Exposure of cells to environmental or chemical stressors will initiate the heat shock response, which is mediated by heat shock proteins. Heat shock proteins are molecular chaperones which are classified by size into six main families: HSP100, HSP90, HSP70, HSP60, HSP40 and the small heat shock proteins (sHsps). The sHsp family members bind to denatured proteins and maintain them in a folding competent state such that they may be refolded by other molecular chaperones. <br /><br /> The present study examined the expression and function of two amphibian sHsps, namely, <em>Rana catesbeiana</em> HSP30 and <em>Xenopus laevis</em> HSP27. Initially, an antisense riboprobe was produced to study the mRNA accumulation of <em>Rana hsp30</em> in cultured tongue fibroblast (FT) cells. Results showed that <em>Rana hsp30</em> mRNA was optimally induced when maintained at 35&deg;C for 2 h. An antibody to the recombinant <em>Rana</em> HSP30 protein was also produced in order to study HSP30 protein accumulation in <em>Rana</em> FT cells. Analysis showed that <em>Rana</em> HSP30 was heat-inducible and accumulated maximally at 4 h when maintained at 35&deg;C and then allowed to recover at 22&deg;C for 2 h. Immunocytochemical analysis indicated that <em>Rana</em> HSP30 protein was present primarily in the nucleus, with diffuse localization in the cytoplasm. Additional immunocytochemical analysis showed that <em>Rana</em> HSP30 remained in the nucleus following heat stress and extended periods of recovery. <br /><br /> The molecular chaperone function of <em>Rana</em> HSP30 was also studied. Recombinant <em>Rana</em> HSP30 was found to inhibit the heat induced aggregation of various target proteins including citrate synthase, luciferase and malate dehydrogenase. Also, no major difference was detected between <em>Rana</em> HSP30 and <em>Xenopus</em> HSP30C in the inhibition of heat-induced aggregation of target proteins. <br /><br /> This study also examined the expression and function of <em>Xenopus laevis</em> HSP27. Analysis of the putative amino acid sequence of the <em>Xenopus hsp27</em> cDNA revealed that it had an identity of 71% with chicken, 65% with zebrafish, 63% with human and 53% with topminnow. Most of the identity was located within the &alpha;-crystallin domain of the protein. Interestingly, <em>Xenopus</em> HSP27 shared only a 19% identity with 2 other <em>Xenopus</em> sHsps, HSP30C and HSP30D. <br /><br /> Western blot analysis using an anti-<em>Xenopus</em> HSP27 antibody revealed that HSP27 was not detectable in cultured kidney epithelial cells. However, examination of early <em>Xenopus</em> embryos revealed that HSP27 was first detected in tadpole embryos (stage 44). Heat-inducible HSP27 was also first detected at this stage. The accumulation pattern of <em>Xenopus</em> HSP27 protein was distinct from <em>Xenopus</em> HSP30, which was heat-inducible at midtailbud stage 26, approximately two and a half days earlier in development. <br /><br /> Analysis of recombinant HSP27 by native pore exclusion limit electrophoresis showed that it formed high molecular weight, multimeric complexes. The molecular chaperone function of HSP27 was assessed by means of thermal aggregation assays employing citrate synthase, luciferase and malate dehydrogenase. <em>Xenopus</em> HSP27 inhibited the heat-induced aggregation of all of these target proteins. This study has revealed that <em>Xenopus</em> HSP27 is a member of the HSP27 subfamily of small heat shock proteins in <em>Xenopus</em> and distinct from the HSP30 family. The accumulation of HSP27 under constitutive and stress-inducible conditions is developmentally regulated. Finally, this sHsp appears to function as a molecular chaperone.

Insulin and cellular stressors both activate p38 MAPK. Insulin protects cardiac tissue in a p38 MAPK-dependent manner. Paradoxically, inhibiting p38 MAPK is also protective. Hsp27 phosphorylation is regulated by p38 MAPK. Insulin was tested in H9c2 cardiomyocytes subjected to media exchange, 6 hours of oxygen-glucose deprivation, and reoxygenation. Insulin suppressed stress-induced phosphorylation of Hsp27 due to media-exchange or oxygen-glucose deprivation. Surprisingly, insulin increased Hsp27 phosphorylation during reoxygenation. Insulin also reduced total p38 MAPK levels. Insulin before oxygen-glucose deprivation prevented both localization of Hsp27 to the nucleus and localization of phospho-p38 MAPK to the cytoplasm. Insulin during oxygen-glucose deprivation caused the localization of phospho-p38 MAPK in the cytoplasm, but did not increase Hsp27 phosphorylation until reoxygenation. In conclusion, insulin may protect before oxygen-glucose deprivation by redirecting phospho-p38 MAPK to the nucleus away from damaging pathways in the cytoplasm and protects during oxygen-glucose deprivation by priming phospho-p38 MAPK to phosphorylate Hsp27. / Insulin was used on a model on H9c2 myotubes to determine the effect of oxygen-glucose deprivation and reoxygenation on the localization and phosphorylation of Hsp27 and p38 MAPK

No description available.

Oxidative stress plays an important role in cardiac diseases, which has been well demonstrated, whereas the role of reductive stress has been poorly investigated. We and others have shown previously that heat shock protein 27 (Hsp27) plays a role as an antioxidant. To investigate whether overexpression of Hsp27 could lead to reductive stress and result in cardiomyopathy, we generated transgenic mice with different expression levels of Hsp27. We observed that transgenic mice with high levels of Hsp27 developed cardiomyopathy. The myopathic hearts were under reductive stress, which was evidenced by an increased ratio of reduced glutathione/oxidized glutathione and a decreased level of reactive oxygen species. In addition, upregulated glutathione peroxidase 1 and decreased iron content were revealed in the myopathic hearts. More importantly, inhibition of glutathione peroxidase 1 significantly attenuated the development of cardiomyopathy. The data indicate that the Hsp27-induced cardiomyopathy could be attributed to, at least in part, upregulation of glutathione peroxidase 1. Our findings suggest that reductive stress plays an important role in the development of cardiomyopathy and that Hsp27 may serve as a potential target for the treatment of patients with cardiomyopathy.

Hsp27 appartient à la famille des protéines dites de survie comme Bcl2 ou la survivine. C’est une protéine anti-apoptotique qui subit une dérégulation de son expression dans de nombreux types tumoraux. Elle est caractérisée comme étant une cible thérapeutique majeure. Au cours de ma thèse, j’ai isolé des peptides stabilisés, dit aptamères, capables d’inhiber fonctionnellement les activités anti-apoptotiques et tumorigènes d’Hsp27. Ces aptamères perturbent la biochimie structurale de Hsp27 et induisent le blocage du cycle cellulaire in vivo. Parallèlement à cette étude, j’ai caractérisé les effets de la déplétion de Hsp27 sur la formation de métastases et de tumeurs osseuses. J’ai aussi montré que la modification du taux de Hsp27 induisait la dégradation de différentes protéines, dites clientes, comme la caspase3, HDAC6 et STAT2. / Hsp27 belongs to the class of survival proteins like Bcl2 or survivin. This protein was well categorized as a major anti apoptotic protein as displaying a high level of expression in lot of tumor types. Moreover, Hsp27 is referenced as a major therapeutic target in cancer. During my PhD, I characterized stable peptides called aptamers, which functionally blocked Hsp27 antiapoptotic and tumorigenic properties. These aptamers disrupted biochemical and structural states of Hsp27 and promoted cell cycle arrest in xenografts. In the same time, I have characterized the effect of Hsp27 depletion in metastasis establishment and bone marrow tumor growth. I have shown that targeting level of Hsp27 induced degradation of several of its client proteins like caspase3, HDAC6 and STAT2

As proteínas de choque térmico (HSPs) são proteínas presentes nas células em condições normais e supra-expressadas em condições de estresse e choque térmico. O laser de diodo tem sido utilizado em diversos tratamentos na odontologia, dentre eles na atenuação de sintomas e na aceleração do reparo de úlceras traumáticas. O objetivo deste estudo foi verificar, através de análise imunoistoquímica da expressão das HSPs (Hsp27 e Hsp 47) e da medição de temperatura por intermédio de câmera termográfica, se o laser de diodo modifica, (e quanto modifica) a temperatura local, bem como se há alteração no padrão de expressão das proteínas citadas em três regiões distintas do fragmento analisado. Para este estudo, foram feitos testes in vitro e in vivo. Para os testes in vivo, foram utilizados 56 ratos, nos quais foi induzida úlcera em ventre lingual. Os animais foram divididos em 4 grupos: GL - grupo ulcerado com posterior irradiação com laser (parâmetros: laser diodo, 0,5 W de potência, pulsado (10 Hz), por 40 segundos (método varredura), 80J/cm2 de energia total (área de 0,25cm2) Má, e a energia por ponto?); GN - grupo ulcerado sem nenhum tratamento posterior; CP - grupo controle sem úlcera e irradiado com laser (mesmos parâmetros de GL); e CN - grupo controle sem úlcera e sem nenhum tratamento posterior. O teste in vitro foi destinado à medição da temperatura durante a irradiação laser, quando utilizaram-se línguas de ratos previamente ulceradas e extirpadas, as quais foram irradiadas com os parâmetros para o teste in vivo. Na superfície irradiada, houve aumento médio de temperatura em torno de 6,7C e, na região mais distante dessa superfície, de 2,7C. Na análise semiquantitativa da expressão imunoistoquímica da Hsp27, observou-se padrão de marcação mais intenso em GL comparando-se a GN e aos demais grupos. Na análise quantitativa da Hsp47, houve maior quantidade de células positivas no GL em relação aos demais grupos, principalmente nas regiões mais próximas da superfície irradiada. Concluiu-se que a irradiação laser provocou aumento de temperatura local, bem como desencadeou padrões de intensidade maiores e diferentes da Hsp27 e da Hsp47 em relação aos demais grupos. Isso indica que o tecido irradiado sofreu maior estresse celular, com resposta tecidual de intensa migração e diferenciação celular, bem como de maior síntese colagênica. / Heat shock proteins (HSPs) are proteins present in cells under normal conditions and over-expressed in terms of stress and thermal shock. The laser diode has been used in various treatments in dentistry, to reduce symptoms and accelerate the repair of traumatic ulcers. The aim of this study was to verify, by means of immunohistochemical expression analysis of HSPs (Hsp27 and Hsp 47) and temperature measurement using a thermographic camera, whether diode laser changes the local temperature (and how much the temperature changes), and whether there is a change in expression pattern of the above-mentioned protein in three distinct regions of the analyzed fragment. For this study, some of the tests were conducted in vitro and others in vivo. For the in vivo tests, 56 rats were used, in which ulcers were induced on the ventral surface of the tongue. The animals were divided into 4 groups: GL-group with ulcer and later laser irradiation (diode laser parameters: 0.5 W power, pulsed (10 Hz), for 40 seconds (defocused), 80J/cm2 total power in area (0.25cm2) GN- group with ulcer and without any further processing; CP- control group without ulcer and with laser irradiation (same parameters as in GL); and GN-control group without ulcer and without any further processing. The in vitro tests were conducted to measure temperature during laser irradiation, using the tongues that had previously been ulcerated in rats, and later removed and irradiated with the same parameters as those used for the in vivo test. In the irradiated surface, there was an increase in mean temperature of around 6.7oC, and in the region more distant from this surface, an increase of 2.7 oC. In the semi-quantitative analysis of immunohistochemical expression of Hsp27, greater expression of Hsp27 was shown in GL in comparison with the other groups in general. In quantitative analysis of Hsp47, there was a larger quantity of positive cells in GL, when compared with the other groups, particularly in the regions closest to irradiated surface. It was concluded that the irradiation laser caused local temperature increase, and also set off greater and different patterns of intensity of Hsp27 and Hsp47, in comparison with those of the other groups. This indicates that the irradiated tissue suffered higher cellular stress, with tissue response of intense migration and cell differentiation, as well as increased collagen synthesis.

Um dos objetivos no tratamento de Diabetes mellitus é aumentar a função, proliferação e sobrevivência de células beta pancreáticas, pois o transplante de ilhotas pancreáticas é severamente limitado pela escassez de doadores de órgãos. Nós mostramos que prolactina recombinante humana (rhPRL) apresenta efeitos benéficos em células beta, inibindo a apoptose. Recentemente reportamos o aumento dos níveis de expressão da proteína antiapoptótica HSP27/25, regulada por rhPRL em ilhotas humanas. Nosso objetivo é explorar o papel de HSP27/25 na citoproteção induzida por prolactina em células beta. Métodos: Células MIN6 parentais e silenciadas para HSP25 foram privadas de soro por 24h e cultivadas na presença ou ausência de rhPRL (300ng/mL). Posteriormente foram tratadas na presença ou ausência de uma combinação de citocinas (TNF-&#945;, INF-&#947 e IL-1&#946;) por diferentes períodos de tempo. A apoptose foi avaliada usando-se análise por citometria de fluxo. Os níveis de expressão gênica de HSP27/25 e membros da família BCL-2, da expressão proteica de membros desta mesma família, além de Caspase-9, Smac-Diablo HSP27/25, HSTF1, pP38 e pSTAT1 foram analisados por Westen blot. Ainda, foram avaliadas a atividade de Caspase-8 e -3, por ensaios fluorimétricos. Após o tratamento com rhPRL e citocinas, a proporção de núcleos fragmentados aumentou em células silenciadas para HSP25 (p<0,05), quando comparadas com células controles. A inibição da atividade de Caspase-3 e -8, tanto quanto os níveis de expressão proteica de caspase- 9, por rhPRL e o aumento da razão de expressão Bcl-2/Bax e BclxL/Bax foram revertidos em células silenciadas (p<0,05). Além disso, a cinética dos níveis de expressão de HSP27/25 e HSTF1 foram estudadas em culturas de ilhotas pancreáticas humanas, mostrando que enquanto HSTF1 apresenta um aumento significativo (p<0,01) nos níveis de expressão proteica após 10min de tratamento com rhPRL, HSP27 alcançou seu nível máximo após 2h do tratamento hormonal. Adicionalmente, foi detectado um aumento significante (p<0,05) nos níveis de fosforilação de STAT1 e de p38, após 10min, atingindo o pico de expressão após 30min (p<0,01) do tratamento com rhPRL. Estes estudos demonstram o papel chave de HSP27/25 sobre os efeitos citoprotetores induzidos por rhPRL, desde que a ausência desta proteína aboliu completamente os efeitos benéficos induzidos por PRL sobre a morte de células beta. Ainda, nós fornecemos pela primeira vez, evidências da corregulação de HSP27 e HSTF1 induzidas por rhPRL em células humanas pancreáticas, que pode estar sendo mediada pela ativação de STAT1 e p38. Coletivamente, nossos resultados podem conduzir para a atenuação da morte de células beta por meio da regulação de uma via de proteção endógena, a qual é independente da modulação do sistema imunológico. / One of the goals in Diabetes mellitus treatment is to enhance pancreatic &#946;-cell function, proliferation and survival since transplantation of pancreatic islets is severely limited by shortage of organ donors. We have shown that recombinant human prolactin (rhPRL) inhibits beta-cell apoptosis. We have recently reported PRL-induced up-regulation of the anti-apoptotic HSP25/27 in human islets. Since the function of HSP25/27 in beta-cells has not been directly studied, we set out to explore the role of HSP25/27 in prolactin-induced beta-cell cytoprotection. Methods: Parental and HSP25 knocked-down Min6 cells were serumstarved for 24h and then cultured in the presence or in the absence of rhPRL (300ng/mL) for different time periods. Apoptosis was evaluated using flow cytometry analysis. Levels of Bcl-2 gene family members, caspase-9, Bcl-2, BclxL, Bax, Smac-Diablo, HSP27/25, HSTF1, pStat- 1,pP38 were studied by western blot. Caspase-3 and -8 activity, were evaluated by fluorimetric assays. Upon cytokines and rhPRL treatment, the proportion of fragmented nuclei was increased in HSP25 silenced cells (p<0.05) when compared to control cells. The inhibition of cytokine-induced capase-3 and -8 activity as well as Bcl-2/Bax and BclxL/Bax ratios and caspase-9 protein levels mediated by rhPRL in wild type cells was reverted in knocked-down cells (p<0.05). Moreover, the kinetics of HSP 25/27 and HSTF1 expression levels studied in primary cultures of human pancreatic islets showed that while HSTF1 presented a significant increase (p<0.01) in protein expression level after 10 min of rhPRL treatment, HSP27 reached its maximum expression level upon 2h of hormonal treatment. Additionally, a significant (p<0.05) increase in both P38 and STAT1 phosphorylation levels were detected after 10min of rhPRL treatment reaching the highest levels upon 30 min (p<0.001) of hormonal treatment. Therefore, we demonstrated a key role for HSP25/27 in rhPRL-induced cytoprotective effects, since the lack of this protein completely abolished the beneficial effects induced by PRL on beta-cell death. Moreover, we provided for the first time, evidence for the co-regulation of HSP27 and HSTF1 induced by rhPRL in human pancreatic cells which could be mediated by activated STAT1 and P38. Collectively, our results could lead to the mitigation of beta-cell death through the up-regulation of an endogenous protective pathway which is independent on the modulation of the immune system.

As proteínas de choque térmico (HSPs) são proteínas presentes nas células em condições normais e supra-expressadas em condições de estresse e choque térmico. O laser de diodo tem sido utilizado em diversos tratamentos na odontologia, dentre eles na atenuação de sintomas e na aceleração do reparo de úlceras traumáticas. O objetivo deste estudo foi verificar, através de análise imunoistoquímica da expressão das HSPs (Hsp27 e Hsp 47) e da medição de temperatura por intermédio de câmera termográfica, se o laser de diodo modifica, (e quanto modifica) a temperatura local, bem como se há alteração no padrão de expressão das proteínas citadas em três regiões distintas do fragmento analisado. Para este estudo, foram feitos testes in vitro e in vivo. Para os testes in vivo, foram utilizados 56 ratos, nos quais foi induzida úlcera em ventre lingual. Os animais foram divididos em 4 grupos: GL - grupo ulcerado com posterior irradiação com laser (parâmetros: laser diodo, 0,5 W de potência, pulsado (10 Hz), por 40 segundos (método varredura), 80J/cm2 de energia total (área de 0,25cm2) Má, e a energia por ponto?); GN - grupo ulcerado sem nenhum tratamento posterior; CP - grupo controle sem úlcera e irradiado com laser (mesmos parâmetros de GL); e CN - grupo controle sem úlcera e sem nenhum tratamento posterior. O teste in vitro foi destinado à medição da temperatura durante a irradiação laser, quando utilizaram-se línguas de ratos previamente ulceradas e extirpadas, as quais foram irradiadas com os parâmetros para o teste in vivo. Na superfície irradiada, houve aumento médio de temperatura em torno de 6,7C e, na região mais distante dessa superfície, de 2,7C. Na análise semiquantitativa da expressão imunoistoquímica da Hsp27, observou-se padrão de marcação mais intenso em GL comparando-se a GN e aos demais grupos. Na análise quantitativa da Hsp47, houve maior quantidade de células positivas no GL em relação aos demais grupos, principalmente nas regiões mais próximas da superfície irradiada. Concluiu-se que a irradiação laser provocou aumento de temperatura local, bem como desencadeou padrões de intensidade maiores e diferentes da Hsp27 e da Hsp47 em relação aos demais grupos. Isso indica que o tecido irradiado sofreu maior estresse celular, com resposta tecidual de intensa migração e diferenciação celular, bem como de maior síntese colagênica. / Heat shock proteins (HSPs) are proteins present in cells under normal conditions and over-expressed in terms of stress and thermal shock. The laser diode has been used in various treatments in dentistry, to reduce symptoms and accelerate the repair of traumatic ulcers. The aim of this study was to verify, by means of immunohistochemical expression analysis of HSPs (Hsp27 and Hsp 47) and temperature measurement using a thermographic camera, whether diode laser changes the local temperature (and how much the temperature changes), and whether there is a change in expression pattern of the above-mentioned protein in three distinct regions of the analyzed fragment. For this study, some of the tests were conducted in vitro and others in vivo. For the in vivo tests, 56 rats were used, in which ulcers were induced on the ventral surface of the tongue. The animals were divided into 4 groups: GL-group with ulcer and later laser irradiation (diode laser parameters: 0.5 W power, pulsed (10 Hz), for 40 seconds (defocused), 80J/cm2 total power in area (0.25cm2) GN- group with ulcer and without any further processing; CP- control group without ulcer and with laser irradiation (same parameters as in GL); and GN-control group without ulcer and without any further processing. The in vitro tests were conducted to measure temperature during laser irradiation, using the tongues that had previously been ulcerated in rats, and later removed and irradiated with the same parameters as those used for the in vivo test. In the irradiated surface, there was an increase in mean temperature of around 6.7oC, and in the region more distant from this surface, an increase of 2.7 oC. In the semi-quantitative analysis of immunohistochemical expression of Hsp27, greater expression of Hsp27 was shown in GL in comparison with the other groups in general. In quantitative analysis of Hsp47, there was a larger quantity of positive cells in GL, when compared with the other groups, particularly in the regions closest to irradiated surface. It was concluded that the irradiation laser caused local temperature increase, and also set off greater and different patterns of intensity of Hsp27 and Hsp47, in comparison with those of the other groups. This indicates that the irradiated tissue suffered higher cellular stress, with tissue response of intense migration and cell differentiation, as well as increased collagen synthesis.

Um dos objetivos no tratamento de Diabetes mellitus é aumentar a função, proliferação e sobrevivência de células beta pancreáticas, pois o transplante de ilhotas pancreáticas é severamente limitado pela escassez de doadores de órgãos. Nós mostramos que prolactina recombinante humana (rhPRL) apresenta efeitos benéficos em células beta, inibindo a apoptose. Recentemente reportamos o aumento dos níveis de expressão da proteína antiapoptótica HSP27/25, regulada por rhPRL em ilhotas humanas. Nosso objetivo é explorar o papel de HSP27/25 na citoproteção induzida por prolactina em células beta. Métodos: Células MIN6 parentais e silenciadas para HSP25 foram privadas de soro por 24h e cultivadas na presença ou ausência de rhPRL (300ng/mL). Posteriormente foram tratadas na presença ou ausência de uma combinação de citocinas (TNF-&#945;, INF-&#947 e IL-1&#946;) por diferentes períodos de tempo. A apoptose foi avaliada usando-se análise por citometria de fluxo. Os níveis de expressão gênica de HSP27/25 e membros da família BCL-2, da expressão proteica de membros desta mesma família, além de Caspase-9, Smac-Diablo HSP27/25, HSTF1, pP38 e pSTAT1 foram analisados por Westen blot. Ainda, foram avaliadas a atividade de Caspase-8 e -3, por ensaios fluorimétricos. Após o tratamento com rhPRL e citocinas, a proporção de núcleos fragmentados aumentou em células silenciadas para HSP25 (p<0,05), quando comparadas com células controles. A inibição da atividade de Caspase-3 e -8, tanto quanto os níveis de expressão proteica de caspase- 9, por rhPRL e o aumento da razão de expressão Bcl-2/Bax e BclxL/Bax foram revertidos em células silenciadas (p<0,05). Além disso, a cinética dos níveis de expressão de HSP27/25 e HSTF1 foram estudadas em culturas de ilhotas pancreáticas humanas, mostrando que enquanto HSTF1 apresenta um aumento significativo (p<0,01) nos níveis de expressão proteica após 10min de tratamento com rhPRL, HSP27 alcançou seu nível máximo após 2h do tratamento hormonal. Adicionalmente, foi detectado um aumento significante (p<0,05) nos níveis de fosforilação de STAT1 e de p38, após 10min, atingindo o pico de expressão após 30min (p<0,01) do tratamento com rhPRL. Estes estudos demonstram o papel chave de HSP27/25 sobre os efeitos citoprotetores induzidos por rhPRL, desde que a ausência desta proteína aboliu completamente os efeitos benéficos induzidos por PRL sobre a morte de células beta. Ainda, nós fornecemos pela primeira vez, evidências da corregulação de HSP27 e HSTF1 induzidas por rhPRL em células humanas pancreáticas, que pode estar sendo mediada pela ativação de STAT1 e p38. Coletivamente, nossos resultados podem conduzir para a atenuação da morte de células beta por meio da regulação de uma via de proteção endógena, a qual é independente da modulação do sistema imunológico. / One of the goals in Diabetes mellitus treatment is to enhance pancreatic &#946;-cell function, proliferation and survival since transplantation of pancreatic islets is severely limited by shortage of organ donors. We have shown that recombinant human prolactin (rhPRL) inhibits beta-cell apoptosis. We have recently reported PRL-induced up-regulation of the anti-apoptotic HSP25/27 in human islets. Since the function of HSP25/27 in beta-cells has not been directly studied, we set out to explore the role of HSP25/27 in prolactin-induced beta-cell cytoprotection. Methods: Parental and HSP25 knocked-down Min6 cells were serumstarved for 24h and then cultured in the presence or in the absence of rhPRL (300ng/mL) for different time periods. Apoptosis was evaluated using flow cytometry analysis. Levels of Bcl-2 gene family members, caspase-9, Bcl-2, BclxL, Bax, Smac-Diablo, HSP27/25, HSTF1, pStat- 1,pP38 were studied by western blot. Caspase-3 and -8 activity, were evaluated by fluorimetric assays. Upon cytokines and rhPRL treatment, the proportion of fragmented nuclei was increased in HSP25 silenced cells (p<0.05) when compared to control cells. The inhibition of cytokine-induced capase-3 and -8 activity as well as Bcl-2/Bax and BclxL/Bax ratios and caspase-9 protein levels mediated by rhPRL in wild type cells was reverted in knocked-down cells (p<0.05). Moreover, the kinetics of HSP 25/27 and HSTF1 expression levels studied in primary cultures of human pancreatic islets showed that while HSTF1 presented a significant increase (p<0.01) in protein expression level after 10 min of rhPRL treatment, HSP27 reached its maximum expression level upon 2h of hormonal treatment. Additionally, a significant (p<0.05) increase in both P38 and STAT1 phosphorylation levels were detected after 10min of rhPRL treatment reaching the highest levels upon 30 min (p<0.001) of hormonal treatment. Therefore, we demonstrated a key role for HSP25/27 in rhPRL-induced cytoprotective effects, since the lack of this protein completely abolished the beneficial effects induced by PRL on beta-cell death. Moreover, we provided for the first time, evidence for the co-regulation of HSP27 and HSTF1 induced by rhPRL in human pancreatic cells which could be mediated by activated STAT1 and P38. Collectively, our results could lead to the mitigation of beta-cell death through the up-regulation of an endogenous protective pathway which is independent on the modulation of the immune system.

La régénération tissulaire est une voie prometteuse de thérapie dans le cadre des maladies dégénératives. Dans ce but sont conçus les microcarriers pharmacologiquement actifs (PAM). Ce sont des microsphères fournissant un environnement adéquat à la survie et la différenciation de cellules souches par la libération d’un facteur de croissance protéique encapsulé.Pour potentialiser l’intérêt des PAM, les microsphères doivent (1) permettre la libération complète et prolongée de la protéine (2) être formulées sans solvant halogéné par un procédé transposable à l’échelle pilote.Deux stratégies sont menées afin d’améliorer la stabilité et la libération de la protéine. La première consiste à utiliser de nouveaux additifs. Une étude bibliographique révèle le potentiel d’additifs protéiques ; leur application a permis d’augmenter significativement l’activité biologique de la protéine libérée. La seconde stratégie consiste à moduler la matrice de copolymère PLGAP188-PLGA. La modification de ses propriétés physicochimiques (Mw, hydrophobie…) a permis d’accéder à la compréhension de la structure des microsphères et d’obtenir une libération continue.Le développement du procédé de fabrication des microsphères sans solvant toxique associe la technique du prilling avec le glycofurol comme solvant. Cette combinaison se heurte à de nombreux verrous technologiques. La mise au point du procédé a été réalisée à l’aide de plans d’expériences. Ils ont conduit à la production de particules grâce à la modélisation des propriétés physicochimiques du milieu de réception et à la prise en compte des différents paramètres du procédé. / Pharmacologically active microcarriers (PAM) have been developed as innovative tools for tissue regeneration. This microspherical platform provided an environment for the survival and the differentiation of stem cells through the release of encapsulated protein growth factor. To improve the therapeutic efficacy of the PAM, the microspheres have to (1) provide the full and sustained release of the protein (2) be formulated without halogenated solvent by a process with an easy scale-up. The protein release has been studied through two strategies. The first one was to look for a preservation of the biological activity of the protein during the release. A literature review highlighted protein additives. Some of them were incorporated into the microspheres and increased significantly the protein release. The second one was the modulation of the matrix copolymer PLGAP188-PLGA. The modification of its properties (MW,hydrophobicity) permitted to reach a continuous release and to understand the structure of the microspheres. The prilling technique and the use of glycofurol provide an easy transferable process without toxic solvent. Experimental designs were performed to overcome the technological barriers. Through the modeling the physicochemical properties of the reception medium and the study of the process parameters, the formulation has been improved to produce acceptable particles.