Les polykétides sont des composés naturels. Ces composés possèdent des rôles thérapeutiques variés tels que antifongiques, antibiotiques, anticancéreux, immunosuppresseurs ou encore anticholestérolémiques. Par conséquent, la recherche de nouvelles structures possédant des bioactivités diverses se révèlent être intéressante. Une stratégie prometteuse pour créer des nouveaux polykétides est l’ingénierie génétique des enzymes synthétisant ces molécules, les polykétide synthases modulaires (PKS), une approche désignée sous le terme de « biologie synthétique ». Pour ce faire, il faut comprendre de façon détaillée le fonctionnement de ces systèmes multienzymatiques. Plusieurs points restent à éclaircir, dont : i) le contrôle de la stéréochimie du polykétide ; et ii) l’interaction des sous-unités composant la PKS. Lors de ma thèse, j’ai identifié deux kétoréductases (KR) qui, introduites dans un contexte modulaire intrinsèquement non-épimérisant, sont capables d’épimériser le méthyle en Cα de façon efficace. Cependant, la modification de la stéréochimie du polykétide ne dépend pas exclusivement des propriétés intrinsèques de la KR mais aussi du contexte modulaire. J’ai également contribué à la réalisation d’un second projet, pour lequel notre équipe a mis en évidence une nouvelle classe de domaine de docking de PKS de type trans-AT présentant une nouvelle topologie. L’un des DD étudié est une protéine intrinsèquement désordonnée dont le repliement est induit par son partenaire. Nous avons caractérisé l’interface complète entre deux sous-unités de PKS de type trans-AT, révélant une chambre de réaction protégée dans laquelle les chaînes de polykétide peuvent croître / Polyketides are natural products which exhibit a variety of therapeutic activities, including anti-fungal, antibiotic, anticancer, immunosuppressant and anti-cholesterolemic properties. Given their medical and economic importance, there is significant interest in identifying new structures with new biological activities. A promising strategy to create such analogues is to genetically engineer the enzymes responsible for synthesizing these molecules, the modular polyketide synthases (PKSs), an approach referred to as ‘synthetic biology’. However, in order to increase the efficacy of this approach, we must understand in detail how the PKS multienzymes work. A number of issues remain to be clarified, including: i) polyketide stereocontrol, ii) the interaction of the component subunits PKS. During my thesis, I identified two ketoreductase (KR) domains which when introduced into an intrinsically non-epimerizing modular context, were able to efficiently epimerise at the Cα of a model polyketide. I also showed that the modular context in which the KR functions has an influence on the ultimate stereochemical outcome. I also made essential contributions to a second project, in which the group identified a novel family of docking domains (DD) in the trans-AT type of PKS which present a novel topology. One of the two model DDs studied is an intrinsically disordered protein whose folding is induced by its partner. Finally, we were able to visualize a complete intersubunit interface within a trans-AT PKS, revealing a protected reaction center in which polyketide chains can grow.

Les modèles de Markov cachés (MMC) par exemple ceux de la librairie Pfam sont des outils très populaires pour l'annotation des domaines protéiques. Cependant, ils ne sont pas toujours adaptés aux protéines les plus divergentes. C'est notamment le cas avec Plasmodium falciparum (principal agent du paludisme chez l'Homme), où les MMC de Pfam identifient peu de familles distinctes de domaines, et couvrent moins de 50% des protéines de l'organisme. L'objectif de cette thèse est d'apporter des méthodes nouvelles pour affiner la détection de domaines dans les protéines divergentes.Le premier axe développé est une approche d'identification de domaines utilisant leurs propriétés de co-occurrence. Différentes études ont montré que la majorité des domaines apparaissent dans les protéines avec un ensemble très réduits d'autres domaines favoris. Notre méthode exploite cette propriété pour détecter des domaines trop divergents pour être identifiés par l'approche classique. Cette détection s'accompagne d'une estimation du taux d'erreur par une procédure de ré-échantillonnage. Chez P. falciparum, elle permet d'identifier, avec un taux d'erreur estimé inférieur à 20%, 585 nouveaux domaines dont 159 familles étaient inédites dans cet organisme ce qui représente 16% du nombre de domaines connus.Le second axe de mes recherches présente plusieurs méthodes de corrections statistiques et évolutives des MMC pour l'annotation d'organismes divergents. Deux types d'approches ont été proposées. D'un côté, nous intégrons aux alignements d'apprentissage des MMC, les séquences précédemment identifiés dans l'organisme cible ou ses proches relatifs. La limitation de cette solution est que seules des familles de domaines déjà connues dans le taxon peuvent ainsi être identifiées. Le deuxième type d'approche contourne cette limitation en corrigeant tous les modèles par une prise en compte de l'évolution des séquences d'apprentissage. Pour cela, nous faisons appel à des techniques classiques de la bioinformatique et de l'apprentissage statistique. Les résultats obtenus offrent un ensemble de prédictions complémentaires totalisant 663 nouveaux domaines supplémentaires dont 504 familles inédites soit une augmentation de 18% à ajouter aux précédents résultats. / Hidden Markov Models (HMMs) from Pfam database for example are popular tools for protein domain annotation. However, they are not well suited for studying highly divergent proteins. This is notably the case with Plasmodium falciparum (main causal agent of human malaria), where Pfam HMMs identify few distinct domain families and cover less than 50% of its proteins. This thesis aims at providing new methods to enhance domain detection in divergent proteins.The first axis of this work is an approach of domain identification based on domain co-occurrence. Several studies shown that a majority of domains appear in proteins with a small set of other favourite domains. Our method exploits this tendency to detect domains escaping to the classical procedure because of their divergence. Detected domains come along with an false discovery rate (FDR) estimation computed with a shuffling procedure. In P. falciparum proteins, this approach allows us identify, with an FDR below 20%, 585 new domains with 159 families that were previously unseen in this organism which account for 16% of the known domains.The second axis of my researches involves the development of statistical and evolutionary methods of HMM correction to improve the annotation of divergent organisms. Two kind of approaches are proposed. On the one hand, the sequences previously identified in the target organism and its close relatives are integrated in the learning alignments. An obvious limitation of this solution is that only new occurrences of previously known families in the taxon can be discovered. On the other hand, we evade this limitation by adjusting HMM parameters by simulating the evolution of the learning sequences. To this end, classical techniques from bioinformatics and statistical learning were used. Alternative libraries offer a complementary set of predictions summing 663 new domains with 504 previously unseen families corresponding to an improvement of 18% to add to the previous results.

L'étude de la composition des protéines en domaines est une étape clé pour la détermination de ses fonctions. Pfam est l'une des banques de domaines les plus répandues où chaque domaine est représenté par un HMM profil construit à partir d'un alignement multiple de protéines contenant le domaine. La méthode classique de recherche des domaines Pfam consiste à comparer la séquence cible à la librairie complète des HMM profils pour mesurer sa ressemblance aux différents modèles. Cependant, appliquée aux protéines d'organismes divergents, cette méthode manque de sensibilité. L'objectif de cette thèse est d'apporter de nouvelles méthodes pour améliorer le processus de prédictions des domaines plus adaptées à l'étude des protéines divergentes. Les premiers travaux ont consisté en l'adaptation et application de la méthode CODD, récemment proposée, à l'ensemble des pathogènes de la base de données EuPathDB. Une base de données nommée EupathDomains (http://www.atgc-montpellier.fr/EuPathDomains/) recensant l'ensemble des domaines connus et ceux nouvellement prédits chez ces pathogènes a été mise en place à l'issue de ces travaux. Nous nous sommes ensuite attachés à proposer diverses améliorations. Nous proposons un algorithme ''CODD_exclusive'' qui utilise des informations d'incompatibilité de domaines pour améliorer la précision des prédictions. Nous proposons également une autre stratégie basée sur l'utilisation de règles d'association pour la détermination des co-occurrences de domaines utilisées dans le processus de certification. La dernière partie de cette thèse s'intéresse à l'utilisation des méthodes profil/profil pour annoter un génome entier. Couplée à la procédure d'annotation par co-occurrence, cette approche permet une amélioration notable en termes de nombre de domaines certifiés et également en termes de précision. / The determination of protein domain composition provides strong clues for the protein function prediction. One of the most widelyused domain scheme is the Pfam database in which each family is represented by a multiple sequence alignment and a profileHidden Markov Model (profile HMM). When analyzing a new sequence, each Pfam HMM is used to compute a score measuring the similarity between the sequenceand the domain. However, applied to divergent proteins, this strategy may miss several domains. This is the case for all eukaryotic pathogens, where noPfam domains are detected in half or even more of their proteins.The main objective of this thesis is to develop methods to improve the sensitivity of Pfam domain detection in divergent proteins. We first adapted the recently proposed CODD method to the whole set of pathogens in EupathDB. A public database named EupathDomains (http://www.atgc-montpellier.fr/EuPathDomains/) gathers known and new domains detected by CODD, along with the associated confidence measurements and the GO annotations.We then proposed other methods to further improve domain detection in these organisms. We proposed ''CODD_exclusive'' algorithm that integrates domain exclusion information to prune false positive domains that are in conflict with other domains of the protein. We also suggested the use of association rules to determine the correlations between domains and used these informations in the certification process.In the last part of this thesis, we focused in the use of profile/profile methods to predict protein domains in a whole genome. Combined with the co-occurrence informations, it achieved high sensitivity and accuracy in predicting domains.

As galectinas, proteínas que compartilham características como afinidade por ?-galactosídeos e a presença de um domínio de reconhecimento ao carboidrato (CRD), tem como importante papel a decodificação de mensagens moleculares. As galectinas são encontradas em uma grande variedade de células e tecidos animais e estão envolvidas em diversos processos celulares relacionados a resposta imune e inflamatória. O grupo tandem-repeat das galectinas é formado por proteínas que apresentam dois CRDs distintos (CRD1 e CRD2) conectados por um peptídeo de ligação, ao qual pertencem as galectinas-4 e -12 humanas, alvos de nosso estudo. Durante o desenvolvimento do presente projeto foram utilizadas abordagens multidisciplinares através de técnicas biofísicas, cristalográficas, computacionais e imunoquímicas. Foi iniciado o estudo de caracterização estrutural da galectina-4 humana (hGal4) e seus domínios hGal4-CRD1 e hGal4-CRD2, de forma independente, através da produção heteróloga das proteínas e ensaios de estabilidade térmica e cristalização. As estruturas cristalográficas dos domínios foram determinadas a 1,48 e 2,46 Å de resolução, respectivamente e utilizadas para a construção de um modelo para a hGal4. Com base nos estudos de dinâmica molecular e estabilidade térmica foi possível propor um modelo de interação entre os CRDs através do peptídeo de ligação. Ensaios de dinâmica da hGal4 com LacNAc sugeriram uma diferença de plasticidade dos CRDs no reconhecimento do ligante. Foram também realizadas incessantes tentativas de desenvolver um protocolo de expressão heteróloga para as isoformas e domínios da galectina-12 humana (hGal12). Como resultado, observamos uma alta tendência à formação de corpos de inclusão e agregados resistentes a desnaturação, além da susceptibilidade ao ataque proteolítico. Os modelos estruturais dos domínios da hGal12 não permitiram identificar nenhuma característica que justificasse o comportamento das proteínas. Foram realizados estudos de localização celular em células HL-60 e foi possível identificar a presença da hGal12 na superfície das gotas de lipídio, organelas responsáveis pelo armazenamento de energia e o processo de catabolismo celular. A alta insolubilidade observada para as isoformas e domínios da hGal12, a sua localização em gotas de lipídeos, a divergência evolutiva da hGal12 quando comparada com outras galectinas do mesmo grupo e incluindo o fato de um dos CRDs não apresentar os requerimentos estruturais básicos para ligar ?-galactosídeos, nos leva a especular se essa proteína estaria, na verdade, sendo expressa como parte de um heterocomplexo proteico, que estabilizaria a estrutura da hGal12 e a endereçaria para as gotas de lipídeo. Em nossa hipótese, o domínio hGal12-CRD2 poderia ter evoluído para favorecer a interação com outros parceiros macromoleculares. Devido ao importante papel desempenhado pelas galectinas em inúmeros processos celulares, os resultados aqui obtidos representam uma importante contribuição no entendimento do papel que as galectinas hGal4 e hGal12 exercem nos diferentes eventos celulares, além de fornecem ferramentas experimentais para desenvolvimento de futuros estudos funcionais. / Galectins are proteins that share characteristics such as affinity for ?-galactosides and the presence of a carbohydrate recognition domain (CRD). They belong to a family of proteins that display the important role of decoding molecular messages. Galectins are found in a variety of cell types and are involved in several biological phenomena related to immune response and inflammation. The tandem-repeat group of galectins consists of proteins with two distinct CRDs (CRD1 and CRD2) connected by a peptide linker, in which belong galectins-4 and -12, targets of our study. During the development of the present project a multidisciplinary approach was used including the use of biophysical, crystallographic, computational and immunochemical techniques. The structural characterization of human galectin-4 (hGal4) and its hGal4-CRD1 and hGal4-CRD2 independent domains was initiated by their heterologous protein production, thermal stability and crystallization assays. The crystallographic structure of domains were determined at 1.48 e 2.46 Å resolution, respectively, and used for constructing a model for hGal4. Based on molecular dynamics and thermal stability studies we propose a model of interaction between CRDs mediated by linker peptide. Dynamics simulation of hGal4 with LacNAc suggested a difference in plasticity between CRDs and ligand recognition. In addition, several attempts have been made towards the development of a protocol for expression of isoforms and independent domains from human galectin-12 (hGal12). As result, we observed a high tendency to body inclusion formation and denaturing resistant aggregates, besides high susceptibility to proteolytic attack. Structural models for the hGal12 domains did not allow the identification of any feature to justify proteins behavior. Cell location studies in HL-60 cells were performed and hGal12 was found to be located on the surface of lipid droplets, organelles responsible for energy storage and catabolism. The high insolubility displayed by isoforms and domains from hGal12, together with its location on lipid droplets, the evolutionary divergence of hGal12 compared to tandem-repeat proteins, and the fact that hGal12-CRD2 does not present the structural requirements for ?-galactosides binding, suggest that hGal12 is, in fact, expressed as part of a protein complex that could both stabilize hGal12 structure and guide it to the lipid droplets. In our hypothesis, the hGal12-CRD2 could have evolved in order to favor the interaction with other macromolecular partners. Due to crucial roles displayed by galectins in innumerous biological process, we believe that our results represent an important contribution for the understanding of hGal4 e hGal12 proteins roles within the cell, besides providing experimental tools for the development of further functional studies.

Neste trabalho estudamos a dinâmica assintótica não linear de algumas equações parabólicas do tipo reação-difusão sob perturbações nos parâmetros e perturbações singulares no domínio do tipo dumbbell. Mais precisamente, trataremos dos atratores provenientes destes problemas, buscaremos compreender a dependência destes conjuntos assintóticos de estados em relação ao parâmetro, investigando a continuidade com taxa dos mesmos. O programa que executaremos para obtenção da taxa de continuidade dos atratores, bem como de toda a estrutura, mostra-nos fortes propriedades de dissipatividade exponencial de alguns semigrupos / In this work we study the asymptotic nonlinear dynamical of some reaction-diffusion parabolic equations under perturbations in parameter and singular perturbations in a dumbbell domain. More precisely, we treat of the attractors from these problems, we seek understand the dependence these asymptotic set of states in relationship the parameter, investigating continuity with rate. The program that we will follow to prove the continuity of the attractors with rate well as the entire structure, we show that these semigroups possess strong exponential dissipative properties

A utilização de redes complexas para a descriçãoi de diversos sistemas naturais e artificias,compreendidos nas mais diversas áreas do conhecimento humano, tem se mostrado uma abordagem poderosa para a redução da complexidade inerente a tais sistemas. Em muitos casos, tal complexidade resulta do número de componentes envolvidos e de suas intrincadas relações. Uma forma de reduzir a complexidade associada a tais sistemas, consiste em identificar e agrupar componentes que possuam características similares. Sendo assim, desenvolvemos nesta tese métodos de identificação de comunidades em redes complexas. Tais métodos se baseiam na ideia de que comunidades surgem quando grupos de vértices possuem um número mais elevado de conexões entre os vértices do mesmo grupo do que com vértices externos à este grupo. Além disso, utilizamos a função modularidade como função objetivo e como forma de avaliação e comparação dos resultados obtidos nesta tese com resultados previamente reportados na literatura. Uma vez estabelecido um método de identificação de comunidades, utilizamos a abordagem de redes complexas para a determinação de domínios estruturais de proteínas. Para tal, criamos redes de contato entre os aminoácidos de uma proteí?na buscando representar apenas as ligações relevantes do ponto de vista topológico. Por meio destas representações, aplicamos os métodos de identificação de comunidades desenvolvidos nesta tese, no intuito de identificar domínios estruturais de cadeias proteicas. Por fim, desenvolvemos um método específico para a identificação de domínios em proteínas com dois domínios sequencias, concluindo desta forma, os objetivos propostos nesta tese. / The use of complex networks for the representation of various natural and artificial sys- tems in the most diverse fields of human knowledge, has proven to be a powerful approach for the reduction of the complexity in the study of such systems. In many cases, this complexity emerges from the number of components of the system and from the intricate relationship between them. A reduction in this complexity is made possible by the iden- tification and grouping of the components of the system with similar characteristics. In this way, we developed in this thesis, methods for community identification in complex networks. Such methods are based on the notion that communities arise when groups of vertices are more densely connected with vertices of their same group, than with ver- tices belonging to other groups in the network. Moreover, the modularity function has been used as an objective function, and as a score for the evaluation and comparison of the results obtained in this thesis with the results reported in the literature of complex networks. Upon the establishment of a method for community detection, we used the framework of complex networks to the determination of structural protein domains. The- refore, we have created contact networks of amino acids of protein chains, focusing on the representation of only the most relevant interactions between them, from a topological point of view. We have applied to these networks the methods for community identi- fication developed in this thesis, aiming to identify the structural domains of proteins. Finally, we have developed a specific method for the identification of protein domains in protein chains with two sequential domains, concluding in this way, the objectives proposed in this thesis.

The paralogous multi-functional adaptor proteins YAP and TAZ are nuclear effectors of the Hippo pathway, a central regulator of developmental organ size control, tissue homeostasis and tumour suppression. YAP/TAZ target the TEAD transcription factor family to promote cell survival and inhibit apoptosis. TEAD proteins contain a DNAbinding domain and a YAP/TAZ interaction domain. PCR analysis of medaka fish TEAD cDNA revealed the presence of alternative TEAD splice-forms with variations at the C-terminus of the DNA-binding domain. Structural analysis indicated the YAPbinding domain of TEAD proteins is folded and globular. NMR spectroscopy showed that the TEAD binding domain of YAP does not contain secondary structure. YAP and TAZ both contain WW domains, which are small protein-protein interaction modules. Two YAP isoforms are known, YAP1 and YAP2 that contain one and two WW domains, respectively. To date, only a single WW isoform of TAZ has been described. PCR analysis of medaka TAZ cDNA identified both single WW and tandem WW isoforms of TAZ. NMR spectroscopy was used to characterise structural, conformational, and peptide binding features of the tandem WW domains from YAP and TAZ. The YAP WW2 solution structure confirms that the domain has the canonical anti-parallel β-sheet WW fold. WW1 of YAP and both WW domains of TAZ undergo conformational exchange. The region linking the two WW domains is flexible and allows interaction of both WW domains with peptides containing single and dual PPxY binding motifs. In addition to YAP and TAZ, tandem WW domains are also present in the core and upstream Hippo pathway proteins Salvador and Kibra. Both proteins contain one atypical WW domain; the tandem WW domains of these two proteins are unstable. Understanding structure and function of Hippo pathway components could contribute to drug development and will also contribute to knowledge of protein folding and interactions.

Non-medical prescribers make an increasing contribution to healthcare across the UK yet little is known about influences on their prescribing decision-making. The aim of this programme of research was to explore and describe prescribing decision-making by non-medical prescribers. A two stage programme of research was carried out. Stage 1 was a systematic review of the social and cognitive influences on prescribing decision-making by non-medical prescribers. Despite a paucity of research, various influences on prescribing decision-making were reported including evidence based guidelines, peer support and patient (or parental) relationships and expectations. While confidence and clinical experience as a practitioner were cited as influences, the lack of prescribing experience and aspects of pharmacological knowledge also impacted on prescribing decision-making, resulting in a cautious approach. Stage 2 of the research employed a phenomenological methodology underpinned by the Theoretical Domains Framework of behavioural determinants (TDF). It comprised three phases. In Phase 1, semi-structured interviews with five nurse prescribers and eight pharmacist prescribers in NHS Grampian explored their experiences and perceptions of influences on their prescribing decision-making, and the impact of these influences. Multiple and sometimes contradictory influences were uncovered. Twelve of the fourteen domains of the TDF were found to be influential along with multi-disciplinary working and experience; optimism and reinforcement did not feature. In Phase 2, these participants recorded reflections on prescribing decisions which they considered noteworthy in relation to their practice, and in Phase 3 participants were interviewed about their reflections. Complexity was a feature of many, in the patients’ clinical or social circumstances or in relation to wider concerns. The same 12 domains were found to be influential as were multi-disciplinary working, experience and complexity. This programme of research has produced original findings which it is hoped will impact on the education, training and practice of these increasingly important prescribers.

Este trabalho trata de estimativas inferiores para o primeiro autovalor de Dirichlet para dom nios multiplamente conexos contidos em variedades riemannianas. Essas estimativas consideram o supremo da curvatura seccional da variedade e a curvatura do bordo do domínio. Para obter os resultados, usa-se uma estimativa C0 para solucões da equação de Poisson. / Lower bounds for the rst Dirichlet eigenvalue are presented. We consider multiply connected domains in riemannian manifolds. The estimates are obtained using hypothesis on the supremum of the manifold's sectional curvature and on the domain's boundary curvature. C0 estimates for solutions of Poissons equation are used to prove the results.

As galectinas, proteínas que compartilham características como afinidade por ?-galactosídeos e a presença de um domínio de reconhecimento ao carboidrato (CRD), tem como importante papel a decodificação de mensagens moleculares. As galectinas são encontradas em uma grande variedade de células e tecidos animais e estão envolvidas em diversos processos celulares relacionados a resposta imune e inflamatória. O grupo tandem-repeat das galectinas é formado por proteínas que apresentam dois CRDs distintos (CRD1 e CRD2) conectados por um peptídeo de ligação, ao qual pertencem as galectinas-4 e -12 humanas, alvos de nosso estudo. Durante o desenvolvimento do presente projeto foram utilizadas abordagens multidisciplinares através de técnicas biofísicas, cristalográficas, computacionais e imunoquímicas. Foi iniciado o estudo de caracterização estrutural da galectina-4 humana (hGal4) e seus domínios hGal4-CRD1 e hGal4-CRD2, de forma independente, através da produção heteróloga das proteínas e ensaios de estabilidade térmica e cristalização. As estruturas cristalográficas dos domínios foram determinadas a 1,48 e 2,46 Å de resolução, respectivamente e utilizadas para a construção de um modelo para a hGal4. Com base nos estudos de dinâmica molecular e estabilidade térmica foi possível propor um modelo de interação entre os CRDs através do peptídeo de ligação. Ensaios de dinâmica da hGal4 com LacNAc sugeriram uma diferença de plasticidade dos CRDs no reconhecimento do ligante. Foram também realizadas incessantes tentativas de desenvolver um protocolo de expressão heteróloga para as isoformas e domínios da galectina-12 humana (hGal12). Como resultado, observamos uma alta tendência à formação de corpos de inclusão e agregados resistentes a desnaturação, além da susceptibilidade ao ataque proteolítico. Os modelos estruturais dos domínios da hGal12 não permitiram identificar nenhuma característica que justificasse o comportamento das proteínas. Foram realizados estudos de localização celular em células HL-60 e foi possível identificar a presença da hGal12 na superfície das gotas de lipídio, organelas responsáveis pelo armazenamento de energia e o processo de catabolismo celular. A alta insolubilidade observada para as isoformas e domínios da hGal12, a sua localização em gotas de lipídeos, a divergência evolutiva da hGal12 quando comparada com outras galectinas do mesmo grupo e incluindo o fato de um dos CRDs não apresentar os requerimentos estruturais básicos para ligar ?-galactosídeos, nos leva a especular se essa proteína estaria, na verdade, sendo expressa como parte de um heterocomplexo proteico, que estabilizaria a estrutura da hGal12 e a endereçaria para as gotas de lipídeo. Em nossa hipótese, o domínio hGal12-CRD2 poderia ter evoluído para favorecer a interação com outros parceiros macromoleculares. Devido ao importante papel desempenhado pelas galectinas em inúmeros processos celulares, os resultados aqui obtidos representam uma importante contribuição no entendimento do papel que as galectinas hGal4 e hGal12 exercem nos diferentes eventos celulares, além de fornecem ferramentas experimentais para desenvolvimento de futuros estudos funcionais. / Galectins are proteins that share characteristics such as affinity for ?-galactosides and the presence of a carbohydrate recognition domain (CRD). They belong to a family of proteins that display the important role of decoding molecular messages. Galectins are found in a variety of cell types and are involved in several biological phenomena related to immune response and inflammation. The tandem-repeat group of galectins consists of proteins with two distinct CRDs (CRD1 and CRD2) connected by a peptide linker, in which belong galectins-4 and -12, targets of our study. During the development of the present project a multidisciplinary approach was used including the use of biophysical, crystallographic, computational and immunochemical techniques. The structural characterization of human galectin-4 (hGal4) and its hGal4-CRD1 and hGal4-CRD2 independent domains was initiated by their heterologous protein production, thermal stability and crystallization assays. The crystallographic structure of domains were determined at 1.48 e 2.46 Å resolution, respectively, and used for constructing a model for hGal4. Based on molecular dynamics and thermal stability studies we propose a model of interaction between CRDs mediated by linker peptide. Dynamics simulation of hGal4 with LacNAc suggested a difference in plasticity between CRDs and ligand recognition. In addition, several attempts have been made towards the development of a protocol for expression of isoforms and independent domains from human galectin-12 (hGal12). As result, we observed a high tendency to body inclusion formation and denaturing resistant aggregates, besides high susceptibility to proteolytic attack. Structural models for the hGal12 domains did not allow the identification of any feature to justify proteins behavior. Cell location studies in HL-60 cells were performed and hGal12 was found to be located on the surface of lipid droplets, organelles responsible for energy storage and catabolism. The high insolubility displayed by isoforms and domains from hGal12, together with its location on lipid droplets, the evolutionary divergence of hGal12 compared to tandem-repeat proteins, and the fact that hGal12-CRD2 does not present the structural requirements for ?-galactosides binding, suggest that hGal12 is, in fact, expressed as part of a protein complex that could both stabilize hGal12 structure and guide it to the lipid droplets. In our hypothesis, the hGal12-CRD2 could have evolved in order to favor the interaction with other macromolecular partners. Due to crucial roles displayed by galectins in innumerous biological process, we believe that our results represent an important contribution for the understanding of hGal4 e hGal12 proteins roles within the cell, besides providing experimental tools for the development of further functional studies.