Contexte : l'aldostérone (Aldo) est impliquée dans la rigidité artérielle et l'insuffisance cardiaque (IC), mais les mécanismes sous-jacents restent méconnus. La galectine-3 (Gal-3), une lectine se liant aux bêta-galactoside, joue un rôle important dans la fibrose et l'IC. Dans cette étude, nous avons recherché si la Gal-3 était impliquée dans la fibrose vasculaire induite par l'Aldo. Méthodes et résultats : Des cellules musculaires lisses vasculaires de rat (CMLVs) ont été stimulées avec de l'Aldo en combinaison avec des antagonistes du récepteur minéralocorticoïde (MR) ou des inhibiteurs de la Gal-3. L'Aldo régule l'expression de la Gal-3 via le MR dans les CMLVs. De plus, la surexpression de la Gal-3 augmente spécifiquement la synthèse de collagène de type I. Les inhibiteurs de la Gal-3 ou sa sous-expression (siRNA) bloquent la synthèse de collagène de type I induite par l'Aldo. Des rats ont été traités avec de l'Aldo + sel combiné avec du spironolactone ou de la pectine de citron modifiée (MCP) pendant 3 semaines. Les rats hypertensifs traités à l'Aldo ont présenté une hypertrophie vasculaire, une fibrose et une augmentation de l'expression aortique de Gal-3. Les traitements avec le spironolactone ou le MCP préviennent tous ces effets. Des souris sauvages (WT) et mutées pour la Gal-3 (KO) ont été traitées avec de l'Aldo pendant 6 heures. Le bolus d'Aldo augmente l'expression de la Gal-3 et du collagène de type I dans l'aorte des souris WT alors qu'aucun changement ne se produit dans les souris KO pour la Gal-3. Conclusions : Nos donnés indiquent que la Gal-3 est indispensable à la réponse fibrotique de l'Aldo dans les CMLVs in vitro et in vivo, suggérant un rôle clef pour la Gal-3 dans la fibrose vasculaire / Background. Aldosterone (Aldo) is involved in arterial stiffness and heart failure (HF), but the mechanisms have remained unclear. Galectin-3 (Gal-3), a beta-galactoside-binding lectin, plays an important role in fibrosis and HF. We here investigated whether Gal-3 is involved in Aldo-induced vascular fibrosis. Methods and Results. Rat vascular smooth muscle cells (VSMCs) were stimulated with Aldo combined with mineralocorticoid receptor (MR) antagonists and Gal-3 inhibitors. Aldo upregulated Gal-3 expression via MR in VSMCs. Moreover, Gal-3 over-expression specifically enhanced collagen type I synthesis. Gal-3 inhibitors or Gal-3 silencing (siRNA) blocked Aldo-induced collagen type I synthesis. Rats were treated with Aldo-salt combined with spironolactone or modified citrus pectin (MCP) for 3 weeks. Hypertensive Aldo-treated rats presented vascular hypertrophy, fibrosis and increased aortic Gal-3 expression. Spironolactone or MCP treatment reversed all the above effects. Wild type (WT) and Gal-3 knock-out (KO) mice were treated with Aldo for 6 hours. Aldo bolus increased aortic Gal-3 and collagen type I expression in WT mice whereas no changes occurred in Gal-3 KO mice. Conclusions. Our data indicate that Gal-3 is required for the fibrotic response to Aldo in VSMCs in vitro and in vivo, suggesting a key role for Gal-3 in vascular fibrosis

A galectina-1 (Gal-1) pertence a uma família de lectinas endógenas que reconhecem ß-galactosídeos e atuam em vários processos biológicos. A Gal-1 pode modular a resposta imunológica por vários mecanismos incluindo o controle da liberação de citocinas pró e anti-inflamatórias e o direcionamento dessa resposta para um padrão do tipo TH2. Apesar da Gal-1 participar de vários processos fisiopatológicos, na literatura não existe relatos sobre o papel dessa lectina em infecções fúngicas. O objetivo deste trabalho foi investigar o impacto biológico da Gal-1 no modelo experimental de histoplasmose murina. Os camundongos (Gal-1-/- e Gal-1+/+) foram inoculados, por via intratraqueal, com uma carga fúngica sub-letal (5x105 leveduras) e a sobrevida desses animais foi avaliada até o 30o dia de infecção. Considerando que o início da mortalidade dos animais ocorreu após duas semanas de infecção, as demais análises foram realizadas em amostras obtidas no 15o dia. O grau de disseminação do H. capsulatum foi analisado pela contagem do número de unidades formadoras de colônia, em partes de pulmões ou baços dos animais infectados. Os cortes de pulmões foram corados por hematoxilina eosina ou por prata (GMS), para investigação histopatológica e quantificação de neutrófilos ou fungos, respectivamente. Nos fluídos bronco-alveolares (BAL) foram realizadas contagens globais e diferenciais de leucócitos. As dosagens de citocinas e de prostagladina E2 foram feitas por ELISA, em homogeneizados de pulmões. A coloração por tetróxido de ósmio foi usada na avaliação da capacidade da Gal-1 de induzir ou modular a formação de corpúsculos lipídicos, in vitro, por componentes do fungo. Nos soros dos animais de experimentação foi determinada a concentração total de nitrito, como indicador da produção de óxido nítrico. A análise dos resultados de sobrevivência indicou que 100% dos animais Gal-1+/+ resistiram à infecção por H. capsulatum; ao contrário, apenas 33% dos animais Gal-1-/- sobreviveram. Os números médios de unidades formadoras de colônias recuperadas no pulmão e no baço de camundongos Gal-1-/- foram de 2,7 e 3,8 vezes maiores do que os obtidos de animais Gal-1+/+, respectivamente. De modo semelhante, os números médios de neutrófilos e fungos no pulmão de animais Gal-1-/-, foram superiores aos valores encontrados nos pulmões de animais grupo Gal-1+/+. Curiosamente, nos homogeneizados pulmonares dos e o dobro da concentração de nitrito total sérico. Além disso, nos homogeneizados de pulmão dos animais Gal-1desafiados com o fungo, detectou-se elevadas concentrações de citocinas do tipo T1 e inflamatórias (IFN-, IL-1 e IL-12) em comparação com amostras de animais selvagens. Em ensaios in vitro, esta lectina não foi capaz de induzir corpúsculos em células do lavado peritoneal de camundongos Gal-1e Gal-1, entretanto inibiu parcialmente a formação induzida por F1 e -glucana. Finalmente, sugerimos que a Gal-1 pode participar da montagem de uma resposta imunológica protetora contra o Histoplasma capsulatum, por modular a liberação de citocinas inflamatórias, síntese de eicosanóides, geração de óxido nitrito; e por controlar a migração e/ou as funções de leucócitos. Além disso, os dados obtidos desse trabalho poderão auxiliar no melhor entendimento da fisiopatologia da histoplasmose e no desenvolvimento de novas estratégias terapêuticas envolvendo o reconhecimento de carboidratos na resposta imunológica. / Galectin-1 (Gal-1) belongs an endogenous lectins family that recognizes -galactoside and participates of various biological activities. This lectin can modulate the innate and adaptative immune responses. Although, Gal-1 participates of various pathophysiological processes, in literature we did not find reports related to the participation of Gal-1 in fungal infections. The aim of this work was to investigate the biological impact of Gal-1 in the experimental histoplasmosis. The mice (GAL-1-/- and GAL-1+/+) were injected (i.t.) with 5 x 105 yeast cell and at 15 days post-infection, BALF cells and lungs cytokine and PGE2 were measured by ELISA. The Recovery of H. capsulatum was made in lung and spleen and the fungal burden was assessed as the CFU per organ. The lung slices were stained by hematoxiline eosin or with Gomoris methanemine silver (GMS) and submitted to histopathological investigation and quantification of neutrophil or fungus, respectively. Total and differential cell counts of the bronchoalveolar lavage (BAL).were performed using diluting solution in Neubauer chamber and Rosenfeld-stained smear. The capacity of Gal-1 to induce or modulate the lipids bodies formation by fungus components was analyzed by staining treated cells with osmium tetroxide. The total nitrite (NO2) concentration in the animals serum was measured by Griess reaction. All H. capsulatum-infected wild type mice survived until 30 days post-infection, whereas only 33% of the Gal-1-/- infected mice died during of this period of observation. At 15 days post-infection, CFU were found to be higher in the spleens or lung from infected-Gal1-/- mice. The number of neutrophils in the lung of the infected-Gal-1-/- mice higher than infected Gal-1+/+ animals. Curiously, H. capsulatum infected Gal-1-/- mice, presented higher levels of PGE2 and TH1 inflammatory cytokines (IFN-, IL-1 e IL-12) in comparison with wild type infected-mice. Adherent peritoneal cells peritoneal derived from Gal-1+/+ and Gal-1-/- mice and treated with Gal-1 did not induce lipid bodies. However, the capacity of F1 e -glucan to induce lipid bodies on the peritoneal cells was inhibited by gal-1 treatment. We suggest that the Gal-1 could participate of the development of a protective immune response to H. capsulatum.

O carcinoma adenóide cístico e o adenocarcinoma polimorfo de baixo grau de malignidade são neoplasias malignas das glândulas salivares que apresentam semelhança nos padrões histológicos, porém com comportamento clínico, tratamento e prognóstico completamente diferentes. A galectina-3 é uma proteína multifuncional da família das lectinas que está envolvida em vários fenômenos biológicos como crescimento celular, adesão celular, diferenciação celular e apoptose. Além disso, tem sido estudada como um marcador de invasão tumoral e metástase. O objetivo deste trabalho foi estudar qualitativamente a expressão imunoistoquímica da galectina-3 em 14 casos de carcinoma adenóide cístico (2 do subtipo tubular, 4 do subtipo sólido e 8 do subtipo cribriforme) e em 12 casos de adenocarcinoma polimorfo de baixo grau de malignidade com padrões histológicos variados, incluindo os padrões lobular, tubular e cribriforme. Espécimes de glândula salivar normal foram também incluídos na amostra. Nas glândulas salivares normais houve forte marcação da galectina-3 no núcleo e no citoplasma das células luminais dos ductos. Nos carcinomas adenóides císticos houve uma maior marcação da galectina-3 no subtipo tubular, localizada apenas nas células luminais das estruturas tubulares. Nos subtipos sólido e cribriforme a marcação foi menor, mas sempre localizada nas células que circundavam espaços luminais. Em todos os casos de carcinomas adenóides císticos estudados a marcação foi predominantemente nuclear. Nos adenocarcinomas polimorfos de baixo grau de malignidade a marcação da galectina-3 foi predominantemente citoplasmática em praticamente todas as células neoplásicas. Diante disso podemos sugerir que, nas neoplasias estudadas, a expressão da galectina-3 parece estar mais relacionada à diferenciação celular do que à progressão tumoral e ao prognóstico. / Adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma are malignant neoplasms of salivary glands which are similar in histologic patterns but very different in clinical behavior, treatment and prognosis. Galectin-3 is a multifunctional protein of a growing family of beta-galactoside-binding animal lectins which is implicated in a variety of biological events such as tumor cell adhesion, proliferation, differentiation and angiogenesis. This protein was found to be implicated in cellular transformation, and a correlation between its expression and cancer progression and metastasis has been described. The aim of this study was to determine the galectin-3 immunoprofile in 14 cases of adenoid cystic carcinoma (2 cases of tubular subtype, 4 cases of solid subtype and 8 cases of cribriform subtype) and in 12 cases of polymorphous low-grade adenocarcinoma with different histologic patterns, included lobular, tubular and cribriform. Moreover, slides of normal salivary glands were included. In normal salivary glands there were strong nuclei and cytoplasmic staining for galectin-3 in ductal luminal cells. Adenoid cystic carcinomas showed specific staining in luminal cells mainly in the nuclei. In the tubular subtype of adenoid cystic carcinoma galectin-3 was strong in the luminal cells of the ductiform structures. The cribriform and solid subtypes showed a few positive cells for galectin-3 only in the luminal cells of small ducts presenting in the cribriform structures and in solid nests respectly. In the cases of polymorphous low-grade adenocarcinoma, independent of the histologic architecture, all tumor cells revealed a positive cytoplasmic reaction with the galectin-3 antibody. Galectin-3 expression seems to be related to cell differentiation more than tumor progression and prognosis in the neoplasms studied.

The work embodied in this thesis pertains to an attempt to understand better, the molecular basis of protein-carbohydrate recognition. For this purpose a systematic study was undertaken, not only of the energetics of lectin-sugar interactions, which serve as molecular recognition prototype of protein-carbohydrate interactions, but also of the complex effects of solvent water molecules surrounding both the species in solution state. The systems chosen for investigation include the specific recognition of sugars by lectins from diverse families, leguminosae and moraceae. The following salient aspects of the molecular recognition process constitute the focus of this thesis: • Effect of site specifically modified, deoxy-, fluorodeoxy-, or methoxy- substituted D-galactopyranoside binding to lectins. Isothermal titration calorimetric (ITC) investigations of the binding of these sugars to a model lectin permitted the correct prediction of the architecture of the primary binding site in the absence of x-ray crystal or NMR structure of the combining site (Ref. 7). The study provided the only unambiguous means of a site specific mapping of the hydrogen-bond donor- acceptor relationship of the monosaccharide within the primary combining site of the lectin. • Novel features of lectin-sugar recognition. Molecular interactions and forces contributing to the stabilization of the saccharides in the primary combining site of lectins. Binding of site specifically modified fluoro- substituted D- galactopyranosides to WBA I led to the demonstration of the involvement of C- F««»H-0 hydrogen bonds in stabilizing the saccharide within the combining site of lectin (Ref. 7). Implication of the novel C-H«**O hydrogen bonds at the specificity determining C-4 position in enabling the methoxy- substituted D- galactopyranoside to be stabilized within the primary binding site of galactose specific lectins WBA I and jacalin. • Development of a novel coupled osmotic-thermodynamic approach for investigating the role of water molecules in determining the specificity of lectin- sugar interactions. The results obtained led to the first direct demonstration of a differential uptake of water molecules accompanying the specific process of recognition of sugars by lectins (Ref 2) • On the origin of enthalpy-entropy compensation, a ubiquitous phenomenon accompanying the thermodynamics of several ligand binding reactions in aqueous solutions in general and the molecular recognition involving all known lectin-sugar interactions, in particular. The results provide the first unequivocal solution state proof of water reorganization as the source of enthalpy-entropy compensation (Ref 3). A new diagnostic test of a true osmotic effect in molecular recognition phenomena was proposed (Ref. 2) and validated (Ref. 3). As an introduction, Chapter 1 is a comprehensive review of literature that touches upon the diverse properties of lectins and our present understanding of their multifarious roles and applications, which has led to their christening, perhaps appropriately, as molecules that mediate the 'social' functions of cells and tissues. Although a challenge it is still, to decipher the "glycocode", it is apparent that the fundamental basis of the recognition function of lectin-sugar interactions is the initial specific binding of the saccharide molecule by the globular proteinaceous lectin molecule. It is imperative, therefore, that an incisive investigation of the origin of specificity of the binding reaction as well as the solvent effects influencing both the interacting species be undertaken for a better understanding of the complete molecular recognition process. Towards this end is introduced in Chapter 1 our present understanding of the results on lectin-sugar interactions from two complementary approaches viz structural, including X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, as well as thermodvnamic ones, which have provided important information on the architecture of the combining sites, the dynamic modes of saccharide recognition and forces involved therein. Despite a detailed knowledge available from such methods, a structure-energetics correlation has persisted as a current challenge of the field. Towards achieving this goal, studies on the energetics of the recognition of sugars by lectins were undertaken, with an aim to better understand the origin of specificity of lectin-sugar interactions. This thesis attempts to provide new insights on some of the possible lacunae precluding structure-energetics correlation and suggests ways to overcome them. Chapter 2 deals with ITC investigation of the effect of deoxy-, fluorodeoxy-, and methoxy- substitutions on the binding of monosaccharides to the primary combining site of the lectin WBA I isolated from the mature seeds of the leguminosae family member Psophocarpus tetragonolobus as well as the moraceae lectin jacalin. These studies provide valuable information on the hydrogen-bond donor-acceptor relationships within the combining site of the lectins wherein the sugar molecule is liganded with the amino-acid residues of the lectin. This study is relevant for understanding the origin of specificity of monosaccharide binding within the primary combining site of the lectins. It has recently become apparent that there is a predisposition in three-dimensional space, of the donor-acceptor pairs within the sugar binding site of the lectins. Hence there appears to be a stereochemical basis of distinguishing the recognition of the donor group vis-a-vis that of the acceptor group and that their spatial disposition determines the specificity of the saccharide recognition. Unambiguous assignment of which of the groups within the hydrogen bonded pairs is a donor and which one is the acceptor assumes greater importance. The ITC measurements of the binding of deoxy-, flurodeoxy-and methoxy-derivatives of D-galactopyranoside (oc-D-Gal) to the basic lectin from winged bean Psophocarpus tetragonolobus, WBA I revealed that each of the ligands bind to WBA I with the same stoichiometry of one per subunit (29 kDa) of WBA I. The binding enthalpies for various derivatives were essentially independent of temperature and showed complementary changes with respect to binding entropies. Replacement of the hydroxyl group by fluorine or hydrogen on C3 and C4 of the galactopyranoside eliminated their binding to the lectin, consistent with C3-OH and C4-OH acting as hydrogen bond donors. The affinity for C2 derivatives of galactose decreased in the order: GalNAc>2MeOGal>2FGal=Gal>2HGal which suggests that both polar and non-polar residues surround the C2 locus of galactose, consistent with the observed high affinity of WBA I towards GalNAc, where the acetamido group at C2 position is probably stabilized by both non-polar interactions with the methyl-group and polar interactions with the carbonyl group. The binding of C6 derivatives followed the order: Gal>6FGal>D-Fuc»6MeOGal=L-Ara indicating the presence of favourable polar interactions with a hydrogen bond donor in the vicinity. Based on these results the hydrogen bond donor-acceptor relationship of the complexation of methyl-a-D-galactopyranoside with the primary combining site of WBA I was proposed (Ref. /), which was subsequently validated by the crystal structure of methyl-a-D-galactopyranoside complexed with WBA I. This chapter also describes the results from ITC studies on the binding of monosaccharides and disaccharides to the lectin jacalin isolated from the mature seeds of the moraceae family member Artocarpus integrifolia. The novel observation about the existence of C-F*«*H-0 and C-H**»O hydrogen bonds in lectin-sugar interactions is also discussed in this chapter. Chapter 3 is a description of the detailed investigation on the role of water molecules in influencing the energetics of lectin-sugar recognition. A novel coupled osmotic-thermodynamic approach was developed to dissect the role of water molecules in determining the recognition of the sugars by lectins. For this purpose, the model system of mannotriose-concanavalin A was used because atomic level structural information on these complexes were available. The work described in this chapter, is the first solution state evidence for the role of water molecules in the specific interaction of carbohydrates with a legume lectin, concanavalin A (Con A) (Ref. 2). Sugar binding to Con A was accompanied by linear changes in the logarithm of binding constants as a function of neutral osmolyte strength, and were described by well defined negative slopes characteristic for each sugar. As these changes were independent of the chemical nature of the osmolyte used, the results were rationalized in terms of a true osmotic effect. It was demonstrated that the specific recognition of the branched trimannoside (3,6-di-0-(a-D-mannopyranosyl)~a-D-mannopyranoside), the individual dimannosidic arms (3-<9-(a-D-mannopyranosyl)-a-D-mannopyranoside, and 6-0-(a-D-marmopyranosyl)-a-D-mannopyranoside) and the monomeric unit D-mannopyranoside by Con A was accompanied by differential uptake of water molecules; 1,3 and 5 respectively. We also observed a conservation of the compensatory behaviour of binding enthalpies and entropies in the presence as well as absence of osmolytes. This provided the first definitive evidence that water-reorganization plays a direct role in effecting the phenomenon of enthalpy-entropy compensation in protein-ligand interactions in general and lectin-sugar interactions in particular, and that the specificity of lectin-sugar recognition is characterized by a differential uptake of water molecules. Chapter 3 also describes the first experimental identification of the origin of enthalpy-entropy compensation (EEC), a ubiquitous phenomenon accompanying the thermodynamics of multifarious biomolecular recognition processes. By coupling direct microcalorimetry with osmotic stress technique, an experimental handle was devised to test the hypothesis that solvent reorganization could be the source of EEC. The results provided an unequivocal demonstration that an osmotic change in water activity alone, at the same temperature and pH, is sufficient to result in the conservation of EEC during the molecular recognition of specific ligands by macromolecules belonging to thermodynamically diverse and unrelated systems, a compelling evidence that the primary source of EEC in aqueous solutions is attributable to reorganization of solvent water molecules, thus validating the test for the role of water reorganization as a source of EEC (Ref. 3). This provides the first definitive evidence for the notion that there is a direct involvement of water molecules in originating the EEC effect. Despite the generality of the results it is urged that several systems be subjected to a vigorous application of the coupled osmotic-thermodynamic approach proposed herein before constituting it as a proof. Suffice to say, it is perhaps heartening that at last one has a handle to test the role of water molecules in effecting EEC in the solution state and appreciate the diverse roles played by water molecules in mediating molecular recognition reactions. The proposal presented in Ref 2, that the strong isoequilibrium relationship of enthalpy with entropy during the recognition of saccharides by Con A studied under osmotic stress, be considered as diagnostic of a true osmotic effect was subsequently validated in a thermodynamically diverse and unrelated system of peptide recognition by monoclonal antibody, the results from which are discussed in an Appendix (A) to this thesis (Ref 4). That the stabilities of these lectins are not hampered in the presence of osmolytes was demonstrated using differential scanning calorimetry (DSC) (Ref 2). During the course of these DSC studies, we discovered an unusual feature in an animal galectin. Despite possessing the legume lectin fold, the 14-kDa S- type lectin exhibits multiple oligomeric states that are influenced profoundly by complementary ligands and surprisingly do not dissociate at the denaturation temperature. These results are discussed in an Appendix (B) to this thesis (Ref. 5). The general discussion and conclusions drawn from this work are summarized in chapter 4. Briefly, the following salient conclusions can be drawn from the work presented in this thesis: 1. Unambiguous assignment of hydrogen-bond donor-acceptor relationship at each of the hydroxyl group of the monosaccharide bound to the lectin belonging to different families has been demonstrated (Refs. 1,6). 2. First report of novel hydrogen bonds in lectin-sugar interactions such as C- F«MH-0 (Ref 1) and C-H^*O hydrogen bonds (Ref 6). 3. Unusual structural stabilities in a galectin with a fold similar to that in legume lectins but with starkly different thermodynamic stabilities (Ref 5). 4. We have demonstrated for the first time in solution state, that water molecules are involved in the specific recognition of sugars by concanavalin A (Ref 2). It appears that lectin-sugar recognition reactions are, in general, mediated by a net uptake of water molecules during the binding process (Ref 7). 5. We have provided the first experimental demonstration that reorganization of water molecules is the source of enthalpy-entropy compensation in molecular recognition processes (Ref 3). 6. We provide evidence for another facet in the recognition of antigens by antibodies, viz water release accompanying the binding reaction (Ref 4). The studies reported in this thesis provide the foundation for embarking on a systematic study not only of the origin of specificity of lectin-sugar recognition but also of the complex roles that water molecules play in mediating these molecular recognition processes. These specific binding reactions wherein non-linear thermodynamics predominates and precludes a direct structure-energetics correlation emphasize the need to account for the effect of solvent water molecules in lectin-sugar interactions in particular and, without any overemphasis, in molecular recognition processes in general.

In this study, we hypothesize that human acidic mammalian chitinase (AMCase) binds and is regulated by the epidermal growth factor receptor (EGFR), and that AMCase interacts with Galectin-3 (Gal-3) to mediate anti-apoptotic functions. We further hypothesize that asthma-associated polymorphisms of AMCase alter chitinase activity and modulate anti-apoptotic effects. We investigated the interactions between AMCase, Gal-3 and EGFR by establishing binding and co-expression in vitro; apoptotic effects were evaluated via Annexin V/Propidium Iodide staining. Molecular cloning was performed to generate single nucleotide polymorphisms (SNPs) of AMCase associated with asthma. Our data showed that co-expression of AMCase and EGFR induces chitinase activity; we found that AMCase and Gal-3 bind each other in vitro, and that they co-localize in the cytoplasm of cells. Co-transfection of AMCase and Gal-3 demonstrates greater anti-apoptotic effect than Gal-3 alone, while recombinant Gal-3 induces apoptosis, which is not blocked by incubation with recombinant AMCase. From these data, we conclude that AMCase is regulated by EGFR, and that AMCase and Gal-3 physically interact, however contrary to our hypothesis, the anti-apoptotic effects of AMCase are unlikely to be mediated by Gal-3. Further exploration of this pathway using SNP constructs generated in this study will shed light on the mechanism of AMCase in asthma.

Protein aggregation is commonly encountered during the manufacture of protein-based bioproducts in processing such as protein expression, purification, refolding, shipping and storage (Volkin and Middaugh, 1992; Brange, 2000). Aggregation may shorten the shelf-life of pharmaceutical proteins (Frokjaer and Otzen, 2005) and induce severe hypersensitivity (Rosenberg, 2006). In addition, several diseases ranging from Alzheimer’s disease to cystic fibrosis are associated with protein aggregation in the form of amyloid fibrils and plaques (Dobson, 1999; Luheshi et al., 2008). Hence, studies on protein aggregation, especially those dealing with high concentrations of proteins, are highly demanded in both academic and industrial laboratories. To address the aforementioned issues, physicochemical factors affecting protein aggregation were investigated systematically in this project. Strategies were developed to inhibit protein aggregation during renaturation and to enhance protein stability against aggregation during and after production, especially when dealing with high protein concentrations. ∆5-3-Ketosteroid isomerase (KSI) was used as a model for aggregation studies during protein renaturation due to its intrinsic aggregation properties. KSI was overexpressed as inclusion bodies (IBs) in Escherichia coli (E. coli). Cost- and time-efficient combination of chemical extraction and one-step affinity purification ensured the production of denatured KSI with high purity at high yield. Several key factors, including protein concentration and ionic strength, were determined to greatly influence KSI aggregation during renaturation. Polymer addition (PEG 3000 and Eudragit S-100) was found to alter KSI aggregation behaviour in a polymer-specific manner, as quantified using reversed phase-high performance liquid chromatography (RP-HPLC) analysis. Light scattering for second virial coefficient (SVC) measurement, surface plasmon resonance (SPR), and microfluidics were applied to study the fundamental mechanism of protein aggregation. Lysozyme was further introduced as a control protein for comparison with KSI. A rapid lumped method was established to measure specific refractive index (∂n/∂c) and SVC values for KSI and lysozyme, which provided quantitative and qualitative information on thermodynamic interactions of molecules in solution. SPR and microfluidics were also used to explore protein aggregation properties. To our best knowledge, it is the first time SPR and microfluidics have been used to investigate protein aggregation behaviour. Both SPR and microfluidics present significant potential for assessing protein aggregation and diagnosis or drug screening of protein aggregation related diseases. The chemical and physical stability of proteins needs to be maintained after successful refolding to ensure an acceptably long shelf life, especially at high protein concentration (Chang and Hermsdorf, 2002). The pharmaceutical effects of lectins on cell growth provided incentive for studies to improve their stability. Human galectin-2 (hGal-2, a homodimeric lectin) was used as a study model in this project. Mutations were introduced at one of the two Cys residues (C57A, C57M, and C57S). Only the C57M variant was highly expressed in bacteria in soluble form. No aggregate of this mutant was detected during 3 weeks of storage. hGal-2 C57M also facilitated site-directed introduction of poly(ethylene glycol) (PEG) into the remaining sulfhydryl group (Cys75). Product analysis revealed rather complete conjugation with one PEG chain per protein subunit in homodimer. Neither secondary structure alteration nor the absence of binding ability to a glycoprotein (asialofetuin) was observed. The results document the feasibility of tailoring a human galectin for enhanced stability against aggregation as well as monoPEGylation, which enables further testing of biological properties including functionality as a growth regulator and the serum clearance rate of hGal-2.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2014-09-18T13:07:03Z No. of bitstreams: 1 Gabriela da Silva Cruz. Avaliação... 2014.pdf: 1237079 bytes, checksum: 7beefa52e21bd18ac4325a1c493249ba (MD5) / Made available in DSpace on 2014-09-18T13:07:04Z (GMT). No. of bitstreams: 1 Gabriela da Silva Cruz. Avaliação... 2014.pdf: 1237079 bytes, checksum: 7beefa52e21bd18ac4325a1c493249ba (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / O Trypanosoma cruzi é um parasita intracelular e agente causador da doença de Chagas, que afeta milhões de pessoas em todo o mundo. Sabe-se que durante os processos de inflamação, regeneração e fibrose desencadeados pelo T. cruzi no hospedeiro há a participação de diversos mediadores e fatores. O objetivo deste trabalho foi avaliar a associação entre polimorfismos de nucleotídeos únicos com as formas clínicas e o grau de fibrose em pacientes com doença de Chagas. Os polimorfismos foram analisados por PCR em tempo real. Foram incluídos no estudo 55 pacientes com diagnóstico de doença de Chagas e classificados de acordo com a forma clínica da doença, sendo que 17 apresentavam a forma indeterminada, 15 a forma cardíaca sem disfunção ventricular e 23 a forma cardíaca com disfunção ventricular. Os genótipos CA dos polimorfismos do gene LGALS3 (rs4644 e rs4652); AG e GG do SOCS3 (rs4969170); CT e TT do IL-28B (rs12979860 e 8099917, respectivamente); AG, AG, CC, AG e AG do CLDN-1 (rs10212165, rs3909582, rs9865082, rs9880018 e rs9848283, respectivamente); e CC do CCL5 (rs2280789) foram estatisticamente mais frequentes em pacientes com a forma cardíaca do que com a forma indeterminada da doença. Com relação ao grau de fibrose, os genótipos CC dos polimorfismos do gene LGALS3 (rs4644 e rs4652); AA do SOCS3 (rs4969170); CC do rs12979860 e TT do rs8099917 do IL-28B; AA do rs10212165, AA, AG e GG do rs3909582, CC e CT do rs9865082, AG e GG do rs9880018 e AA do rs9848283 do gene CLDN1; e CC do CCL5 (rs2280789) foram estatisticamente mais frequentes em indivíduos com fibrose cardíaca <15% quando comparados com o grupo com fibrose ≥15%. Diante do exposto concluimos que os polimorfismos analisados podem ser úteis como futuros biomarcadores para estadiamento e conduta terapêutica em pacientes com doença de Chagas. / Trypanosoma cruzi is an intracellular parasite and the agent that causes Chagas disease, which affects millions of people worldwide. Several factors and mediators are known to actively participate in the inflammation, fibrosis and tissue regeneration, which is triggered by T. cruzi within the host. The aim of this study was to evaluate the association of single nucleotide polymorphisms with clinical forms and rate of fibrosis in Chagas disease patients. The polymorphisms were analyzed by real-time PCR. The study consisted of 55 Chagas disease patients that were classified according to the clinical form of the disease, including 17 patients presenting the indeterminate form, 15 patients presenting the cardiac form without ventricular dysfunction and 23 patients presenting the cardiac form with ventricular dysfunction. The genotypes of CA of LGALS3 gene polymorphisms (rs4644 and rs4652); AG and GG of SOCS3 (rs4969170); CT and TT of IL-28B (rs12979860 and 8099917, respectively); AG, AG, CC, AG and AG of CLDN-1 (rs10212165, rs3909582, rs9865082, rs9880018 and rs9848283, respectively); and CC of CCL5 (rs2280789) were significantly more frequent in patients presenting the cardiac form compared to patients presenting the indeterminate form. Regarding the degree of fibrosis, the CC genotype of polymorphisms of the genes LGALS3 (rs4644 and rs4652); AA of SOCS3 (rs4969170); CC of rs12979860 and TT of rs8099917 of the IL-28B; AA of rs10212165 and AA, AG and GG of rs3909582, CC and CT of rs9865082, AG and GG of rs9880018 and AA of rs9848283 of the gene CLDN1; and CC of CCL5 (rs2280789) were statistically more frequent in patients presenting <15% cardiac fibrosis when compared to patients presenting fibrosis ≥15%. Taken together, our results suggest that the polymorphisms analyzed may be useful biomarkers for therapeutic management of patients with Chagas disease.

Head and neck squamous cell carcinomas are still challenging despite progress in the oncological treatment. Study of the molecular biology allows to deeply characterize tumor properties and to predict the prognosis for affected patients. Nowadays there are many drugs clinically tested in the group of targeted therapy medicine Experimental work comprised both in vitro and in situ assays, being performed thanks to the collaboration between a number of departments of the 1st Faculty of Medicine of the Charles University in Prague, Academy of Sciences of the Czech Republic, Institute of Hematology and Blood Transfusion and Faculty of Veterinary Medicine of the Ludwig-Maxmillian University Munich. Galectin-1 is important inductor of the myofibroblasts/cancer associated fibroblasts. These fibroblasts are regarded as negative prognostic markers thanks to their capability of invasive cancer cells induction. On the other hand, Galectin-9 is not present in the carcinoma and in the case of dysplasia, its expression indicate aberrant features together with aberrant expression of keratin 14 and 19. Except from galectins using as prognostic markers, we focused on the galectins as a therapeutics instruments as well. Presented work with mutant variants of galectin-2 proved their effect on both pharmacodynamics and...

nÃo hà / INTRODUÃÃO: Estudos epidemiolÃgicos sugerem que o carcinoma de tireoide seja a neoplasia maligna mais frequente nos pacientes acromegÃlicos. Atà este momento nÃo hà relatos de estudos avaliando marcadores moleculares do carcinoma papilÃfero de tireoide (CPT) nessa populaÃÃo. OBJETIVOS: Avaliar a associaÃÃo entre acromegalia, presenÃa da mutaÃÃo BRAFV600E, marcadores imunohistoquÃmicos (galectina-3 e IGF-1) e caracterÃsticas clÃnico-patolÃgicas em pacientes acromegÃlicos com CPT. MATERIAL E MÃTODOS: Trata-se de um estudo transversal realizado no perÃodo de janeiro/09 a dezembro/2011, onde 11 pacientes acromegÃlicos com CPT, provenientes de 5 centros brasileiros de referÃncia no tratamento da acromegalia foram comparados com 45 pacientes com CPT sem acromegalia. Foram estudadas variÃveis clÃnicas e histopatolÃgicas do CPT. Utilizou-se cortes histolÃgicos de CPT emblocados em parafina para o estudo da mutaÃÃo BRAFV600E e para a anÃlise imunohistoquÃmica dos marcadores IGF-1 e galectina-3. Na anÃlise utilizou-se os testes t de student e do qui-quadrado (software SPSS, versÃo 13.0 para Windows) (p<0,05). RESULTADOS: A idade mÃdia dos pacientes acromegÃlicos com CPT foi de 61,5  6,02 anos, sendo 72,7% do sexo feminino. O tempo mÃdio de diagnÃstico da acromegalia foi de 7,7  3,90 anos, sendo o intervalo entre o diagnÃstico da acromegalia e do CPT, em mÃdia, 3,4  2,71 anos. Os nÃveis sÃricos de IGF-1 dos acromegÃlicos ao diagnÃstico do CPT foi de 417,0 ng/mL. NÃo houve diferenÃa quanto ao estadiamento TNM (Tumor, Nodule, Metastasis) e Ãndice prognÃstico AMES (Ages, Metastasis, Extent, Size) entre os grupos. Houve maior prevalÃncia da mutaÃÃo BRAFV600E (90,9% vs 55,6%; p=0,039) e de forte imunoexpressÃo para IGF-1 (88,9% vs 38,1%; p= 0,017) nos acromegÃlicos. NÃo houve diferenÃa na expressÃo de galectina-3 entre os grupos. CONCLUSÃO: Neste trabalho, pela primeira vez se mostrou uma alta prevalÃncia da mutaÃÃo BRAFV600E em CPT de acromegÃlicos, muito superior à descrita na populaÃÃo com CPT neste e em estudos anteriores (cerca de 40%). Contudo essa mutaÃÃo nÃo se mostrou associada a um fenÃtipo mais agressivo do tumor, o que diverge dos achados em populaÃÃo nÃo acromegÃlica com CPT. Conclui-se que a acromegalia à possivelmente associada à mutaÃÃo BRAFV600E em pacientes acromegÃlicos com CPT. Novos estudos serÃo necessÃrios para definir os mecanismos responsÃveis por tal associaÃÃo. / INTRODUCTION: Epidemiological studies suggest that thyroid carcinoma is the most common malignant neoplasm in acromegalic patients. At this moment there are no reports of studies evaluating molecular markers of papillary thyroid carcinoma (PTC) in this population. OBJECTIVES: The present work aimed to evaluate the association between acromegaly, expression of the mutation BRAFV600E, immunohistochemical markers (galectin-3 and IGF-1), and clinical-pathological characteristics in acromegalic patients with PTC. MATERIALS AND METHODS: This is a cross-sectional study conducted from January/09 to December/2011, where 11 acromegalic patients with CPT, from 5 Brazilian centers of reference in the treatment of acromegaly were compared with 45 patients with acromegaly without PTC. We evaluated clinical and histopathological variables of PTC. We used histological PTC embedded in paraffin for mutation study BRAFV600E and immunohistochemical analysis of markers IGF-1 and galectin-3. In the analysis we used the Student t test and chi-square test (SPSS software, version 13.0 for Windows) (p <0.05). RESULTS: The average age of acromegalic patients with PTC was 61.5  6.02 years and 72.7% were female. The average time of diagnosis of acromegaly was 7.7  3.90 years, and the interval between diagnosis of acromegaly and PTC was an average 3.4  2.71 years. The serum levels of IGF-1 in the diagnosis of acromegaly PTC was 417.0 ng / mL. There was no difference in the TNM (Tumor, Nodule, Metastasis) and AMES prognostic index (Ages, Metastasis, Extent, Size) between groups. There was a higher prevalence of the BRAFV600E mutation (90.9% vs 55.6%, p = 0.039) and stronger immunohistochemical expression for IGF-1 (88.9% vs 38.1%, p = 0.017) in acromegaly. There was no difference in the expression of galectin-3 between the groups. CONCLUSION: This work for the first time showed a high prevalence of mutations in BRAFV600E in PTC of acromegalic patients superior to those described in the population with PTC in this and previous studies (approximately 40%). However, this mutation was not associated with a more aggressive tumor phenotype, which differs from the findings in acromegalic population without PTC. We conclude that acromegaly is possibly associated to a mutation BRAFV600E in acromegalic patients with CPT. Further studies are needed to define the mechanisms responsible for this association.

Aim: The overarching aim of the thesis was to explore the occurrence and clinical importance of two manifestations of myocardial injury; unrecognized myocardial injury (UMI) and altered levels of cardiac biochemical markers in patients with stable coronary artery disease (CAD). Methods: A prospective multicenter cohort study investigated the prevalence, localization, size, and prognostic implication of UMI in 235 patients with stable CAD. Late gadolinium enhancement cardiovascular magnetic resonance (LGE-CMR) imaging and coronary angiography were used. The relationship between UMI and severe CAD and cardiac biochemical markers was explored. In a substudy the short- and longterm individual variation in cardiac troponins I and T (cTnI, cTnT) and N-terminal pro-B-type natriuretic peptide (NT-proBNP) were investigated. Results: The prevalence of UMI was 25%. Subjects with severe CAD were significantly more likely to exhibit UMI than subjects without CAD. There was a strong association between stenosis ≥70% and presence of UMI in the myocardial segments downstream. The presence of UMI was associated with a significant threefold risk of adverse events during follow up. After adjustments UMI was associated with a nonsignificant numerically doubled risk. The levels of cTnI, NT-proBNP, and Galacin-3 were associated with the presence of UMI in univariate analyses. The association between levels of cTnI and presence of UMI remained significant after adjustment. The individual variation in cTnI, cTnT, and NT-proBNP in subjects with stable CAD appeared similar to the biological variation in healthy individuals. Conclusions: UMI is common and is associated with significant CAD, levels of biochemical markers, and an increased risk for adverse events. A change of &gt;50% is required for a reliable short-term change in cardiac troponins, and a rise of &gt;76% or a fall of &gt;43% is required to detect a long-term reliable change in NT-proBNP.