Οι Heat Shock Proteins (Hsps) αποτελούν πολλές διαφορετικές οικογένειες που επάγονται προς απάντηση μιας ευρείας σειράς φυσιολογικών και περιβαλλοντικών ερεθισμάτων. Μια τέτοια πρωτεΐνη, η οποία επάγεται ιδιαίτερα κατά τη διάρκεια της απάντησης σε stress, είναι η Hsp27, με ΜΒ:27kDa, της οποίας η έκφραση φαίνεται να σχετίζεται με αυξημένη επιβίωση των κυττάρων όταν επιδρούν σε αυτά κυτταροτοξικά ερεθίσματα. Έχει φανεί ότι εμποδίζει τον κυτταρικό θάνατο μέσω αλληλεπίδρασης με ποικιλία παραγόντων που προκαλούν απόπτωση. Η Hsp27 είναι μια μοριακή πρωτεΐνη chaperone με ικανότητα να αλληλεπιδρά με μεγάλο αριθμό πρωτεϊνών. Πρόσφατα στοιχεία έχουν δείξει ότι η Hsp27 ρυθμίζει την απόπτωση, χάρη στην ικανότητα που έχει να διαντιδρά με μόρια ¨κλειδιά¨ στο μονοπάτι μεταβίβασης σημάτων της απόπτωσης και ιδιαίτερα, με εκείνα που ενέχονται στην ενεργοποίηση των κασπασών. Οι άλλοι δυο ρόλοι της Hsp27 κατά τη διάρκεια του stress που αποσκοπούν στην κυτταροπροστασία, είναι αφενός να αλληλεπιδρά και να σταθεροποιεί τον κυτταροσκελετό, αφετέρου να ρυθμίζει το ενδοκυττάριο επίπεδο των τοξικών ριζών οξυγόνου. Όταν τα κύτταρα βρίσκονται σε stress τα επίπεδα της Hsp27 είναι γενικώς χαμηλά και αυτή υπάρχει κυρίως με τη μορφή μεγάλων ολιγομερών. Κατά τη διάρκεια της απάντησης στο stress έχουμε αύξηση στα επίπεδα έκφρασης της Hsp27, που συνδυάζεται με μια αναδιοργάνωσή της λόγω φωσφορυλίωσης. Η φωσφορυλίωση μπορεί να συμβεί σε τρεις διαφορετικές θέσεις που εντοπίζονται σερίνες (Ser15, Ser78, Ser82). Η φωσφορυλίωση της Hsp27 καταλήγει σε ανακατανομή των μεγάλων ολιγομερών σε μικρότερα σύμπλοκα. ΣΚΟΠΟΣ: Αντικείμενο της μελέτης μας είναι ακριβώς η έκφραση των φωσφορυλιωμένων μορφών της Hsp27 με μονοπύρηνα κύτταρα περιφερικού αίματος φυσιολογικών ενηλίκων και οι διαφορές που εμφανίζονται όταν τα κύτταρα βρίσκονται in vitro σε βασικές συνθήκες, σε συνθήκες επίδρασης θερμικού stress και σε συνθήκες επίδρασης του παράγοντα TNF-a. ΥΛΙΚΑ ΚΑΙ ΜΕΘΟΔΟΙ: Εφαρμόστηκε η τεχνική της SDS-PAGE ηλεκτροφόρησης και επακόλουθα της WESTERN BLOTTING για την ανίχνευση των πρωτεϊνών. Τα πρώτα πειράματα πραγματοποιήθηκαν σε πρωτεϊνικό εκχύλισμα της κυτταρικής σειράς λευχαιμικών κυττάρων U937 με και χωρίς τη χορήγηση Doxorubicin.Τα επόμενα πειράματα πραγματοποιήθηκαν σε εκχύλισμα πρωτεϊνών μονοπύρηνων κυττάρων περιφερικού αίματος προελεύσεως από ένα τυχαίο δείγμα 6 ενηλίκων ανδρών-γυναικών ηλικίας από 20-30 ετών. Τα τελευταία πειράματα πραγματοποιήθηκαν σε πρωτεϊνικό εκχύλισμα μονοπύρηνων κυττάρων περιφερικού αίματος, το οποίο προερχόταν από ένα τυχαίο δείγμα 7 φυσιολογικών ανδρών-γυναικών ηλικίας 20-30 ετών. Τα κύτταρα αυτά είχαν χωριστεί σε τρεις υποπληθυσμούς ανάλογα με τις συνθήκες που είχαν υποβληθεί: βασικές συνθήκες, συνθήκες επίδρασης θερμικού stress και συνθήκες επίδρασης TNF-a. Ο αρχικός διαχωρισμός των μονοπύρηνων κυττάρων περιφερικού αίματος έγινε με Ficoll. ΑΠΟΤΕΛΕΣΜΑΤΑ: Τα πρώτα πειράματα έγιναν σε πρωτεϊνικό εκχύλισμα κυττάρων της σειράς U937 τα οποία είχαν δεχθεί την επίδραση TNF-a. Η Western Blotting ανάλυση ανέδειξε την παρουσία φωσφορυλιωμένης Hsp27 για τη θέση Ser78,αλλά όχι για τις θέσεις Ser15 και Ser 82. Όταν όμως η ίδια μελέτη πραγματοποιήθηκε σε πρωτεϊνικό εκχύλισμα της ίδιας σειράς που είχαν δεχθεί την επίδραση Doxorubicin αναδείχθηκε η παρουσία φωσφορυλιωμένης Hsp27 για τις θέσεις Ser15 και Ser82. Τα πειράματα που πραγματοποιήθηκαν σε πρωτεϊνικό εκχύλισμα μονοπύρηνων κυττάρων υγιών ενηλίκων, τα οποία δεν είχαν υποστεί απολύτως καμμία επίδραση, ανέδειξαν την παρουσία και των τριών μορφών φωσφορυλιωμένης Hsp27 καθώς και της ολικής μη φωσφορυλιωμένης μορφής σε όλα τα δείγματα. Στα τελευταία πειράματα που έγιναν σε πρωτεϊνικό εκχύλισμα μονοπύρηνων κυττάρων περιφερικού αίματος -που είχαν διαιρεθεί σε τρεις πληθυσμούς ανάλογα με τις συνθήκες που είχαν επιδράσει- ανέδειξαν τα εξής: Η φωσφορυλιωμένη μορφή της Hsp27 για τη θέση Ser78 και τη θέση Ser82 επαγόταν και στους τρεις υποπληθυσμούς κυττάρων, δηλαδή όταν αυτά βρισκόντουσαν υπό βασικές συνθήκες, υπό συνθήκες θερμικού shock και υπό συνθήκες επίδρασης TNF-a. Όσον αφορά τη φωσφορυλιωμένη μορφή της Hsp27 για τη θέση Ser15 διαπιστώθηκε ότι επαγόταν περισσότερο η έκφρασή της σε συνθήκες επίδρασης TNF-a και θερμικού shock από ότι στις βασικές συνθήκες. Η ολική Hsp27, η μη φωσφορυλιωμένη, δεν αναδείχθηκε σε κανέναν από τους τρεις υποπληθυσμούς κυττάρων, σε όσα πειράματα πραγματοποιήθηκαν. ΣΥΖΗΤΗΣΗ: Από τα αποτελέσματα των πρώτων πειραμάτων στα κύτταρα της σειράς U937 προέκυψε η βάσιμη υποψία ότι η επίδραση κάποιου στρεσσογόνου παράγοντα είχε διαφορετική επίδραση στην Hsp27,ανάλογα με το ποιος ήταν αυτός και για πόσο διάστημα επιδρούσε. Επομένως σε διάφορες συνθήκες ανευρίσκονται διαφορετικοί συνδυασμοί της Hsp27, γιατί ακριβώς εξυπηρετούν τις απαιτούμενες διαφορετικές, ενδεχομένως, λειτουργίες αντίστοιχα. Η παρουσία όλων των μορφών της Hsp27 στα μονοπύρηνα κύτταρα περιφερικού αίματος υγιών ενηλίκων όταν αυτά δεν είχαν υποστεί καμμία εξωγενή επίδραση, οδήγησε στο συμπέρασμα ότι ενδεχομένως όλες αυτές οι μορφές της πρωτεΐνης να βρίσκονται σε μια δυναμική κατάσταση εναλλαγής στα κύτταρα ανάλογα με τις ενδογενείς επιδράσεις που δέχονται ανά πάσα στιγμή. Τέλος ,η παρουσία των φωσφορυλιωμένων μορφών της Hsp27 μόνο για τις θέσεις Ser78 και Ser82 σε βασικές συνθήκες αποτελεί σημαντική ένδειξη ότι σε αυτήν την περίπτωση επάγονται περισσότερο τα μεγάλα ολιγομερή της πρωτεΐνης. Αντίθετα, σε συνθήκες επίδρασης θερμικού shock και TNF-a, επάγονται και τα μικρότερα σύμπλοκα της Hsp27, αφού ανευρίσκεται επιπλέον και η φωσφορυλιωμένη μορφή της και για τη θέση Ser15. / -

Solid tumours are a hostile tissue environment in which the cells are exposed to many stresses including hypoxia. One consequence of hypoxic conditions is an increase in extracellular levels of the purine nucleoside adenosine, which enhances tumour cell migration. This is achieved in part through an increase in the levels of the chemokine receptor CXCR4, which along with its ligand CXCL12, is a key player in breast cancer metastasis. The cellular response to stress is mediated by a family of proteins known as heat-shock proteins (HSPs). The small heat shock protein 27 (HSP27) has been implicated in changes in cancer cell migration. I have therefore studied the regulation of HSP27 in human breast cancer cells by conditions that normally exist in the stressful tumor environment. My project specifically aimed to establish whether changes in HSP27 are linked to hypoxia, adenosine levels and alterations in the CXCL12-CXCR4 migratory pathway.

No description available.

Células dendríticas são as principais células apresentadoras de antígeno e apresentam alterações em pacientes com câncer. As vias de sinalização ERK 1/2 e p38 MAPK participam da diferenciação de DCs derivadas de monócitos (Mo-DCs). A exposição ao sobrenadante tumoral (ST) da linhagem MCF-7 levou à diminuição de CD1a e aumento de CD14 (frequência), além do aumento de IL-6 e IL-10. A inibição da via ERK1/2 MAPK corrigiu a expressão de CD14 e corrigiu parcialmente a produção das citocinas. A inibição da via p38 MAPK corrigiu a expressão de CD1a e CD14 e diminuiu parcialmente a produção das citocinas. Identificamos a proteína de choque térmico HSP27. A exposição à HSP27 não levou às alterações observados quando as células foram expostas ao ST. Por fim, em Mo-DCs de pacientes com câncer de mama o tratamento com o inibidor da p38 MAPK diminuiu a expressão de CD86 e HLA-DR. Portanto, os resultados deste trabalho sugerem que a inibição da via p38 MAPK não parece ser uma abordagem interessante na manipulação de Mo-DCs de pacientes com carcinoma ductal invasivo de mama. / Dendritic cells are the main presenting cells and present alterations in cancer patients. The signaling pathways p38 and ERK1/2 MAPK participate of monocyte-derived dendritic cells (Mo-DCs) differentiation. Exposition to MCF-7s supernatant (TS) decreased CD14 and CD1a expression (frequency) while enhanced IL-6 and IL-10 production. Inhibition of ERK1/2 MAPK reverted CD14 expression and partially reverted cytokines production. Inhibition of p38 MAPK reverted CD1a and CD14 expression and partially reverted cytokines production too. We identified the heat shock protein HSP27. Exposition to HSP27 did not cause the observed alterations seen when the cells were exposed to TS. Lastly, treatment of Mo-DCs from breast cancer patients with the p38 inhibitor decreased CD86 and HLA-DR expression. Therefore, the data presented in this study suggest that p38 MAPK inhibition does not appear to be an interesting approach in the manipulation of Mo-DCs from breast cancer patients.

O envelhecimento populacional está em constante crescimento, sendo acompanhado por uma maior incidência de doenças crônicas e neurodegenerativas como Alzheimer, Parkinson e Huntington. Sabe-se que parte destas doenças são causadas pelo acúmulo de proteínas mal-enoveladas, e estudos têm apontado que os componentes do sistema ubiquitina-proteassomo possuem um potencial alvo terapêutico para doenças relacionadas ao processo senil do envelhecimento. A chaperona molecular HSP27 é capaz de aumentar a atividade catalítica do proteassomo e a degradação de proteínas ubiquitinadas, além de atuar em situações nocivas para célula, como choque térmico e estresse oxidativo. Em vista disso, o presente projeto visou analisar o efeito da superexpressão de HSP27 humana, sob o controle de nove promotores mutantes TEF na longevidade de S. cerevisiae. MATERIAL E MÉTODOS: Os ensaios realizados envolveram a determinação da longevidade cronológica e replicativa, ensaio de termotolerância, resistência ao estresse oxidativo e determinação da atividade proteassomal de S. cerevisiae. RESULTADOS: Os resultados obtidos indicam que a superexpressão de HSP27 prolonga a longevidade replicativa e melhora a resistência celular contra choque térmico e estresse oxidativo, além de aumentar a atividade proteasomal. Contudo, essas melhorias só ocorreram em certos níveis de expressão de HSP27, sendo TEF8 o promotor mutante que conferiu os melhores resultados. DISCUSSÃO E CONCLUSÃO: Esses resultados sugerem que a função de HSP27 implica no conceito de hormese, em que níveis ideais puderam ser determinados em S. cerevisiae, e este fato poderá ser importante para interferir na longevidade, e corroborar para o futuro desenvolvimento de fármacos que poderiam controlar a expressão gênica da proteína e modular os fenótipos observados na senescência em humanos. / INTRODUCTION: Population ageing is constantly increasing, accompanied by a higher incidence of chronic and neurodegenerative diseases such as Alzheimer\'s, Parkinson\'s and Huntington\'s. It is known that some of these diseases are caused by accumulation of unfolded proteins, and studies have pointed out that the components of the ubiquitinproteasome system have a potentially therapeutic target for diseases related to the senile aging process. The molecular chaperone HSP27 is able to increase the catalytic activity of the proteasome and degradation of ubiquitinated proteins, besides acting in situations harmful to the cell, such as heat shock and oxidative stress. In view of this, the present study aimed to analyze the effect of overexpression of human HSP27, under the control of nine TEF mutant promoters on longevity of S. cerevisiae. MATERIAL AND METHODS: The assays involved the determination of chronological and replicative longevity, thermotolerance, resistance to oxidative stress and determination of proteasomal activity of S. cerevisiae. RESULTS: The results indicated that the overexpression of HSP27 prolongs a replicative longevity and improves the cellular resistance to heat shock and oxidative stress, besides increasing the proteasomal activity. However, these improvements only occurred at optimal levels of HSP27, wherein mutant promoter TEF8\' obtained the best performance. DISCUSSION AND CONCLUSIONS: These results suggest that the function of HSP27 implies in the concept of hormesis, in which ideal levels could be determined in S. cerevisiae, and this fact may be important to interfere in the longevity, and corroborate for the future development of drugs that could control the expression of the protein and modulate the observed phenotypes in senescence in humans.

La myélofibrose (MF) est la plus agressive des néoplasies myéloprolifératives (NMP). Elle porte à elle seule le plus mauvais pronostic pour les patients puisqu’elle s’accompagne d’une fibrose de la moelle osseuse évoluant vers une insuffisance médullaire. Les inhibiteurs de la kinase JAK2 ont apporté de nouveaux espoirs pour le traitement des NPM mais leurs effets ont été essentiellement bénéfiques sur les symptômes et non sur la fibrose elle-même ni sur le cours de la maladie. Plus récemment, la protéine de choc thermique 90 (HSP90) - connue pour stabiliser JAK2 - est apparue comme une cible thérapeutique prometteuse pour les NMP. Cependant, les inhibiteurs de la HSP90 ont montré une toxicité importante accompagnée d’une expression compensatoire des HSPs inductibles (i.e HSP70, HSP27), connues pour favoriser l’émergence de phénomène de résistance. Par ailleurs, des études ont montré que HSP27 était fortement exprimée chez les patients présentant une fibrose pulmonaire idiopathique ou rénale montrant l’importance de HSP27 dans les processus fibrotiques. Sur la base de l’ensemble de ces données, nous avons évalué d’une part l'efficacité chez l’animal d'un oligonucléotide inhibiteur spécifique de HSP27 appelé OGX-427 (en essai clinique dans plusieurs cancers). D’autre part, nous avons déterminé le niveau d’expression intra- et extracellulaire de HSP27 chez des patients atteints de MF. L'effet de l'OGX-427 a été évalué dans deux modèles murins de myélofibrose, laquelle est induite soit par la sécrétion excessive de thrombopoïétine (TPOhigh) soit par la mutation JAKV617F. Nous avons mis en évidence dans les souris traitées par l’OGX-427, une réduction de la taille de la rate, de la prolifération mégacaryocytaire et de l’hématopoïèse extramédullaire par rapport aux souris contrôles, révélant ainsi un effet bénéfique de l’inhibition de HSP27 sur la progression de la maladie. De toutes récentes observations complémentaires à ce travail ont également montré une diminution de la fibrose réticulinique dans la moelle osseuse de souris JAKV617F. Au niveau moléculaire, nous démontrons que l'effet prolifératif induit par la voie de signalisation exacerbée - JAK2/STAT5 - est régulé par HSP27 via des interactions directes. Pour finir, nous avons détecté une augmentation de l'expression de HSP27 aussi bien dans les progéniteurs circulants CD34+ que dans le sérum des patients atteints de NMP avec MF. Ce travail révèle pour la première fois le rôle intra et extracellulaire de HSP27 dans la physiopathologie de la MF et le bénéfice thérapeutique potentiel de l’utilisation des inhibiteurs de HSP27 dans cette maladie. / Myelofibrosis (MF) is the most aggressive myeloproliferative neoplasms (MPN) with the highest degree of morbidity and mortality, including progressive bone marrow fibrosis resulting into bone marrow failure. JAK2 kinase inhibitors have been successfully used for a few years in MPN and more particularly for MF treatment. Nevertheless, their beneficial effects are mainly restricted on symptoms and not on the course of the disease. Recently, heat shock protein 90 (HSP90) - known to stabilize JAK2 - has been reported as a promising therapeutic target in MPN. However HSP90 inhibitors show toxicity and induce the expression of stress-inducible proteins such as HSP70 and, most likely HSP27 as previously shown in other cancers. In addition, we and others have shown that HSP27, was strongly expressed in patients with idiopathic pulmonary or kidney tubulointerstitial fibrosis, underlying a relevant role of HSP27 in fibrotic processes. Taking into account both the beneficial effects of HSP inhibitors in leukemia and in MPN, and the possible implication of HSP27 in fibrosis, we have evaluated here the status of HSP27 in MF patient’s samples and assess the effectiveness of an HSP27 oligonucleotide inhibitor called OGX-427 in murine models. We report here the effect of OGX-427 in two murine models of thrombopoietin- and JAKV617F-induced myelofibrosis. OGX-427 limited the progression of the disease associated with a reduction of spleen weight and of megakaryocytic expansion. And more recently, our additional results show a decrease of reticulin fibrosis in JAK2V617F’s bone marrow. We show that HSP27 regulates JAK2/STAT5 proliferative effect through direct interactions, and we report an increase expression of HSP27 both in CD34+ circulating progenitors and in the serum of patients with NMP with fibrosis. Taking altogether, this work supports that extra and intracellular HSP27 plays a key role of in the pathophysiology in MF and highlight the potential therapeutic benefit of HSP27 inhibitors in this disorder.

La recherche des marqueurs biologiques de la tendreté a fait l’objet de nombreux travaux chez les animaux producteurs de viande et en particulier les bovins. A l’issue de ces études, une expression différentielle de la protéine Hsp27 entre des groupes de tendreté extrême a été mise en évidence. Cette protéine est présente à un « carrefour » biologique de l’interactome lié à la tendreté. Comprendre les mécanismes d’action de la protéine Hsp27 dans la tendreté de la viande bovine est l'un des défis de recherche dans le domaine de la production de viande. Dans cette optique, mon travail de thèse (2010-2013) avait pour objectif d’analyser le rôle de Hsp27 dans le développement du muscle et son implication dans le déterminisme des caractéristiques des tissus liés à la qualité de la viande. La première étape de ce travail a consisté à produire un modèle de souris présentant une inactivation du gène de la protéine Hsp27 (KO HspB1) et d’analyser leur phénotype comparativement à des témoins. Les souris KO HspB1 sont viables, fertiles et ne présentent aucune anomalie majeure, mais ont un format plus petit que celui de leurs témoins. L’analyse de leurs caractéristiques musculaires par une technique immunohistoligique mise au point spécifiquement (Publication 1) n’a pas révélé de différences. Au niveau ultrastructural, l'observation du muscle des souris par microscopie électronique à transmission a révélé des différences ultrastructurales entre les deux génotypes à T0 post-mortem avec des écarts entre les myofibrilles très espacées chez les souris KO HspB1 et un appareil contractile musculaire moins organisé. Ces différences sont encore plus marquées à T72 heures post-mortem. Ainsi le phénotype musculaire fin des souris KO HspB1 est plus altéré (Publication 2). Une analyse bio-informatique a été réalisée dans l'objectif de compléter la liste des interacteurs de la protéine Hsp27 et des gènes cibles de l’invalidation d’HspB1 susceptibles de participer à des différences de structure du muscle et de la tendreté. Les partenaires ou cibles prédits de Hsp27 sont des protéines impliquées dans différentes fonctions, comme des Heat shock proteines, des régulateurs de l'apoptose, des facteurs de traduction, des protéines du cytosquelette et des antioxydants. Les abondances de 15 protéines ont été quantifiées par Western-bloting dans deux muscles (m. Soleus, m. Tibialis). Elles sont modifiées chez les souris dépourvues de Hsp27 principalement dans le muscle le plus oxydatif. Cette étude démontre l'existence de liens fonctionnels entre Hsp27 et ses cibles prédites qui pourraient participer au phénotype fin des souris (Publication 3). Pour compléter cette étude, une analyse protéomique du muscle Tibialis anterior a été menée en utilisant la technique d’électrophorèse bidimensionnelle couplée à la spectrométrie de masse. La comparaison des protéomes spécifiques de ces deux génotypes a permis de mettre en évidence des profils d’expression différents pour plusieurs protéines. Elle confirme l’effet muscle spécifique du KO et révèle un lien avec le métabolisme du calcium et des Hsps différentes de celles mises en évidence dans le muscle oxydatif (Publication 4). L'ensemble des données issues de cette étude réalisée dans une espèce modèle apporte des connaissances nouvelles susceptibles d’éclairer sur les mécanismes moléculaires impliqués dans l’établissement de la tendreté de la viande bovine. Elle suggère que le statut en Hsp, les processus apoptotiques et la protection contre le stress oxydatif contribuent à l'évolution de l'ultrastructure post-mortem des muscles et à la tendreté de la viande. Ces nouvelles connaissances seront validées ultérieurement sur muscle bovin. / Thanks to genomics, we have previously identified markers of beef tenderness, and computed a bioinformatic analysis that enabled us to build an interactome in which we found Hsp27 at a crucial node. Understanding the role of Hsp27 in the development of muscle and in the determinism of beef tenderness is one of the research challenges in meat production. In this context, my pHDthesis (2010-2013) aimed to analyze the role of Hsp27 in muscle development and its involvement in the determination of the characteristics related to the quality of the meat tissue. In this study, we generated mice devoid of Hsp27 protein by homologous recombination of the HspB1 gene as an animal model. The HspB1-/ - mice were viable and fertile, showing no apparent abnormality but a smaller than their control format. The muscle structure of animals was examined by optical microscopy and transmission electron microscopy. The first approach, made by a developed immunohistochemical classification (Publication 1), did not reveal any differences in the characteristics of muscle fibers (contractile and metabolic type, shape, perimeter, cross-sectional area) but a trend for a higher proportion of small fibers. Different myosin heavy chains electrophoretic profiles were also observed in HspB1-/- mice. At the ultrastructural level, examination of the myofibrillar material showed destructured myofibrils and higher gaps between myofibrils in HspB1-/-, and a greater disintegration of myofibrils at 72h postmortem (Publication 2). We have used a network-based approach for understanding the contribution of Hsp27 to tenderness through the prediction of its interactors related to tenderness. We have revealed the direct interactors of Hsp27. The predicted partners of Hsp27 included proteins involved in different functions e.g. members of Hsp families, regulators of apoptosis, translation factors, cytoskeletal proteins and antioxidants. The abundances of 15 proteins were quantified by Western blotting in two muscles of HspB1-null mice and their controls. We observed changes in the amount of most of the Hsp27 predicted targets in mice devoid of Hsp27 mainly in the most oxidative muscle (Soleus. Our study demonstrates the functional links between Hsp27 and its predicted targets. It suggests that Hsp status, apoptotic processes and protection against oxidative stress are crucial for post-mortem muscle metabolism, subsequent proteolysis, and therefore for beef tenderness (Publication 3). To complete this study, we performed a proteomic analysis of m. Tibialis anterior (glycolytic muscle), using 2D gel electrophoresis, to detect changes in protein abundance subsequent to the invalidation of HspB1 gene. This study confirms the muscle specific effect of HspB1 invalidation and reveals a new list of Heat shock proteins different from those highlighted in oxidative muscle and relationships with calcium (Publication 4). All together, these results provided from a model species showed the very important role of Hsp27 for muscle ultrastructure and revealed its implication in different muscle biological pathways. This provided new elements for understanding the crucial role for Hsp27 in the modulation of the tenderizing process of muscle during meat ageing that will be further examined in beef.

HSP27, the Drosophila homolog of mammalian HspB1, is a nuclear sHsp that is both stress induced and developmentally regulated with a conserved cyto-protective function. It is multiply phosphorylated in vivo through an unconfirmed mechanism at unidentified residues. The effect of phosphorylation on its localization, oligomerization, and function is also not well understood. Here we report a genetic investigation into the role of Hsp27 in Drosophila development, and a preliminary investigation into the effect of phosphorylation on HSP27 localization and function in Drosophila S2 cells. Through a proteomic screen, a pro-apoptotic role for Hsp27 in embryonic developmentally regulated programmed cell death was suggested and supported by RNAi experiments, but not replicated using Hsp27null mutant stocks. These stocks were complicated by the intriguing appearance of multiple background mutations. Specific developmental defects in transgenic lines overexpressing phospho-mutant isoforms were then investigated. These too were subject to multiple independent incidences of background genetic mutation, which we believe may be related to Hsp27 mis-expression. We also studied the endogenous expression and localization pattern of HSP27 in stressed and unstressed Drosophila S2 cells. We found evidence that wild-type protein localization is influenced by stress. Finally, we took a first step toward understanding how phosphorylation might regulate HSP27 localization by examining the effect of targeted mutations of serine residues (S58, S71, and S75) on the localization pattern of exogenous HSP27. By characterizing the expression of endogenous and overexpressed HSP27 in Drosophila cells, we provide a foundation for future investigation into the regulated localization and function of HSP27 that can be extended to address the regulatory mechanisms that govern the protective capacities and oligomeric properties of phosphorylated HSP27 in Drosophila.

Células dendríticas são as principais células apresentadoras de antígeno e apresentam alterações em pacientes com câncer. As vias de sinalização ERK 1/2 e p38 MAPK participam da diferenciação de DCs derivadas de monócitos (Mo-DCs). A exposição ao sobrenadante tumoral (ST) da linhagem MCF-7 levou à diminuição de CD1a e aumento de CD14 (frequência), além do aumento de IL-6 e IL-10. A inibição da via ERK1/2 MAPK corrigiu a expressão de CD14 e corrigiu parcialmente a produção das citocinas. A inibição da via p38 MAPK corrigiu a expressão de CD1a e CD14 e diminuiu parcialmente a produção das citocinas. Identificamos a proteína de choque térmico HSP27. A exposição à HSP27 não levou às alterações observados quando as células foram expostas ao ST. Por fim, em Mo-DCs de pacientes com câncer de mama o tratamento com o inibidor da p38 MAPK diminuiu a expressão de CD86 e HLA-DR. Portanto, os resultados deste trabalho sugerem que a inibição da via p38 MAPK não parece ser uma abordagem interessante na manipulação de Mo-DCs de pacientes com carcinoma ductal invasivo de mama. / Dendritic cells are the main presenting cells and present alterations in cancer patients. The signaling pathways p38 and ERK1/2 MAPK participate of monocyte-derived dendritic cells (Mo-DCs) differentiation. Exposition to MCF-7s supernatant (TS) decreased CD14 and CD1a expression (frequency) while enhanced IL-6 and IL-10 production. Inhibition of ERK1/2 MAPK reverted CD14 expression and partially reverted cytokines production. Inhibition of p38 MAPK reverted CD1a and CD14 expression and partially reverted cytokines production too. We identified the heat shock protein HSP27. Exposition to HSP27 did not cause the observed alterations seen when the cells were exposed to TS. Lastly, treatment of Mo-DCs from breast cancer patients with the p38 inhibitor decreased CD86 and HLA-DR expression. Therefore, the data presented in this study suggest that p38 MAPK inhibition does not appear to be an interesting approach in the manipulation of Mo-DCs from breast cancer patients.

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.