Yes / Articular cartilage, a connective tissue, contains chondrocytes and glycosaminoglycans (GAGs) which aid in water retention, providing the tissue with its magnificent ability to prevent friction, withstand loads and absorb compressive shocks however, cartilage, does not have the ability to regenerate and repair. Osteoarthritis (OA) is a progressive degenerative disease, which includes reduction of cartilage thickness between two bones in a joint, causing painful bone-to-bone contact. OA affects over 8 million people in the UK alone. , and as the primary causes are unknown, available treatments including surgical and non-surgical techniques which only reduce the symptoms created by the disorder instead of providing a cure. This project focused on utilizing TGF-β1, a cytokine found in elevated amounts in healthy cartilage when compared to degraded cartilage, in order to observe the effects of the growth factor on both healthy and osteoarthritic chondrocytes. The healthy and the osteoarthritic chondrocytes were cultured in two different media (DMEM with and without TGF- β1) before utilizing the SpectraMax M2/M2e plate reader to observe and analyze the effect of TGF-β1 on water retention properties of cells. This has been achieved by quantifying the GAG content using DMMB dye. Results showed that although TGF-β1 did displayed an increase in glycosaminoglycan synthesis, the statistical increase was not vast enough for the alternative hypothesis to be accepted; further experimentation with TGF-β1, alongside other cytokines within the growth factor family is needed to perceive the true influence of the growth factor on un cured degenerative diseases. It was concluded that both the healthy and osteoarthritic cells treated with TGF-β1 absorbed considerably more DMMB in comparison to the cells, suggesting that TGF-β1 indeed works to aid in water retention. TGF-β1 is a key factor to be exploited when constructing treatments for osteoarthritis

Yes / Osteoarthritis (OA) is a degenerative disease leading to the breakdown of the hyaline cartilage between a varieties of diarthrodial joints such as the knee joint, carpals of the wrist and etc. When the cartilage is affected by trauma or wear and tear, Osteolysis may occur; broken debris of cartilage found within the synovial fluid may be recognised as a pathogen and therefore, the body’s autoimmune response will directly target the cartilage for destruction. Cytokines are proteins/peptides of glycoproteins that are secreted by cells and are involved in interaction and communication between cells. Transforming Growth Factors Beta 1 (TGF-β1) is one of well-known cytokines and had shown many effects on cellular biology including simulation or inhibition of cell proliferation, differentiation, production of extracellular matrix (ECM), remodelling, and producing both hormones and growth factors. On the other hand, Photonics recently play an important role for treatment of OA. The main aim of this review article is to investigate the effect of TGF-β1 in treatment of OA. Other important aim of this work is to explore the broad applications of optics and photonics in biomedical applications including treatment of OA. Biomedical applications of photonics have broad aspects including laser, carbon nanotubes (CNTs), quantum dots (QDs) and graphene and photodynamic therapy (PDT) which discussed in this review paper.

Ce travail a été mené afin d’étudier l’effet du FGF 2 et du TGF-β1 sur les étapes précoces de la régénération dentinaire en utilisant la micro-encapsulation de ces facteurs dans une matrice pour les protéger et contrôler leur libération et ensuite l’application des microparticules obtenues en coiffage pulpaire direct dans un modèle de culture de dents entières. Ce travail consiste d’abord à l’optimisation des moyens techniques mis en oeuvre pour réaliser l'encapsulation du TGFβ1, FGF-2 à l'aide de l'acide poly (lactique-glycolique) PLGA. Les études de la caractérisation colloïdal et physico chimique des microparticules montre que les microparticules gardent leurs caractéristiques physicochimiques après séchage et resuspension dans l’eau. La procèdes optimisé a été ensuite utilisé pour encapsuler les facteurs de croissance. L’encapsulation de FGF-2 et TGF-β1 a été obtenue avec une taille, une efficacité d’encapsulation et une profile de libération adaptés au type d’application choisi. Les études biologiques ne montrent aucun effet toxique des particules sur les fibroblastes pulpaires. Les facteurs de croissance ont gardé leur activité biologique spécifique. Un modèle de culture de dent entier humain a été utilisé pour réaliser l’application de nos microparticules comme un matériau de coiffage dentaire pour confirmer leurs activités biologiques ex-vivo. Ces microparticules peuvent être utiles dans les études des étapes précoces de la régénération dentinaire, l'activation et la migration des cellules progénitrices de la pulpe dentaire / The purpose of this work was to investigate the effect of FGF 2 and TGF-β1 on the early steps of dentin regeneration using microencapsulation of theses factors into a microparticles matrix to ensure growth factors protection and to provide bioactive sustained release in contact with dental pulp cells and then the application of the obtained microparticles in direct pulp capping using a culture model of entire tooth. This work involves the optimization of technical means used to achieve encapsulation of TGFβ1, FGF-2 using the poly (lactic-glycolic acid) PLGA. Physicochemical and colloidal characterization of microspheres shows that the microparticles retain their physicochemical characteristics after drying and re-suspended in water. The double emulsion method was used to separately encapsulate (FGF-2) and (TGFβ1). Microparticles morphology, loading, shelf life, potential toxicity and release kinetics were studied. Then the proliferation of dental pulp cells was examined in contact with microparticles. Biological studies show no toxic effect of particles on pulp fibroblasts. Growth factors have kept their specific biological activity. A culture model of human entire tooth was used to achieve the application of microparticles as a dental direct capping material to confirm their biological activities ex vivo. These microparticles can be useful in studies of early steps of dentin regeneration, activation and migration of progenitor cells in dental pulp

Epithelial to Mesenchymal Transition (EMT) is a dynamic process by which a distinct change in the phenotype and function of epithelial cells render them as mesenchymal cells. Characteristics of mesenchymal cells include the ability to invade, increased migratory kinetics and heightened resistance to apoptosis. Therefore, there is a strong need to fully understand the mechanism for the induction of EMT in pathological conditions such as carcinoma progression. Recent advances highlight two pivotal contributors, soluble growth factor (gf) signals, and mechanical signals, in the process. However, to date, no clear mechanism exists linking the two in epithelial transdifferentiation. Transforming Growth Factor-β1 (TGF-β1), a gf known to induce EMT in breast cancer formation, induces EMT on rigid surfaces and apoptosis on compliant surfaces. It is our belief that a combination of mechanical signals, gf signals, and the type of extracellular matrix (ECM) proteins assembled by cells together drive the process of EMT. Here we investigated the role of the ECM protein fibronectin (FN) in EMT. Upon assembly into elastic, insoluble fibrils through cell-generated forces which become larger on stiffer surfaces, FN is able to serve as a gf delivery system. We examined the following hypothesis: Increased tissue stiffness drives FN assembly, which exposes cryptic binding sites for various gfs, such as TGF-β1, and creates a high concentration of these gfs at the cell surface, which in turn drives EMT. In this project we investigated three aims: (1) evaluate the effect of inhibiting FN fibrillogenesis and gf localization on TGF-β1-induced EMT, (2) assess the effect of TGF-β1 concentration on spatial patterning of ECM dynamics, cell phenotype and adherens junctional force, and (3) probe the role of the FN matrix in TGF-β1-induced spatial patterning of EMT. Results showed that both inhibition of FN fibril assembly and blocking the gf binding site on fibrils significantly attenuated the downstream effects of EMT. In microcontact patterns of epithelial colonies, increasing gf concentration led to spatial patterning of FN fibrils, cell phenotype and cell-cell junctional force. Elimination of FN fibrils effectively attenuated TGF-β1-induced spatial patterning. The knowledge acquired through these studies serves as an addition to an increasingly important body of work aimed at elucidating how physical changes within the microenvironment regulate physiology and pathology. By establishing a novel mechanism by which gf signaling induces EMT through interaction with the extracellular matrix, this research serves to combat the development and initiation of pathological phenomena, such as metastasis.

Sex steroids are inevitable in women. However, long-term exposure to sex steroids increases the risk of breast cancer. A complete understanding of sex steroid control of the breast and how it relates to breast cancer risk is still lacking. Angiogenesis and proteolytic enzyme activity are crucial for the process by which tumors evolve into a vascularized, invasive phenotype. Matrix metalloproteinases are potent matrixdegrading enzymes that affect several steps in tumor progression including angiogenesis. In the female reproductive organs, sex steroids regulate angiogenesis and MMP activity, yet little is known how sex steroids affect these crucial events in normal and malignant breast tissue. This thesis elucidates a link between sex steroids, MMP activity, and angiogenesis. It is shown that estradiol down-regulates while tamoxifen up-regulates the protein expression and activity of MMP-2 and MMP-9 in human breast cancer cells in vitro and in human breast cancer xenografts in vivo. The results further suggest that a biological consequence of this regulation may be modulation of tumor angiogenesis. The net effect of adding tamoxifen to estradiol treatment was an increase in extracellular levels of the endogenous angiogenesis inhibitor endostatin and decreased levels of the tumor promoter TGF-β1 compared to estradiol treatment only. This was accompanied by reduced vasculature and decreased tumor growth. Similarly, a regulatory effect of estradiol and tamoxifen on endostatin generation was observed in normal human breast tissue by whole-tissue culture and microdialysis in human breast tissue in situ. In conclusion, the results presented in this thesis suggest previously unknown mechanisms of action of estradiol and tamoxifen in breast cancer and in normal human breast tissue, and novel means by which estradiol may tip the scale to favor angiogenesis. This knowledge may be important for the understanding of sex steroid dependent breast carcinogenesis and in the future development of tissue-specific preventive as well as therapeutic strategies against breast cancer.

Mesenchymal stem cells (MSCs) are bone marrow derived multipotent cells with the ability to self-renew and differentiate into multiple connective cell lineages. In vivo, MSCs travel from the bone-marrow to the inflammatory sites and actively participate in remodeling and regeneration process under the influence of soluble growth factors. Due to these inherent properties, MSCs have emerged as an ideal candidate for diverse regenerative therapeutic applications. The development of MSC-based therapies requires in vitro expansion of MSCs; however, MSC expansion results in phenotypical changes that have limited its efficacy upon reintroduction in vivo. In order to increase the efficacy of MSC-based therapeutics, it is critical for us to improve the current understanding of MSC interactions with its niche specific factors and explore new methods to enhance MSC function in vivo. We used tumor conditioned media, which contains soluble factors secreted by tumor cells in culture (TCM), and inflammatory niche-specific soluble factors, such as platelet derived growth factor (PDGF) and transforming growth factor-β1 (TGF-β1), to characterize the mechanical response of MSCs. The intracellular mechanical properties of MSCs were dramatically altered in response to soluble factors and MSCs displayed cytosolic stiffening in response to TCM and TGF-β1. Although PDGF treated cells did not elicit any mechanical response, blocking PDGF signaling with a small molecule inhibitor reversed the stiffening response in TGF-β1 treated cells, indicating crosstalk between these two pathways is essential in TGF-β1 mediated cell stiffening. Furthermore, a genome-wide microarray analysis revealed TGF-β1 dependent regulation of cytoskeletal actin-binding protein (ABP) genes. Actin crosslinking and bundling protein genes, which regulate cytosolic rheology through changes in semiflexible actin polymer meshworks, were upregulated with TGF-β1 treatment. Since TGF-β1 treatment profoundly altered the MSC phenotype after relatively short exposure times, we sought to understand if pretreated cells could sustain these enhanced characteristics leading to higher efficacy in vivo. We found that MSCs pretreated with TGF-β1 displayed enhanced adhesive properties while maintaining the expression profile of surface adhesion molecules even after removal of stimulus. Additionally, pretreated MSCs exposed to lineage specific induction media, demonstrated superior differentiation potential along multiple lineages. Based on the large number of sustained changes, TGF-β1 pretreated cells were used to treat full thickness skin wounds for in vivo wound healing model to determine their therapeutic efficacy. TGF-β1 pretreated MSCs increased wound closure rate and displayed enhanced migration of MSCs towards the center of the wound compared to the control cells. In conclusion, soluble factor pretreated MSCs with altered mechanical properties displayed significantly improved cell functions leading to highly efficient tissue regeneration in vivo. Mechanical priming of MSCs with niche specific factors prior to transplantation can become a viable strategy to maximize their therapeutic potential.

Submitted by TATIANA EMY KOIKE null (tatikoike@yahoo.com) on 2016-03-10T11:09:02Z No. of bitstreams: 1 Encadernaçao Mestrado UNESP- Tatiana Emy Koike.pdf: 1535953 bytes, checksum: 25ca75a268f1c6bf7d7ca80e833b9cbd (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-03-10T20:19:49Z (GMT) No. of bitstreams: 1 koike_te_me_prud.pdf: 1535953 bytes, checksum: 25ca75a268f1c6bf7d7ca80e833b9cbd (MD5) / Made available in DSpace on 2016-03-10T20:19:49Z (GMT). No. of bitstreams: 1 koike_te_me_prud.pdf: 1535953 bytes, checksum: 25ca75a268f1c6bf7d7ca80e833b9cbd (MD5) Previous issue date: 2016-02-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Muscle injuries are often cause due to the practice of sports and recreational activities. Because of its high incidence, it is important to research the treatments that promote quality in the muscle and shorter repair process. To evaluate the effect of the combination of the therapeutic resources of Cryotherapy and Therapeutic Ultrasound in the treatment of muscle damage by impact. 55 Wistar rats was separate into groups, Acute Injury (AI), Injury (I), Cryotherapy (CR), Therapeutic Ultrasound (TU), Cryotherapy and Therapeutic Ultrasound (CRTU). All animals were anesthetize and muscle damage due to impact by the release of a load of 200 g at 30 cm. Then received treatments as allocated group and CR protocol using ice bag for 20 minutes and TU for five minutes with an intensity of 0.5W / cm2 and the frequency of 1MHz. Euthanasia was performed by intraperitoneal administration of overdose of Xylazine and Ketamine. The collection of the gastrocnemius muscle for the Body and Muscle mass analysis, histological analysis and fractal dimension of inflammation and collagen gene quantification of mRNA (TNF-α and TGF-β1). Data analysis was performed using SPSS for Windows 22. The Shapiro-Wilk test to verify the normality of the data was performed. When data showed normal, we used t test for paired samples test and one-way ANOVA followed by Tukey’s post-test. When it violated the normality of the data, followed by the Kruskal- Wallis test with Dunn’s post-test. For all analyzes was adopted the significance level of 5%. Among all groups, the CRTU lose less body and muscle mass, improved morphometry, besides presenting collagen reduction by DF compared to AI and CR (p <0.05). With regard to the inflammatory process CRTU group showed a significant reduction of DF in relation to the AI groups (p = 0.001), I (p = 0.001) and CR (p = 0.007), and TU reduced the DF significantly relative to AI groups (p = 0.001), I (p = 0.001) and CR (p = 0.036). The reduction of TNF-α was significant in TU group compared with AI groups (p = 0.008); I (p = 0.032) and CR (p = 0.046) and TGF- β1 in the CR group compared to AI (p = 0.001) and I (p = 0.006), in the TU group compared to AI (p = 0.049) and CRTU compared to AI (p = 0.023). The combination treatment was superior to the results presented by the isolated treatments in the muscle repair process. Observed by reducing the loss of body and muscle mass, improved histological appearance and reduction of collagen. / As lesões musculares são frequentemente ocasionadas em decorrência da prática de atividades esportivas e recreativas. Devido sua alta incidência, é importante pesquisar os tratamentos que promovam qualidade no processo de reparação muscular e menor duração. Avaliar o efeito da combinação dos recursos terapêuticos de Crioterapia e Ultrassom Terapêutico no tratamento de lesão muscular por impacto. 55 ratos wistar foram separados em Grupos Lesão Aguda (LA), Lesão (L), Crioterapia (CR), Ultrassom Terapêutico (US), Crioterapia e Ultrassom Terapêutico (CRUS). Todos os animais foram anestesiados e submetidos à lesão muscular por impacto pela liberação de uma carga de 200g a 30 cm de altura. Em seguida receberam os tratamentos conforme grupo alocado, sendo o protocolo de CR por meio de bolsa de gelo durante 20 minutos e o US durante cinco minutos com intensidade de 0,5W/cm2 e frequência de 1MHz. A eutanásia foi realizada por administração intraperitoneal de superdosagem de Xilazina e Ketamina, para subsequente coleta do músculo gastrocnêmio destinado às análises de massa Corporal e Muscular, análises Histológica e Dimensão Fractal do processo inflamatório e de colágeno, Quantificação gênica de RNAm (TNF-α e TGF-β1). A análise dos dados foi realizada utilizando o programa estatístico SPSS 22 for Windows. Foi realizado o teste de Shapiro-Wilk para verificação da normalidade dos dados. Quando os dados apresentaram normalidade, foi utilizado teste T para amostras pareadas e teste de Anova one-way, seguido pelo pós-teste de Tukey. Quando violada a normalidade dos dados, seguiu-se com o teste de Kruskall-Wallis, com pós-teste de Dunn. Para todas as análises foi adotado o nível de significância de 5%. Dentre todos os grupos, o CRUS perdeu menos massa corporal e muscular, melhora da morfometria, além de apresentar redução de colágeno pela DF em comparação aos LA e CR (p < 0,05). Com relação ao processo inflamatório, grupo CRUS apresentou redução significante da DF em relação aos grupos LA (p = 0,001), L (p = 0,001) e CR (p = 0,007), e o US reduziu a DF significativamente em relação aos grupos LA (p = 0,001), L (p = 0,001) e CR (p = 0,036). A redução de TNF-α foi significante no grupo US comparado com grupos LA (p = 0,008); L (p = 0,032) e CR (p = 0,046), e TGF-β1 no grupo CR em comparação aos LA (p = 0,001) e L (p = 0,006), no grupo US em comparação ao LA (p = 0,049), e CRUS em comparação aos LA (p = 0,023). A associação de tratamentos foi superior aos resultados apresentados pelos tratamentos isolados no processo de reparação muscular. Observado pela redução da perda de massa corporal e muscular, melhora do aspecto histológico e redução de colágeno.

Patients with classical vitiligo lose partially their protecting inherited pigment. The cause of the disease is still unknown. Despite massive epidermal oxidative / nitrative stress and signs for DNA-damage in the skin and in the plasma, these patients have no higher prevalence for sun induced non-melanoma skin cancer and increased photo-damage. Protection and DNA-repair have been attributed to a functioning up-regulated wild type p53 / p21 cascade in association with up-regulated p76 MDM2. As some patients with cutaneous melanoma develop depigmentations away from their primary tumour site post surgical excision, it became of our interest, whether this melanoma associated leucoderma (MAL) is the same as classical vitiligo. The purpose of this thesis was two-fold. In part I, we wanted to further substantiate the reasons behind the constantly up-regulated wild-type functioning p53 / p21 cascade in classical vitiligo utilising a panel of proteins with direct and / or indirect action on p53 regulation, including p21, p76MDM2, MDM4/MDM4phospho, SPARC, VEGF-A and TGF-β1. In part II, we wanted to characterize MAL and compare this peculiar leucoderma with classical vitiligo using the same protein panel and methodologies. To achieve our goals, we used in vivo FT-Raman spectroscopy, in vitro cell cultures, in vitro and in situ immuno-fluorescence labelling, Western blot, dot blot and computer modelling techniques. Our data showed distinct differences between classical vitiligo and MAL. Our results in MAL exhibited a concentration dependent protein expression gradient between the basal / suprabasl layers and the upper layers of the epidermal compartment using catalase, ONOO-, p53, p21, MDM4, p76MDM2, TGF-β1 and VEGF-A expression gradient. Moreover, we document for the first time the presence of a nitrated non-fuctional SPARC protein in classical vitiligo which is absent in MAL. Although we show in vivo considerable ROS / RNS- mediated stress in MAL and classical vitiligo documented by FT-Raman spectroscopy, Western blot and in situ immuno-fluorescence, our results prove that MAL and classical vitiligo are two distinct entities.

Fibrosis is a disease where the normally transitory wound healing response enters a chronic state. Bleomycin and Adenovector models of pulmonary fibrosis have implicated TGF-β1 in this disease. Concern regarding a synergistic combination of TGF-β1 with an adaptive immune response within the Adenovector model prompted its use within mice devoid of T Lymphocytes, Balb/c SCIDs. The lack of an adaptive immune response within these mice did not affect the severity of fibrogenesis, as compared to Balb/c data in a hydroxyproline assay. TGF-β1 is a pluripotent cytokine with key roles in wound healing, immune regulation, and development, making it a dangerous molecule to therapeutically modulate directly. Future strategies will likely focus on downstream fibrotic molecules uninvolved in immune regulation, such as CTGF. While CTGF has been associated with fibrosis and is likely activated by TGF-β1, no conclusive evidence is available within an animal model. TGF-β1 stimulates cells by binding its receptor and signaling through the Smad signal transduction pathway. Smad3 knockout mice were used to examine the regulation of CTGF by TGF-β1, and study its role in pulmonary fibrosis. We show that these mice produce dramatically less CTGF in response to TGF-β1 than littermates expressing Smad3, and they show protection against TGF-β1 induced pulmonary fibrosis, using the Adenovector system. TGF-β1 can alter lung development, and is thought to be a causative agent in Bronchopulmonary Dysplasia, a disease affecting immature lungs. Utilizing the Adenovector system, we developed a neonatal rat model of BPD that closely resembles the human disease, providing researchers with a system to study the disease course. TGF-β1 is part of a family of growth factors, of which TGF-β3 is also a member. What role TGF-β3 plays in pulmonary fibrosis has not been evaluated. To allow future in vivo studies on the effect of TGF-β3 on lung morphology, we constructed a replication deficient Adenovector expressing constitutively active TGF-β3. / Thesis / Master of Science (MSc)

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.