Submitted by Amanda Silva (amanda.osilva2@ufpe.br) on 2015-03-11T13:42:17Z No. of bitstreams: 2 TESE Adriana da Silva Andrade Pereira.pdf: 6828087 bytes, checksum: 9d66e9700be0a70e927b955ee06cb850 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-11T13:42:17Z (GMT). No. of bitstreams: 2 TESE Adriana da Silva Andrade Pereira.pdf: 6828087 bytes, checksum: 9d66e9700be0a70e927b955ee06cb850 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2014-02-17 / CNPq; CAPES / Schistosoma mansoni, é um dos mais importantes parasitas que infectam seres humanos. No nordeste do Brasil, a esquistossomose é historicamente endêmica e considerada como um problema de saúde pública. No estado de Pernambuco, particularmente no litoral e zona da Mata, predomina a cepa São Lourenço da Mata (SLM). Estudos ultraestruturais desta cepa até então ainda não foram realizados, embora análises morfométricas e morfológicas de outras cepas existentes no Brasil já tenham sido publicadas na literatura. Os vermes são bem adaptados ao hospedeiro, tendo a sua longevidade vista como uma conseqüência do eficaz escape do sistema imune. Na circulação sanguínea os vermes devem obter lipídios a partir do hospedeiro, já que eles não sintetizam tais compostos, sendo as lipoproteínas plasmáticas a possível fonte de tais lipídios, além de poderem atuar mascarando o reconhecimento do verme pelo sistema imune. Neste trabalho, microscopia eletrônica de varredura (SEM) foi utilizada para a análise morfológica e morfométrica de cercárias e vermes adultos de S. mansoni cepa SLM, bem como para estudar a interação da lipoproteína de baixa densidade (LDL) com o tegumento do parasito. Também foram usadas diferentes técnicas para a localização e identificação das proteínas envolvidas no processo de absorção e/ou transporte de lipoproteínas no verme adulto de S. mansoni. As cercárias foram obtidas de caramujos Biomphalaria glabrata e os vermes adultos de camundongos machos Mus musculus e Swiss, ambos infectados pela cepa SLM de S. mansoni. Os resultados da SEM mostraram que os corpos da cercárias são cobertos por espinhos, com uma ventosa ventral, uma ventosa oral com receptores sensoriais, e um par de glândulas de penetração na cabeça. Seu comprimento total variou de 174 a 290μm. O comprimento dos vermes adultos machos foi de 4mm e das fêmeas 5mm. O comprimento da região anterior do macho foi de 470μm e 271μm para o sexo feminino. Todos os parâmetros foram realizados em dez amostras. Os valores encontrados na morfometria da cepa SLM foram menores que em outras cepas de S. mansoni já descritas. Através da microscopia eletrônica de transmissão (TEM), os experimentos de imunomarcação mostraram partículas de ouro localizadas dentro do tegumento, na região muscular e espículas dos machos e em torno das células vitelínicas das fêmeas. A utilização de técnicas tais como imunoblotting e “liquid chromatography–mass spectrometry” (LC-MS) proporcionaram a identificação no S. mansoni de duas formas diferentes da chaperona Hsp 70, proteínas envolvidas na interação com a apo-B e o seu transporte para o retículo endoplasmático, sendo responsável também pela ruptura das interações clatrina-clatrina na endocitose humana. Com a descoberta destas proteínas, novos estudos serão necessários para esclarecer o papel funcional das mesmas no S. mansoni.

As Heat Shock Proteins (HSPs) ou proteínas do choque térmico são encontradas em todas as células e são classificadas de acordo com seu peso molecular. Dentre elas encontram-se as de 27, 60, 70, 90 e 110 kDa, sendo as mais estudadas no contexto da reprodução as da família 60 e 70. Essas proteínas são ditas como chaperoninas, em razão do seu importante papel no dobramento e desdobramento de outras proteínas celulares sem alterar sua conformação final, e são expressas frente a qualquer tipo de estresse como calor, vírus, bactéria, hormônios, diferenciação celular, etc, e influenciam nas respostas imune inata e adquirida. A placenta também expressa essas proteínas, uma vez que é um órgão de intenso estresse e diferenciação celular durante toda a gestação. Nesse estudo, busca-se avaliar a expressão ou não dessas proteínas na placenta bovina e para isso foram utilizadas 30 amostras de diferentes animais em estágios distintos de gestação, fixadas em formol tamponado a 10% e processadas pela técnica de imuno-istoquímica. O mesmo numero de amostras foi também processado para a análise de imuno-microscopia eletrônica de transmissão pelas técnicas de \"freeze-substitution\" e marcação por pós-embebição. Na imuno-istoquímica, as HSPs 60 e 70 foram localizadas nos trofoblastos, epitélio materno e células binucleadas. A expressão da HSP 60 foi maior no início declinando no segundo e terceiro terço. Já a expressão da HSP 70 manteve-se praticamente constante, evidenciando a forte expressão dessa proteína durante todo o período. Na análise de imuno-microscopia eletrônica de transmissão, ambas as famílias foram localizadas nas células binucleadas (núcleo, citoplasma e vesículas) e epitélio materno (núcleo e citoplasma) em todos os terços gestacionais. O perfil das proteínas estudadas na placenta bovina foi diferente quando comparada à placenta humana, pois nesta última, a intensidade da expressão para a HSP 70 diminuiu com o decorrer da gestação enquanto para a HSP 60 foi constante durante todo a gestação. Provavelmente essas diferenças podem estar relacionadas ao fato dessas amostras terem sido coletadas de mulheres com gravidez interrompidas e também pelo tipo de placentação distinta. A bovinocultura de corte é de extrema importância para a econômica para o Brasil e se faz necessário o conhecimento de fatores que possam melhorar suas características reprodutivas. Dessa forma os resultados obtidos nesse estudo contribuirão certamente de subsídio para experimentos futuros sobre o papel das Heat Shock Proteins na placenta bovina. / Heat Shock Proteins (HSP) can be found in any kind of cell. These proteins are classified according to their molecular weight and their known families include the HSP 27, 60, 70, 90 and 110 kDa. Among these, HSP 60 and 70 are the ones of interest in reproduction. They were known as chaperonines because of their capacity to fold and unfold other proteins into the cell, without changing their own conformation. They are expressed during several stress conditions likes virus and bacteria infections, hormones, heat, cellular differentiation, etc, and also take part signalizing for innate and acquired immune responses. Heat shock proteins are expressed in several tissues and organs, including the placenta. In this study we have evaluated the expression of these proteins in the bovine placenta, using thirty samples from different animais with distinct gestational periods, fixed in 10% formalin and processed for immunohistochemistry. The same numbers of samples were processed for immunoelectron microscopy using freeze-substitution and post embedding labeling techniques. The immunohistochemistry results show the expression of HSP 60 and 70 in trophoblasts, maternal epithelia and binucleated cells. The HSP 60 expression was higher in the beginning of gestation, becoming lower during the second and third trimester. Heat shock protein 70 expression were practically constant throughout the gestation. The immunoelectron microscopy analysis revealed that both HSP 60 and 70 were located in the cytoplasm and nucleio binucleated cells and maternal epithelia from the beginning to the end of pregnancy. The immunolocalization of HSP 60 and 70 in the bovine placenta were distinct from the ones found in studies on women, probably due to the differences of the placentation type and to the fact that those samples were collected from abnormal or discontinuous pregnancy. Beef production in Brazil is an important economical activity and studies to improve the bovine reproductive characteristics are necessary and must be expended, therefore our results certainly contributes for further studies on HSP function during pregnancy in this species.

As Heat Shock Proteins (HSPs) ou proteínas do choque térmico são encontradas em todas as células e são classificadas de acordo com seu peso molecular. Dentre elas encontram-se as de 27, 60, 70, 90 e 110 kDa, sendo as mais estudadas no contexto da reprodução as da família 60 e 70. Essas proteínas são ditas como chaperoninas, em razão do seu importante papel no dobramento e desdobramento de outras proteínas celulares sem alterar sua conformação final, e são expressas frente a qualquer tipo de estresse como calor, vírus, bactéria, hormônios, diferenciação celular, etc, e influenciam nas respostas imune inata e adquirida. A placenta também expressa essas proteínas, uma vez que é um órgão de intenso estresse e diferenciação celular durante toda a gestação. Nesse estudo, busca-se avaliar a expressão ou não dessas proteínas na placenta bovina e para isso foram utilizadas 30 amostras de diferentes animais em estágios distintos de gestação, fixadas em formol tamponado a 10% e processadas pela técnica de imuno-istoquímica. O mesmo numero de amostras foi também processado para a análise de imuno-microscopia eletrônica de transmissão pelas técnicas de \"freeze-substitution\" e marcação por pós-embebição. Na imuno-istoquímica, as HSPs 60 e 70 foram localizadas nos trofoblastos, epitélio materno e células binucleadas. A expressão da HSP 60 foi maior no início declinando no segundo e terceiro terço. Já a expressão da HSP 70 manteve-se praticamente constante, evidenciando a forte expressão dessa proteína durante todo o período. Na análise de imuno-microscopia eletrônica de transmissão, ambas as famílias foram localizadas nas células binucleadas (núcleo, citoplasma e vesículas) e epitélio materno (núcleo e citoplasma) em todos os terços gestacionais. O perfil das proteínas estudadas na placenta bovina foi diferente quando comparada à placenta humana, pois nesta última, a intensidade da expressão para a HSP 70 diminuiu com o decorrer da gestação enquanto para a HSP 60 foi constante durante todo a gestação. Provavelmente essas diferenças podem estar relacionadas ao fato dessas amostras terem sido coletadas de mulheres com gravidez interrompidas e também pelo tipo de placentação distinta. A bovinocultura de corte é de extrema importância para a econômica para o Brasil e se faz necessário o conhecimento de fatores que possam melhorar suas características reprodutivas. Dessa forma os resultados obtidos nesse estudo contribuirão certamente de subsídio para experimentos futuros sobre o papel das Heat Shock Proteins na placenta bovina. / Heat Shock Proteins (HSP) can be found in any kind of cell. These proteins are classified according to their molecular weight and their known families include the HSP 27, 60, 70, 90 and 110 kDa. Among these, HSP 60 and 70 are the ones of interest in reproduction. They were known as chaperonines because of their capacity to fold and unfold other proteins into the cell, without changing their own conformation. They are expressed during several stress conditions likes virus and bacteria infections, hormones, heat, cellular differentiation, etc, and also take part signalizing for innate and acquired immune responses. Heat shock proteins are expressed in several tissues and organs, including the placenta. In this study we have evaluated the expression of these proteins in the bovine placenta, using thirty samples from different animais with distinct gestational periods, fixed in 10% formalin and processed for immunohistochemistry. The same numbers of samples were processed for immunoelectron microscopy using freeze-substitution and post embedding labeling techniques. The immunohistochemistry results show the expression of HSP 60 and 70 in trophoblasts, maternal epithelia and binucleated cells. The HSP 60 expression was higher in the beginning of gestation, becoming lower during the second and third trimester. Heat shock protein 70 expression were practically constant throughout the gestation. The immunoelectron microscopy analysis revealed that both HSP 60 and 70 were located in the cytoplasm and nucleio binucleated cells and maternal epithelia from the beginning to the end of pregnancy. The immunolocalization of HSP 60 and 70 in the bovine placenta were distinct from the ones found in studies on women, probably due to the differences of the placentation type and to the fact that those samples were collected from abnormal or discontinuous pregnancy. Beef production in Brazil is an important economical activity and studies to improve the bovine reproductive characteristics are necessary and must be expended, therefore our results certainly contributes for further studies on HSP function during pregnancy in this species.

MSc (Microbiology) / Department of Microbiology / See the attached abstract below

No description available.

In this Master’s Thesis Research the results can be summarized from two major tasks: (1) In our first task, we utilized our two protein system (BAG-1 and HSP 70) as part of beta testing of a computational software 1 that can take three dimensional x-ray crystallography information about protein complexes and predict the strength of atom –atom interactions between amino-acid residues Open Contact predicts binding hotspots that can be used to identify short amino acid chains or peptides that mimic that particular binding segment of the larger protein. These peptides are called pepidyl-biomimetics. The peptide can potentially act as an antagonist drug by binding to the hotspot on protein A before protein B of the A-B complex can form. Two potential peptide candidates were identified. In particular, a helical peptide was discovered that demonstrated a variety of different types of atom-atom interactions. (2) Our second task is to experimentally test the helical peptide for its ability to block the binding that occurs between the 70-kilodalton Heat Shock Protein (HSP-70) and the Bcl-2 Associated Athanogene (BAG-1) Protein. As reviewed here, the binding between HSP-70 and BAG-1 elicits a cascade of cellular events that maintain high cancer growth rates and a greatly increased resistance to chemotherapy. In addition, BAG-1 has been implicated in a number of onco-signal pathways, as reviewed here, and its inhibition alone is believed to act as an agent against cancer cell growth

Les urodèles amphibiens, dont fait partie l’axolotl (Ambystoma mexicanum), ont la capacité de régénérer leurs organes et membres suite à une amputation, tout au long de leur vie. La patte est l’organe dont le processus de régénération est le mieux caractérisé et ce dernier est divisé en deux phases principales. La première est la phase de préparation et commence immédiatement suite à l’amputation. Elle renferme des étapes essentielles au processus de régénération comme la guérison de la plaie et la formation d’une coiffe apicale ectodermique. Par la suite, les fibroblastes du derme et certaines cellules musculaires vont revenir à un état pluripotent via un processus appelé dédifférenciation cellulaire. Une fois dédifférenciées, ces cellules migrent et s’accumulent sous la coiffe apicale pour former le blastème. Lors de la phase de redéveloppement, les cellules du blastème se divisent puis se redifférencient pour régénérer la partie amputée. Fait intéressant, la régénération d’un membre ou la guérison d’une plaie chez l’axolotl ne mène jamais à la formation d’une cicatrice. Afin d’en apprendre plus sur le contrôle moléculaire de la régénération, les gènes Heat-shock protein-70 (Hsp-70) et Transforming growth factor-β1 (Tgf-β1) ont été sélectionnés. Ces gènes jouent un rôle important dans la réponse au stress et lors de la guérison des plaies chez les mammifères. HSP-70 est une chaperonne moléculaire qui est produite pour maintenir l’intégrité des protéines cellulaires lorsqu’un stress se présente. TGF-β1 est une cytokine produite suite à une blessure qui active la réponse inflammatoire et qui stimule la fermeture de la plaie chez les amniotes. Les résultats présentés dans cette thèse démontrent que Hsp-70 est exprimé et régulé lors du développement et de la régénération du membre chez l’axolotl. D’autre part, nos expériences ont mené à l’isolation de la séquence codante pour Tgf-β1 chez l’axolotl. Nos résultats montrent que Tgf-β1 est exprimé spécifiquement lors de la phase de préparation dans le membre en régénération. De plus, le blocage de la voie des Tgf-β avec l’inhibiteur pharmacologique SB-431542, lors de la régénération, mène à l’inhibition du processus. Ceci démontre que la signalisation via la voie des Tgf-β est essentielle à la régénération du membre chez l’axolotl. / Urodele amphibians, such as the axolotl (Ambystoma mexicanum), have the unique ability, among vertebrates, to perfectly regenerate many parts of their body throughout their life. Among the complex structures that can be regenerated, the limb is the most widely studied. Limb regeneration is divided in two main phases. The preparation phase, which begins right after amputation, includes wound healing and the formation of an apical ectodermal cap. During this phase, dermal fibroblasts and muscle cells will lose their characteristics and become pluripotent through a process called cellular dedifferentiation. The dedifferentiated cells migrate and accumulate under the apical ectodermal cap to form the blastema. During the redevelopment phase, the cells in the blastema proliferate and redifferentiate to regenerate the lost structures. It is interesting to highlight the fact that regeneration never leads to scar formation in the axolotl. In order to learn more about the molecular control of limb regeneration, the genes Heat-shock protein-70 (Hsp-70) and Transforming growth factor-β1 (Tgf- β1) were selected for their important roles in stress response and wound healing in mammals. HSP-70 is a molecular chaperone which is produced to protect cellular proteins when the cell faces a stress. TGF-β1 is a cytokine produced after wounding that activates the inflammatory response and stimulates wound closure in amniotes. Results presented in this thesis show that Hsp-70 is expressed and regulated during limb development and regeneration in the axolotl. We were also able to isolate the cDNA coding for axolotl Tgf-β1 and our results show that this gene is expressed specifically during the preparation phase of limb regeneration. Treatment of regenerating axolotls with a specific inhibitor of Tgf-β signalling, SB-431542, led to complete inhibition of regeneration. This directly implies that Tgf-β signalling is essential for limb regeneration in axolotl.

O ψευδάργυρος -Zn ελέγχει τον μεταβολισμό των λιπιδίων και γλυκόζης μέσω Ζn-μεταγραφικών παραγόντων. Δίαιτες υψηλών λιπαρών οδηγούν στην ενδοκυτταρική συσσώρευση ελευθέρων ριζών, μειώνουν την μεταγραφική έκφραση των εκκαθαριστών τους και την ενεργότητα αντιοξειδωτικών ενζύμων και οδηγούν σε μεταβολικό σύνδρομο. Οι πρωτεϊνες του θερμικού shock (Heat shock proteins-HSP) παίζουν σημαντικό ρόλο στην αντίσταση στο κυτταρικό stress ως προσαρμογή ,μετά από έκθεση, σε διάφορα ερεθίσματα. ΣΚΟΠΟΣ : Μελέτη της επίδρασης του διατροφικού Ζn και των Hsp70,στην μεταβονομική των λιπιδίων αίματος και ήπατος ποντικών ΥΛΙΚΟ-ΜΕΘΟΔΟΙ :Ενήλικα αρσενικά ποντίκια: 1. Wild type (Wt) Hybrid (F1/F1) C57Bl/6 x CBA και 2. Transgenic (Tg) human HSP70 overexpressing mice, από ηλικίας ενός μηνός εντάχθηκαν στις παρακάτω διατροφές ,επί 10 εβδομάδες. Διατροφή Αριθμός Wt Αριθμός Tg Chow Diet (30 Zn) 6 10 High Fat Diet (3 Zn) 13 12 High Fat Diet (30 Zn) 9 8 High Fat Diet (300 Zn) 12 12 Σύνθεση των τριών διαιτών υψηλών λιπαρών -HFD: 3mg, 30mg, 300mg Zn /kg τροφής (mucedoola s.r.l Ιtaly-55% Cal από λιπαρά στοιχεία), Chow : δίαιτα ελέγχου Βιοχημικές αναλύσεις: Cholesterol ,Triglycerides ,HDL-cholesterol, Glucose ,Insulin Μετρήσεις μετάλλων και αντιοξειδωτικών παραγόντων: SOD (ερυθρά αιμοσφαίρια ) TAC (ορός αίματος) Zn , Cu (ερυθρά αιμοσφαίρια και ιστός ήπατος) Μεταβονομική ανάλυση και αξιολόγηση λιπιδίων :Εκχύλιση ορού αίματος και ηπατικού ιστού για λήψη Φάσματος 1H-NMR ΣΥΜΠΕΡΑΣΜΑΤΑ 1. Η HF δίαιτα αύξησε σημαντικά ,το τελικό βάρος και τον ρυθμό αύξησης βάρους ,τη χοληστερόλη -CL και την λιποπρωτεϊνη υψηλής πυκνότητας-ΗDL ,την ινσουλίνη –Ins, στα WT & Tg και μείωσε την ολική αντιοξειδωτική ικανότητα-TAC στο πλάσμα και την δραστηριότητα της σουπεροξείδιο-δισμουτάσης-SOD στα ερυθροκύταρα των WΤ και Tg 2. Ο επαρκής διατροφικός Zn (30mg/kgτροφής) φαίνεται να αποτελεί κρίσιμο παράγοντα διαμόρφωσης προστατευτικού ηπατολιπιδαιμικού προφίλ, ποντικών σε υπερλιπιδική δίαιτα. Η αύξηση ή μείωση του Zn σε HFDς φαίνεται να μειώνει την TAC του πλάσματος σε WΤ και Tg ενώ η δραστικότητα της SOD φαίνεται ανάλογη των επιπέδων του Zn Η ανεπάρκεια ή περίσσεια Zn αυξάνει τα αθηρογόνα SFA και μειώνει τα αντι-αθηρογόνα λιπίδια, PC, DU, LA DIAL ,UFA στις HDL, nonHDL και το ήπαρ, στα WΤHFD και στα ΤgHFD 3. Οι HSP70 : Τα διαγονιδιακά ζώα που φέρουν το γονίδιο hHsp70, φαίνεται ότι είτε σε έλλειψη είτε σε περίσσεια Ζn, που συνήθως συνοδεύoνται από μεταβολικό σύνδρομο, κατορθώνουν να διατηρούν καλλίτερους δείκτες λιπιδικού προφίλ σε σχέση με τα WT ζώα. Τα Τg εμφάνισαν σημαντικά χαμηλότερη CL ,TG , HDL σε σύγκριση με τα WT ενώ η HSP70 αύξησε σημαντικά την ενεργότητα της ολικής SOD στα ερυθρά αιμοσφαίρια. Η HSP70 μείωσε τα αθηρογόνα SFA και αύξησε τα αντιαθηρογόνα λιπίδια DIAL, DU, LA, UFA, SM στις HDL, nonHDL , και στο ήπαρ ποντικών που εκτέθηκαν σε HFD και σε όλες τις συγκεντρώσεις Zn. / Zinc –Zn controls lipid and glucose metabolism via Zn-transcription factors. High fat diets –HFDs lead to intracellular free radicals accumulation, decrease the transcriptional expression of their scavengers and anti-oxidative enzymes’ activity leading finally to metabolic syndrome. Heat shock proteins-HSPs play a significant role in cellular resistance response , as an adaptation mechanism, after exposure to various stimuli. SCOPE: To study the effects of nutritional Zn and HSP70s on the metabonomics of serum and liver lipids in mice MATERIALS-METHODS: Male mice: 1. Wild type (Wt) Hybrid (F1/F1) C57Bl/6 x CBA και 2. Transgenic (Tg) human HSP70 overexpressing mice, one month old, were subjected for 10 weeks to the following diets : Diet Number of Wt Number of Tg Chow Diet (30 Zn) 6 10 High Fat Diet (3 Zn) 13 12 High Fat Diet (30 Zn) 9 8 High Fat Diet (300 Zn) 12 12 Composition of the three HF Diets: 3mg, 30mg, 300mg Zn /kg τροφής (mucedoola s.r.l Ιtaly-55% Cal from greasy ingredients),Chow :control diet Biochemical analyses: Cholesterol ,Triglycerides ,HDL-cholesterol, Glucose ,Insulin Metals and antioxidative factors levels: SOD (red blood cells )TAC (blood serum) Zn , Cu (red blood cells and liver tissue) Lipids metabonomics : Blood serum and liver tissue extracts for 1H-NMR spectrum analysis and evaluation CONCLUSIONS 1. HFDiet significantly increased the rate of mice body weight gaining, as well as, cholesterol-CL, high density lipoproteins-HDL, insulin, in WT and Tg mice and decreased plasma total antioxidant capacity-TAC and red blood cell super oxide dismutase –SOD activity , in WT and Tg mice 2.Suficient nutritional Zn (30 mg/kg food) prevails as a crucial modulator of a protective hepato-lipidaemic profil of mice in high fat diet. Zinc deficiency or excessiveness , in HFDiets , decreases plasma TAC in WT and Tg mice, while SOD activity shows proportional to Zn levels. Zinc deficiency or excessiveness increases the atherogenic SFA and decreases the anti –atherogenic lipids : PC, DU, LA DIAL ,UFA, in HDL, nonHDL and liver, in WΤ-HFD and Τg-HFD mice 3. HSP70s : Transgenic mice over-expressing hHsp70 gene and exposed either to Zn deficiency or Zn excessiveness , both driving usually to metabolic syndrome , reveal significantly better lipid profile indicators, comparing to WT mice. Tg mice revealed significantly lower CL , TG , HDL levels compared to WT, while HSP70 in Τg mice significantly increased total SOD activity in red blood cells. HSP70 also decreased the atherogenic SFAs and increased the anti-atherogenic lipids DIAL, DU, LA, UFA, SM in HDL, nonHDL and in the liver of mice exposed to HFDiets and all Zn concentrations

Les urodèles amphibiens, dont fait partie l’axolotl (Ambystoma mexicanum), ont la capacité de régénérer leurs organes et membres suite à une amputation, tout au long de leur vie. La patte est l’organe dont le processus de régénération est le mieux caractérisé et ce dernier est divisé en deux phases principales. La première est la phase de préparation et commence immédiatement suite à l’amputation. Elle renferme des étapes essentielles au processus de régénération comme la guérison de la plaie et la formation d’une coiffe apicale ectodermique. Par la suite, les fibroblastes du derme et certaines cellules musculaires vont revenir à un état pluripotent via un processus appelé dédifférenciation cellulaire. Une fois dédifférenciées, ces cellules migrent et s’accumulent sous la coiffe apicale pour former le blastème. Lors de la phase de redéveloppement, les cellules du blastème se divisent puis se redifférencient pour régénérer la partie amputée. Fait intéressant, la régénération d’un membre ou la guérison d’une plaie chez l’axolotl ne mène jamais à la formation d’une cicatrice. Afin d’en apprendre plus sur le contrôle moléculaire de la régénération, les gènes Heat-shock protein-70 (Hsp-70) et Transforming growth factor-β1 (Tgf-β1) ont été sélectionnés. Ces gènes jouent un rôle important dans la réponse au stress et lors de la guérison des plaies chez les mammifères. HSP-70 est une chaperonne moléculaire qui est produite pour maintenir l’intégrité des protéines cellulaires lorsqu’un stress se présente. TGF-β1 est une cytokine produite suite à une blessure qui active la réponse inflammatoire et qui stimule la fermeture de la plaie chez les amniotes. Les résultats présentés dans cette thèse démontrent que Hsp-70 est exprimé et régulé lors du développement et de la régénération du membre chez l’axolotl. D’autre part, nos expériences ont mené à l’isolation de la séquence codante pour Tgf-β1 chez l’axolotl. Nos résultats montrent que Tgf-β1 est exprimé spécifiquement lors de la phase de préparation dans le membre en régénération. De plus, le blocage de la voie des Tgf-β avec l’inhibiteur pharmacologique SB-431542, lors de la régénération, mène à l’inhibition du processus. Ceci démontre que la signalisation via la voie des Tgf-β est essentielle à la régénération du membre chez l’axolotl. / Urodele amphibians, such as the axolotl (Ambystoma mexicanum), have the unique ability, among vertebrates, to perfectly regenerate many parts of their body throughout their life. Among the complex structures that can be regenerated, the limb is the most widely studied. Limb regeneration is divided in two main phases. The preparation phase, which begins right after amputation, includes wound healing and the formation of an apical ectodermal cap. During this phase, dermal fibroblasts and muscle cells will lose their characteristics and become pluripotent through a process called cellular dedifferentiation. The dedifferentiated cells migrate and accumulate under the apical ectodermal cap to form the blastema. During the redevelopment phase, the cells in the blastema proliferate and redifferentiate to regenerate the lost structures. It is interesting to highlight the fact that regeneration never leads to scar formation in the axolotl. In order to learn more about the molecular control of limb regeneration, the genes Heat-shock protein-70 (Hsp-70) and Transforming growth factor-β1 (Tgf- β1) were selected for their important roles in stress response and wound healing in mammals. HSP-70 is a molecular chaperone which is produced to protect cellular proteins when the cell faces a stress. TGF-β1 is a cytokine produced after wounding that activates the inflammatory response and stimulates wound closure in amniotes. Results presented in this thesis show that Hsp-70 is expressed and regulated during limb development and regeneration in the axolotl. We were also able to isolate the cDNA coding for axolotl Tgf-β1 and our results show that this gene is expressed specifically during the preparation phase of limb regeneration. Treatment of regenerating axolotls with a specific inhibitor of Tgf-β signalling, SB-431542, led to complete inhibition of regeneration. This directly implies that Tgf-β signalling is essential for limb regeneration in axolotl.