga@gmail.com) on 2017-08-31T16:15:53Z No. of bitstreams: 1 Tese Doutorado Ana Claudia Silva Braga.pdf: 4552779 bytes, checksum: 456de4a6fbfd60347292d8755d6c8c47 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-09-01T14:16:37Z (GMT) No. of bitstreams: 1 braga_acs_dr_sjrp.pdf: 4552779 bytes, checksum: 456de4a6fbfd60347292d8755d6c8c47 (MD5) / Made available in DSpace on 2017-09-01T14:16:37Z (GMT). No. of bitstreams: 1 braga_acs_dr_sjrp.pdf: 4552779 bytes, checksum: 456de4a6fbfd60347292d8755d6c8c47 (MD5) Previous issue date: 2017-08-04 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O vírus da hepatite C (HCV) causa a doença da Hepatite C e estima-se que cerca de 3% da população mundial esteja infectada com o vírus. A infecção por HCV promove a alteração na expressão de várias proteínas celulares. Estudos têm demonstrado que muitas proteínas de choque térmico (HSPs) possuem um perfil de expressão alterado na presença do vírus e algumas HSPs interagem diretamente com proteínas do HCV. Assim, o presente estudo teve como objetivo avaliar in vitro os níveis de expressão de proteínas de choque térmico na presença e ausência de HCV. Com este propósito, células de hepatoma humano Huh7.5 e células Huh7.5 infectadas com o vírus (HCV JFH-1) foram submetidas à extração de RNA e síntese de cDNA. A expressão diferencial de 84 HSPs e chaperonas foi avaliada por qPCR Array. Os resultados demonstram que cinco genes apresentaram expressão aumentada (em Log2 2), enquanto outros cinco apresentaram expressão reduzida. Para validar estes resultados os 10 genes diferencialmente expressos foram testados por qPCR em três modelos celulares para o HCV: células contendo replicon subgenômico do HCV (SGR-JFH-1), células infectadas com JFH-1 (ambos do genótipo 2a) e células contendo o replicon subgenômico S52 (genótipo 3). O gene HSPB8 mostrou expressão aumentada nos três modelos testados, condizente com os resultados obtidos por qPCR Array. Em seguida, promovemos o silenciamento de HSPB8 e foi observado um aumento na replicação viral. Em contraste, quando aumentamos a expressão de HSPB8, o HCV teve uma diminuição na taxa de replicação. O mesmo procedimento foi adotado para o gene DNAJC5B, validado no modelo viral genótipo 3, e o HCV mostrou padrão de replicação semelhante ao observado para o gene anterior. Esses resultados sugerem que HSPB8 pode atuar como um fator intracelular contra a replicação do vírus da hepatite C e DNAJC5B apresenta a mesma função, mas específico para o genótipo 3. Também avaliamos interações diretas com proteínas do HCV e os resultados demonstraram uma interação física entre a proteína NS4B de HCV e HSPB8. Esses resultados podem contribuir para uma melhor compreensão dos mecanismos envolvidos na replicação do HCV. / Hepatitis C virus (HCV) causes Hepatitis C disease and it is estimated that about 3% of world population are infected with the virus. HCV infection promotes alteration in the expression of several cellular proteins. Studies have shown that many heat shock proteins (HSPs) have an altered expression profile in the presence of the virus and some HSPs interact directly with HCV proteins. Thus, the present study aimed to evaluate in vitro the expression levels of heat shock proteins in the presence and absence of HCV. With this purpose, human hepatoma Huh7.5 cells and Huh7.5 cells infected with the virus (HCV JFH-1) were subjected to RNA extraction and cDNA synthesis. The differential expression of 84 HSPs and chaperones was assessed by qPCR Array. The results demonstrate that five genes showed increased expression (over Log2 2), while five other presented reduced expression. To validate these results, the 10 differentially expressed genes were tested by real-time PCR in three different HCV cell culture models: subgenomic HCV replicon cells (SGR-JFH-1), JFH-1 infected cells (both genotype 2a) and subgenomic S52 cells (genotype 3). The HSPB8 gene showed increased expression in all of three tested models, consistent with qPCR Array results. Then we promoted the silencing of HSPB8 and observed an increase in viral replication. In contrast, when we increased an expression of HSPB8, HCV had a decrease in replication rate. The same procedure was adopted for the DNAJC5B, validated in the viral model genotype 3, and HCV showed replication pattern similar to that observed for the previous gene. These results suggest that HSPB8 may act as an intracellular factor against hepatitis C virus replication and DNAJC5B have the same function, but genotype 3 specific. We also evaluated direct interactions with HCV proteins and the results demonstrated a physical interaction between the HCV NS4B protein with HSPB8. These results can contribute for a better understanding of the mechanisms involved in HCV replication. / FAPESP: 2013/17253-9

HSP22 et HSP70 sont des protéines d'origine humaine appartenant à la famille des Heat Shock<br />Proteins. Cette thèse décrit des travaux visant à déterminer l'implication de ces deux protéines humaines de<br />choc thermique HSP70 et HSP22, dans les voies de réparation de l'ADN après stress. Ces protéines<br />possèdent, comme leur nom le sous-entend, la capacité d'être surexprimées par la cellule après choc<br />thermique mais aussi après une grande variété d'autres stress et dans de nombreuses maladies (Tavaria M et<br />al, 1996). Pour mieux connaître ces protéines, une étude structurale a été initiée. Les structures<br />tridimensionnelles de ces protéines ne sont pas connues ou seulement partiellement (Osipiuk J et al, 1999).<br />Les essais de cristallisation des protéines furent arrêtés après l'obtention de clones. Des prédictions de<br />structures secondaires et tertiaires (obtenues à l'aide de la bioinformatique) concernant HSP70-1 (partie Cterminale)<br />et HSP22 furent alors réalisées, montrant une organisation en feuillets β pour le domaine de liaison<br />au substrat de HSP70-1 et pour le domaine α-cristallin de HSP22. Les résultats obtenus dans le cadre de<br />l'étude des HSP dans les voies de la réparation de l'ADN ont montré que les HSP70 inductibles par le stress<br />(HSP72 et HSP70-1) sont impliquées dans ces voies cellulaires après différents stress (rayonnement X et UV,<br />champ magnétique, péroxyde d'hydrogène, cis-platine). HSP72 et HSP70-1 sont phosphorylées par les<br />kinases apparentées à la famille des Phosphatydil-Inositol-3-kinases ATM, ATR et DNA-PK (Sarkaria JN et<br />al, 1998) après radiations ionisantes et réalisent à ce moment un déplacement en deux phases entre le<br />cytoplasme et le noyau ; elles sont impliquées dans les stades précoces de la réparation. La même démarche<br />de recherche a été adoptée pour HSP22. HSP22 apparaît comme étant impliquée dans les voies de la<br />réparation de l'ADN aussi et plus particulièrement dans les voies de la réparation des cassures double brin<br />après irradiation par rayonnement X (Paull TT et Gellert M, 1998 ; Lobrich M et Jeggo PA, 2005). HSP22<br />forme des foci dans les cellules âgées suggérant un rôle pour HSP22 dans le vieillissement cellulaire chez<br />l'humain comme chez la mouche drosophile (Morrow G et al 2004). Une nouvelle méthode d'imagerie<br />Diffraction Enhanced Imaging (DEI ; Chapman D et al, 1997), visant à l'acquisition d'une information<br />histologique, développée au synchrotron ESRF, a été testée d'un point de vue radiobiologique sur des cultures<br />primaires de fibroblastes et de chondrocytes. L'irradiation par cette méthode n'a pas présenté de différence en<br />comparaison avec une irradiation par une source conventionnelle de cellules humaines issues de culture<br />primaire, avec la même dose de rayonnement X (Rothkamm K et Lobrich M, 2003). Dans les cellules<br />glioblastomales de rat, la réponse HSP70 est augmentée dans les conditions de la thérapie « synchrotron PATPlat<br />» (c'est-à-dire en cas de photoactivation du platine à l'aide du rayonnement synchrotron (Corde S et al,<br />2003 ; Biston MC et al, 2004)).