Over 100 years ago, X-ray crystallography gave scientists a window to the atomic world with varied applications in biology, chemistry and physics among other subjects. Macromolecular crystallography is now considered an essential tool for solving the three-dimensional structure of proteins and understanding their physiological role at the atomic level. As crystal growth remains a bottleneck in crystallography, various other techniques are often employed to help understand the protein structure and function. These methods range from simple analysis of the protein sequence to experiments such as dynamic light scattering, isothermal titration calorimetry, activity assays, and analytical ultracentrifugation. The additional knowledge gained about proteins from these methods can then assist in the modification of the protein to facilitate its crystallisation. The structural biology of proteins belonging to two diverse families; Galectins and Sirtuins, both involved in the regulation of cancer, was studied in this thesis. Galectins are evolutionarily conserved and ubiquitously present animal lectins with a high affinity for -galactose containing oligosaccharides. To date, 15 mammalian galectins have been identified. Their involvement in cell–cell and cell–matrix interactions has highlighted their importance in signal transduction and other intracellular processes. Human Galectin-7 (hGal-7) is a 16 kDa prototype galectin which is involved in the stimulation and development of cancer. The crystal structure of native hGal-7 and its complex with galactose and lactose have been reported. In this study, cross-linking of hGal-7 by glycodendrons and the resulting clustering and lattice formation have been studied. For this purpose, the high resolution X-ray structures of hGal-7 in complex with carbohydrate-based multivalent dendrons have been elucidated and analysed. Also discussed in this thesis are preliminary binding affinity results obtained using isothermal calorimetry. Supramolecular assembly formation was also assessed using dynamic light scattering. These experiments reveal how multivalent glycodendendrons interact with and form cross-links with hGal-7 molecules. Understanding how these dendrimeric compounds interact with hGal-7 would help in the design of new tools to investigate the recognition of multivalent carbohydrates by lectins and their resulting role in aggregation processes in tumour embolisation and survival. Sirtuins are NAD+-dependent deacylases that are involved in the regulation of diverse biological functions such as ageing, metabolism and stress resistance, in normal cellular physiology. Their role in ageing and ageing-related diseases, including cancer and neurodegenerative diseases among others, has received much attention and sirtuins have been extensively studied to help in extension of human lifespan. Seven members of the sirtuin family (SIRT1-7) are known, which are diversely sub-localised in the cell. A myriad of questions regarding their deacylation activity, their interplay and their role in various diseases still remain unanswered. In this study, structural biology techniques have been used to understand the role of SIRT1, SIRT2 and SIRT7. The cloning, expression, purification and crystallisation of these sirtuins are presented in this thesis. Various supporting techniques used to confirm the identity and activity of the proteins are also discussed. A brief discussion of the methods that can be employed to overcome various barriers in structural biology is also presented in this thesis. Elucidating the structure of full length sirtuins would help in the development of highly selective modulators of sirtuins to aid in the understanding of their role in ageing and ageing-related diseases.

Les galectines forment une famille de lectines solubles impliquées dans de multiples processus. Elles sont caractérisées par la présence d’un domaine de liaison aux carbohydrates conservé au cours de l’évolution et une affinité particulière pour les β-galactosides. Au cours de ce projet doctoral, nous nous sommes intéressés à galectine-7, une lectine exprimée spécifiquement dans les épithéliums pluristratifiés, comme l’épiderme. Grâce aux modèles de souris invalidées pour galectine-7 ou surexprimant galectine-7, notre équipe a précédemment montré que cette protéine était impliquée dans l’adhérence entre les cellules de l’épiderme et dans la migration collective, deux processus clés de la progression tumorale et de la cicatrisation épidermique. Cependant, les mécanismes moléculaires sous-jacents restent à élucider.En combinant différentes approches, nous avons pu déterminer que le retard de migration observé en absence de galectine-7 pouvait s’expliquer, du moins en partie, par une diminution de la coordination et du comportement collectif des kératinocytes en migration. De plus, nos données montrent que galectine-7 interagit directement avec le domaine extracellulaire de la E-cadhérine, un des composants majeurs des jonctions adhérentes et une protéine clé dans la migration collective. De façon surprenante, cette interaction ne fait pas intervenir de groupements carbohydrates. Tentant de préciser le rôle de galectine-7 au niveau des jonctions adhérentes, nous avons identifié une nouvelle fonction de galectine-7 dans la stabilisation de la E-cadhérine à la membrane plasmique. De manière intéressante, l’augmentation du renouvellement de la E-cadhérine à la membrane plasmique causée par l’extinction de galectine-7 est également couplée à une baisse de la force de l’adhérence intercellulaire. Enfin, nos expériences indiquent que cette nouvelle fonction de galectine-7 requiert une activité lectine fonctionnelle, suggérant l’implication d’un autre acteur glycosylé dans ce mécanisme de régulation de la E-cadhérine par galectine-7.En conclusion, ce doctorat a permis de préciser le rôle de galectine-7 dans la migration collective et de découvrir une fonction non-décrite auparavant de galectine-7 dans la régulation de la dynamique de la E-cadhérine. Cette modulation de la E-cadhérine par galectine-7 pourrait permettre à la cellule de s’adapter aux perturbations de l’environnement, comme c’est le cas au cours de la migration collective. En effet, les galectines étant des molécules avec une capacité de redistribution rapide, ce sont de bons candidats pour créer des réponses adaptatives / Galectins composed a family of soluble lectins implicated in multiple processes. They are characterized by the presence of a carbohydrate recognition domain evolutionary conserved and an affinity for β-galactosides containing sugars. During this thesis, we focused on a protein called galectine-7 whose expression is restricted to stratified epithelia such as the epidermis. Using mouse models with altered expression of galectin-7, our team previously showed that this protein participates in intercellular adhesion and collective cell migration, two key processes in tumour progression and epidermal wound healing. However, the underlying mechanisms remain to be elucidated. Combining different approaches, we discovered that the migration delay observed in the absence of galectin-7 during wound healing could be explained, at least in part, by a reduction of cell coordination and collective cell behaviour of migrating keratinocytes. Moreover, our data showed that galectin-7 directly interact with E-cadherin, a key component of adherent junctions and a major player in collective migration. Surprisingly, this binding did not involve carbohydrate groups. Aiming to precise the role of galectin-7 at adherent junctions, we identified a new function of galectin-7 in the stabilisation of E-cadherin at the plasma membrane. Interestingly, the increased of E-cadherin turnover caused by galectin-7 extinction is also associated to a decreased of the strength of adherent junctions-based intercellular adhesion. Eventually, our experiments indicated that this previously unknown function of galectin-7 required a functional lectin activity, suggesting the involvement of an additional glycosylated actor in this regulation mechanism of E-cadherin dynamics by galectin-7.In conclusion, this thesis allowed to precise the role of galectin-7 in collective cell migration and revealed a novel function of galectin-7 in the regulation of the E-cadherin stability at the plasma membrane. This modulatory effect of galectin-7 on E-cadherin could provide the cell a possible adaptive response to environmental perturbations, as during collective cell migration. Indeed, galectins, because they exhibit rapid redistribution capacities, are good candidates to create adaptive responses

Galectins are proteins with 15 known members found in nearly all living organisms. They share a conserved CRD that binds beta-galactoside sugars, and functions to cross-link glycoproteins as well as glycolipid receptors on the surface of cells to initiate biological responses. Functional studies on the extracellular and intracellular roles of galectins implicate them in cell adhesion, chemoattraction and migration as well as growth, differentiation and apoptosis. Therefore, studies were conducted to identify functional roles of galectin 15 (LGALS15) during the periimplantation period of pregnancy in the sheep. The first study was designed to develop and characterize primary ovine trophectoderm cell lines for the study of the biological functions of LGALS15. Once characterized, these cell lines were used to investigate the role of LGALS15 in trophectoderm gene expression, development, growth, and survival. Two primary trophectoderm cell lines (oTr1 and oTrF) were developed, and they had characteristics similar to in vivo conceptus trophectoderm relative to gene expression, morphology, and migration and proved suitable as an in vitro model to investigate functional roles of LGALS15. The second study investigated LGALS15 function in trophectoderm cell adhesion. A dose-dependent increase in oTr cell attachment to LGALS15 was found that could be inhibited by cyclic GRGDS, but not GRADS, peptides. Mutation of the LDVRGD integrin binding sequence of LGALS15 to LADRAD decreased its ability to promote oTr cell attachment, whereas mutation of the CRD had little effect. LGALS15 induced formation of robust focal adhesions in oTr cells that were abolished by mutation of the LDVRGD sequence. The third study tested the hypothesis that LGALS15 is a secreted regulator of trophectoderm development and gene expression, as well as growth, migration, and apoptosis of trophoblast. LGALS15 moderately increased cellular proliferation, partially inhibited staurosporine elicited apoptosis, stimulated migration that was dependent on Jun N-terminal kinase (JNK), and initiated differential gene expression of oTr cells. Collectively, these results support the hypothesis that LGALS15 has a biological role in the peri-implantation stage of early pregnancy in the ovine uterus and stimulates trophectoderm cell gene expression, migration and attachment via integrin binding and activation which are critical to blastocyst elongation and implantation.

A família das galectinas compreende um grupo de lectinas cujos domínios de reconhecimento de carboidratos (CRDs) possuem afinidade específica para ß-galactosídeos. Estas se encontram amplamente distribuídas em células normais e neoplásicas de diferentes organismos e estão envolvidas em uma grande diversidade de mecanismos celulares. As galectinas têm sido foco de estudos recentes, principalmente pelo seu envolvimento em processos inflamatórios e neoplásicos, entretanto, muitas perguntas sobre as interações com diferentes carboidratos, a especificidade destas interações e o papel específico das galectinas em inflamação, adesão celular, progressão tumoral e metástase permanecem ainda sem resposta. O presente projeto focou os estudos estruturais dos domínios de reconhecimento de carboidratos (CRDs) da galectina-4 humana (HGal-4). Nosso trabalho envolveu a clonagem, expressão, purificação dos domínios de reconhecimento de carboidratos (CRD-I e CRD-II) de forma independente. O domínio CRD-I da HGal-4 foi cristalizado e sua estrutura determinada por técnicas de cristalografia de raios-X a 2 Å de resolução. A estrutura cristalográfica do domínio CRD-I da galectina-4 humana possue duas folhas- compostas de seis fitas (S1- S6) e cinco fitas (F1-F6) enoveladas na forma de um -sanduiche. Uma comparação estrutural entre membros da classe das galectinas mostra que este enovelamento global dos CRDs é conservado e que e a diferença em especificidade pelos carboidratos observado pelas diferentes galectinas é consequência de mutações pontuais de aminoácidos. Os resultados obtidos no desenvolvimento do presente projeto serão utilizados como uma ferramenta importante para o entendimento de processos celulares que envolvem a galectina-4 humana, como inflamação, progressão celular e metástase, e conseqüentemente, contribuir para o planejamento de novas estratégias de diagnóstico e tratamento de neoplasias. / The galectin family comprises a group of lectins where the carbohydrate-recognition domains (CRDs) display specific affinity for ß-galactosides. They are widely distributed in normal and neoplasic cells of different organisms and are involved in a great diversity of cellular mechanisms. The galectins have been focus of recent studies, mainly for their involvement in inflammatory and neoplasic processes, however, many questions about the interactions with different carbohydrates, the specificity of these interactions and the specific role of the galectins in inflammation, cell adhesion, tumor progression and metastasis remain unanswered.The present project focused the strctural studies of human galectin-4 (HGal-4) carbohydrate-recognition domains (CRDs). Our work involved the independent cloning, heterologous expression and purification of both carbohydrate-recognition domains (CRD-I and CRD-II). The HGal-4 CRD-I domain has been successfully crystallized and its structure solved by X-ray crystallography techniques at 2 Å resolution. The crystallographic structure of HGal-4 CRD-I domain comprises two -sheets containing six (S1- S6) and five strands (F1-F6) each packed as a -sandwich domain. A structural comparison among members of galectin class of proteins shows that this folding is highly conserved and that the difference in specificity for carbohydrate molecules is consequence of punctual aminoacid mutations. Our results will be used as an important tool towards a better understanding of cellular processes such as inflammation, cell progression and metastasis, and consequently, contribute for the development of new strategies for diagnosis and treatment of neoplasies.

A família das galectinas compreende um grupo de lectinas cujos domínios de reconhecimento de carboidratos (CRDs) possuem afinidade específica para ß-galactosídeos. Estas se encontram amplamente distribuídas em células normais e neoplásicas de diferentes organismos e estão envolvidas em uma grande diversidade de mecanismos celulares. As galectinas têm sido foco de estudos recentes, principalmente pelo seu envolvimento em processos inflamatórios e neoplásicos, entretanto, muitas perguntas sobre as interações com diferentes carboidratos, a especificidade destas interações e o papel específico das galectinas em inflamação, adesão celular, progressão tumoral e metástase permanecem ainda sem resposta. O presente projeto focou os estudos estruturais dos domínios de reconhecimento de carboidratos (CRDs) da galectina-4 humana (HGal-4). Nosso trabalho envolveu a clonagem, expressão, purificação dos domínios de reconhecimento de carboidratos (CRD-I e CRD-II) de forma independente. O domínio CRD-I da HGal-4 foi cristalizado e sua estrutura determinada por técnicas de cristalografia de raios-X a 2 Å de resolução. A estrutura cristalográfica do domínio CRD-I da galectina-4 humana possue duas folhas- compostas de seis fitas (S1- S6) e cinco fitas (F1-F6) enoveladas na forma de um -sanduiche. Uma comparação estrutural entre membros da classe das galectinas mostra que este enovelamento global dos CRDs é conservado e que e a diferença em especificidade pelos carboidratos observado pelas diferentes galectinas é consequência de mutações pontuais de aminoácidos. Os resultados obtidos no desenvolvimento do presente projeto serão utilizados como uma ferramenta importante para o entendimento de processos celulares que envolvem a galectina-4 humana, como inflamação, progressão celular e metástase, e conseqüentemente, contribuir para o planejamento de novas estratégias de diagnóstico e tratamento de neoplasias. / The galectin family comprises a group of lectins where the carbohydrate-recognition domains (CRDs) display specific affinity for ß-galactosides. They are widely distributed in normal and neoplasic cells of different organisms and are involved in a great diversity of cellular mechanisms. The galectins have been focus of recent studies, mainly for their involvement in inflammatory and neoplasic processes, however, many questions about the interactions with different carbohydrates, the specificity of these interactions and the specific role of the galectins in inflammation, cell adhesion, tumor progression and metastasis remain unanswered.The present project focused the strctural studies of human galectin-4 (HGal-4) carbohydrate-recognition domains (CRDs). Our work involved the independent cloning, heterologous expression and purification of both carbohydrate-recognition domains (CRD-I and CRD-II). The HGal-4 CRD-I domain has been successfully crystallized and its structure solved by X-ray crystallography techniques at 2 Å resolution. The crystallographic structure of HGal-4 CRD-I domain comprises two -sheets containing six (S1- S6) and five strands (F1-F6) each packed as a -sandwich domain. A structural comparison among members of galectin class of proteins shows that this folding is highly conserved and that the difference in specificity for carbohydrate molecules is consequence of punctual aminoacid mutations. Our results will be used as an important tool towards a better understanding of cellular processes such as inflammation, cell progression and metastasis, and consequently, contribute for the development of new strategies for diagnosis and treatment of neoplasies.

Liver progenitor cells (LPCs) respond to hepatic injury when hepatocyte division is impaired in chronic or severe injury. The LPCs are intimately surrounded by myofibroblasts, macrophages and laminin, thus constituting a potential progenitor cell niche. Laminin has been proposed to maintain LPCs in an undifferentiated state within the LPC niche. LPCs differentiate once they leave the laminin niche. However, mechanisms regulating this process have not been completely investigated. I hypothesized that cell membrane proteins which are implicated in intergin activation and mediation of cell adhesion to laminin such as galectin-3 and CD98 may be involved in this mechanism. Galectin-3 is a carbohydrate-binding protein which plays an important role in various cell functions, including cell growth, proliferation, adhesion, and differentiation. Galectin-3 has been reported to bind integrins and regulates β1 mediated adhesion to ECM. In addition, galectin-3 may also indirectly mediate β1 integrin activation by binding to and activating the heterodimeric transmembrane amino acid transporter CD98. However a role for galectin-3 in regulating LPC behavior has not been demonstrated. In this thesis, the mechanisms of galectin-3 mediating LPC proliferation and differentiation were investigated in an experimental model of LPC induction, the CDE diet, by using mutant mice lacking the gene encoding galectin-3. I have found galectin-3 is important for LPC induction and proliferation in vivo. In addition, galectin-3 is crucial for the LPC proliferation but is a negative regulator of LPC differentiation in vitro in a laminin dependent manner, suggesting that galectin-3 is required for LPC to maintain in an undifferentiated state on laminin. Moreover, the 2 extracellular binding activity of galectin-3 is important for LPC proliferation and adhesion to laminin. Furthermore, in the absence of galectin-3, LPCs down-regulate cyclin-D1 and the cyclin inhibitors p21 and p16 are elevated. Finally I suggest that integrin-β1 and CD98 are involved in regulating LPC proliferation. There is an increasing literature examining the role of LPC niche in regulating LPC behavior. My work suggests that galectin-3 is required for the expansion of LPCs in the injured adult liver. Galectin-3 enhances LPC adhesion to laminin. Galectin-3 is a crucial factor for LPCs to maintain in an undifferentiated state on laminin. My findings not only emphasize the requirement of LPCs to interact with their extracellular environment to expand but also propose that galectin-3 is a key signalling intermediary in the LPC niche, regulating homeostatic balance between proliferation and differentiation of LPCs, thus controlling regeneration.

Galectin-3, a unique member of the growing family of β-galactoside binding lectins, contains a single carbohydrate recognition domain and a glycine rich N-terminal domain through which it can form oligomers and functions to cross-link both carbohydrate and non-carbohydrate ligands. Galectin-3 is widely expressed in adult tissues, particularly on and secreted by activated macrophages and monocytes. Galectin-3 has been implicated in many facets of the inflammatory response including neutrophil and macrophage activation and function. In this thesis I have examined the role of galectin-3 during fibrosis, alternative activation of macrophages and pneumonia. Galectin-3 expression is upregulated in established human fibrotic liver disease and in a mouse model of liver fibrosis induced by carbon tetrachloride. Galectin-3 expression is temporally and spatially related to the induction and resolution of experimental hepatic fibrosis in this model. In addition, disruption of the galectin-3 gene markedly attenuates liver and kidney fibrosis, induced by unilateral ureteric obstruction, with reduced collagen deposition and myofibroblast activation. Results suggest that galectin-3 may promote fibrosis by stimulating myofibroblast activation by a transforming growth factor-β (TGF-β)-independent mechanism. Recent reports suggest that alternative macrophage activation is one of the key steps toward the progression of fibrosis. Disruption of the galectin-3 gene specifically restrains interleukin-4 (IL-4)/IL-13-induced alternative macrophage activation in vitro. My results suggest that the key mechanism required for activation of an alternative macrophage phenotype is an IL-4-stimulated galectin-3 feed back loop which directly activates CD98 causing sustained phosphatidylinositol 3-kinase (PI3-K) activation. The gram-positive Streptococcus pneumoniae (S. pn) is the leading cause of community acquired pneumonia worldwide, resulting in high mortality. Galectin-3-/- mice demonstrate a clearance defect of S. pn with increased septicaemia and a greater extent of lung damage compared to wild type mice. This phenotype is markedly reduced in pneumonia induced by the gram-negative Escherichia coli (E.coli). I have shown that presence of galectin-3 reduces the severity of pneumonia induced by S. pn and this is achieved through a number of processes: 1) Galectin-3 has bactericidal properties towards S. pn in vitro. 2) Galectin-3-/- macrophages show reduced production of nitrite following incubation with both S. pn and E. coli and hence a reduction in bacterial killing. 3) Galectin-3 activates neutrophils to produce reactive oxygen species which enhances the bactericidal activity of neutrophils. 4) Activation of neutrophils by galectin-3 augments phagocytosis of bacteria. 5) Finally, initial data suggests that galectin-3-/- neutrophils apoptose more readily than wild type neutrophils in vitro and galectin-3-/- macrophages phagocytose apoptotic neutrophils less efficiently compared to wild type. In vivo this would result in an accumulation of dying cells in the lung. The damage these apoptotic cells would have on the lung tissue may enable the bacteria to enter the blood stream resulting in sepsis. In summary, in response to chronic tissue injury, persistant upregulation of galectin-3 causes myofibroblast and alternative macrophage activation, thus enhancing collagen deposition and scarring. However during an acute S. pn infection, galectin-3 plays a benefitial role to aid the clearance of bacteria through a variety of processes. Therefore, galectin-3 plays a critical role in a variety of inflammatory disorders.

La galectine-3 (Gal-3), protéine de type lectine, de 29-35 kDa, étudiée comme marqueur d’aggressivité dans les gliomes, présente des caractéristiques biologiques importantes justifiant son étude dans le domaine du mélanome. En effet, la Gal-3 est une protéine qui peut se lier à la laminine, tout comme l’intégrine α6/β1 dont l’expression est réduite dans le mélanome. L’expression de cette intégrine peut d’ailleurs être modulée par la Gal-3 comme récemment montré dans des lignées cellulaires de cancer du sein (BT-549) et de glioblastome (U373). Le mélanome, véritable problème de santé publique qui est susceptible d’atteindre 1 individu sur 75 dans nos contrées, reste un tumeur mal comprise avec des évolutions parfois incertaines, et des traitements dont l’efficacité est limitée. Le diagnostic histologique du mélanome lui-même peut parfois représenter une difficulté pour le clinicien et l’expert pathologiste ou dermatopathologiste. La couleur (hyperpigmentation d’un lésion pigmentée), dont l’évaluation d’ailleurs reste subjective à défaut de standardisation, ne peut à elle seule signer la malignité d’une lésion pigmentée. Globalement l’évolution d’un patient est prédite par l’indice de Breslow qui traduit en mm l’épaisseur de la tumeur. Si cet indice dépasse 1mm, le risque métastatique augmente, justifiant la réalisation de bilans extensifs de suivi. Ceci dit, certains mélanomes épais peuvent ne pas présenter de caractéristiques d’aggressivité, alors que des mélanomes fins sont parfois mortels. L’identification de marqueurs moléculaires est donc impérative, tant pour développer des stratégies thérapeutiques ciblées, que pour affiner le diagnostic et le pronostic d’un patient. Après avoir mis en évidence par immunohistochimie une expression de Gal-3 par les mélanocytes, nous avons démontré une surexpression de cette protéine par les mélanocytes tumoraux. Nous avons démontré également sur des lésions primitives qu’à l’aggressivité mesurée selon l’indice de Breslow correspondait une diminution de cette surexpression. Cette observation a pu être confirmée par un modèle de greffe orthotopique chez la souris nude. Nous nous somme intéressés par la suite à la détection de la protéine dans le sérum, et nous avons constaté, un taux élevé de Gal-3 dans le sérum de patients en stade métastatique avancé, ce taux élevé pouvant s’expliquer tant par la charge tumorale que par la présence d’une inflammation, d’ailleurs bien connue chez le patient cancéreux en stade avancé. Le rôle antiapoptotique de la Gal-3 nous a alors amené à préciser la valeur prédictive et pronostique de cette protéine. L’hypothèse d’une potentielle action bénéfique sur la réponse immunitaire des patients atteints de mélanome qui ont été vaccinés a été rejetée. La Gal-3 sérique s’est révélée comme facteur de mauvais pronostic chez les patients métastatiques, et une analyse multivariée avec la définition d’une valeur « cut-off » de 10 ng/ml a permis de montrer une valeur pronostique indépendante, supérieure à la S100B et à la CRP.

O melanoma canino é uma neoplasia frequente em cães, tem um caráter maligno, invasivo, com potencial metastático e, neste contexto, estudos acerca do envolvimento das galectinas se justifica para ampliar o conhecimento do microambiente tumoral desta neoplasia. As galectinas são proteínas da família das lectinas animais que apresentam domínios de reconhecimento de carboidratos e podem estar localizadas no núcleo, no citoplasma, na superfície de células e secretadas em diversos tecidos. Acredita-se que principalmente a galectina-1 (gal-1) e a galectina-3 (gal-3) estejam associadas à transformação neoplásica, sobrevivência da célula neoplásica, angiogênese, evasão do sistema imune e formação de metástases. A gal-1 está principalmente relacionada com a transformação tumoral e evasão do sistema imune. A gal-3 está principalmente associada com a angiogênese, desenvolvimento de metástases pelo aumento da motilidade e adesão entre as células neoplásicas e adesão entre as células neoplásicas e o endotélio, além de contribuir para a evasão do sistema imune. O objetivo do estudo foi verificar o padrão de expressão de gal-1 e gal-3 nos diferentes graus de agressividade do melanoma canino, além de avaliar a concentração sérica de gal-3 e comparar com cães clinicamente saudáveis. Foram analisadas a expressão de gal-1 e gal-3 em 30 fragmentos de melanoma canino, seis fragmentos de melanocitoma e nove fragmentos de linfonodos metastáticos. Foi realizada a dosagem sérica de gal-3 em 30 cães com melanoma e comparada a 10 cães clinicamente saudáveis. O melanoma canino expressou gal-1 principalmente no citoplasma e expressou um padrão variável de gal-3 no citoplasma e no núcleo. Em relação à expressão de gal-3 observou-se que conforme a agressividade do melanoma houve diminuição da frequência de células com marcação citoplasmática e um aumento da intensidade de marcação nuclear com concomitante diminuição da frequência de células com marcação nuclear. Os cães com melanoma apresentaram aumento dos níveis séricos de gal-3 antes da exérese da neoplasia quando comparados aos animais clinicamente saudáveis, mostrando o seu potencial uso como biomarcador do melanoma. / Canine melanoma is a frequent neoplasm in dogs. It has a malignant, invasive and metastatic potential. In this context, studies about the involvement of galectins are justified to increase the knowledge of melanoma tumor microenvironment. Galectins are proteins of the animal lectins family, that display carbohydrate recognition domains and may be located in the nucleus, cytoplasm, cell surface, as well as secreted in various tissues. Galectin-1 (Gal-1) and galectin-3 (Gal-3) are associated with neoplastic transformation, neoplastic cell survival, angiogenesis, immune system evasion, and metastasis formation. Gal-1 is mainly related to tumor transformation and immune system evasion. Gal-3 is mainly associated with angiogenesis, development of metastasis by increased motility and adhesion between neoplastic cells and adhesion between neoplastic cells and endothelium, while contributing to the evasion of the immune system. The aim of the study was to ascertain the expression pattern of Gal-1 and Gal-3 in different severity degrees of canine melanoma, as well as to evaluate the serum concentration of Gal-3 and to compare with clinically healthy dogs. Gal-1 and Gal-3 expression was analyzed in 30 canine melanoma fragments, six melanocytoma fragments and nine fragments of metastatic lymph nodes. Serum Gal-3 was measured in 30 dogs with melanoma and compared to 10 clinically healthy dogs. Canine melanoma expressed Gal-1 primarily in the cytoplasm and presented a variable pattern of Gal-3 in the cytoplasm and nucleus. Regarding the expression of Gal-3, it was observed that according to melanoma severity, there was a decrease in the percent frequence of cells with cytoplasmic labeling and an increase in the nuclear marking intensity with concomitant decrease in the percent frequency of nuclear-labeled cells. Dogs with melanoma had increased serum levels of Gal-3 before the excision of the neoplasia when compared to the clinically healthy animals, showing its potential use as a melanoma biomarker.

A galectina-1 (Gal1) é uma lectina, altamente conservada, que reconhece ?- galactosídeos, e está envolvida na regulação da tolerância da imunidade celular e na homeostase. Dados da literatura mostram que esta lectina endógena é amplamente expressa em locais de inflamação e na tumorigênese, participando diretamente dos processos de adesão celular, crescimento tumoral, metástase e angiogênese, ressaltando a relevância de sua detecção em amostras biológicas, e sugerindo que a inibição dirigida da Gal1 pode resultar em benefícios no tratamento de distúrbios inflamatórios e em novas estratégias terapêuticas antitumorais. Entretanto, ainda são escassos os dados sobre inibidores de Gal1 com real impacto terapêutico no bloqueio da atividade biológica dessa lectina. Os aptâmeros são oligonucleotídeos de cadeia simples (DNA ou RNA), que podem se ligar a uma vasta diversidade de alvos, tais como íons, peptídeos, proteínas, moléculas orgânicas e inorgânicas, com alta afinidade e especificidade. Os aptâmeros são selecionados a partir de bibliotecas com sequências randômicas de oligonucleotídeos fita simples (ssDNA) constituídos por uma região central variável, flanqueada por duas regiões de interação com primers para amplificação das sequências via PCR. Esse processo de seleção é denominado de Evolução Sistemática de Ligantes por Enriquecimento Exponencial (SELEX). Neste trabalho foram selecionados e caracterizados aptâmeros de DNA que se ligam a Gal1 humana recombinante e inibem sua atividade lectínica. O processo de seleção dos aptâmeros foi feito através de uma variação da metodologia SELEX, desenvolvida neste trabalho e aqui denominada de \"single vial selection\" (SVS), na qual todas as etapas de seleção dos aptâmeros ocorreram em um único recipiente, de forma rápida e eficiente, evitando etapas cromatográficas, que geralmente são utilizadas no SELEX. Análises com a técnica de termoflúor (TSA) e espectroscopia de fluorescência intrínseca do triptofano permitiram confirmar que os aptâmeros, de fato, se ligam a Gal1, mas em um sítio afastado do CRD. Ensaios de hemaglutinação mostraram que os aptâmeros selecionados conseguiram inibir a ligação da Gal1 com as glicanas da superfície celular, bloqueando a atividade lectínica da proteína. Assim, esse conjunto de resultados mostram que foi possível o desenvolvimento de uma nova classe de inibidores da Gal1 baseada em aptâmeros de DNA, a partir de uma nova metodologia de SELEX, e que não atuam através dos mecanismos clássicos de bloqueio da atividade lectínica via CRD, abrindo nossas possibilidades no desenvolvimento de estratégias diagnósticas e terapêuticas envolvendo esta proteína. / Galectin-1 (Gal1) is a highly conserved lectin that recognizes ?-galactosides, involved in the regulation of cellular immunity tolerance and homeostasis. Data from the literature show that this endogenous lectin is widely expressed in sites of inflammation and tumorigenesis, directly participating in cell adhesion processes, tumor growth, metastasis and angiogenesis, highlighting the relevance of its detection in biological samples, and suggesting that its direct inhibition may result in benefits in the treatment of inflammatory disorders and in novel antitumor therapeutic strategies. However, data on Gal1 inhibitors with real therapeutic impact in blocking the biological activity of this lectin are still scarce. Aptamers are single-stranded oligonucleotides (DNA or RNA), which can bind to a wide variety of targets, such as ions, peptides, proteins, organic and inorganic molecules, with high affinity and specificity. The aptamers are selected from pools of random single-stranded oligonucleotide (ssDNA) sequences consisting of a variable central region, flanked by two sites of primers interaction for PCR amplification. This selection process is called Systematic Evolution of Ligands by EXponential enrichment (SELEX). In this work, DNA aptamers that bind to recombinant human Gal1 and inhibit their lectin activity have been selected and characterized. The aptamers selection process was done through a variation of SELEX methodology, developed in this work and here called \"single vial selection\" (SVS), in which all stages of aptamers selection occurred in a single container, quickly and efficient, avoiding chromatographic steps, which are usually used in SELEX. Analyzes by Thermofluor (TSA) method and intrinsic tryptophan fluorescence spectroscopy have confirmed that aptamers actually bind to Gal1, but at a site away from the CRD. Hemagglutination assay showed that selected aptamers succeeded in inhibiting the Gal1 binding to cell surface glycans, blocking the protein lectin activity. Thus, this set of results showed that it was possible to develop a new class of Gal1 inhibitors based on DNA aptamers and on a new SELEX methodology, that does not act through the classic blocking mechanisms of lectin activity via CRD, opening new possibilities for the development of diagnostic and therapeutic strategies involving this protein.