Ao longo do processo de progressão tumoral, se observa alteração na expressão de glicoconjugados contendo oligossacarídeos N-ligados. Uma das formas mais comuns de glicosilação aberrante observada em células transformadas e em tumores humanos é representada por (poli)lactosaminas presentes em oligossacarídeos N-ligados. Estes glicanos são ligantes de galectina-3. Com o objetivo de identificar a expressão e distribuição dos ligantes de galectina-3 associados a processos fisiopatológicos, como a transformação maligna, desenvolvemos uma proteína quimérica, a galectina-3 conjugada a fosfatase alcalina (Gal-3/FA). Observamos que a Gal-3/FA possui a mesma especificidade de galectina-3 e que pode ser usada como sonda em ensaios de overlay e ensaios de imunoistoquímica. Entre os ligantes de galectina-3 identificamos a ?1 integrina, mediador de processos biológicos dependentes da interação célula-matriz como a migração celular. Linhagens de células de origem mesenquimal derivadas de tumores induzidos com metilcolantreno de animais selvagens (linhagens S11 e S12) e nulizigoto (linhagem ?12) para o gene da galectina-3 foram estabelecidas. Avaliamos a capacidade migratória dessas células e os nossos resultados mostraram que células que expressam galectina-3 são mais migratórias em superfícies de laminina-1. Este dado sugere que a galectina-3 seja um modulador positivo do processo de migração celular em superfícies de laminina-1. No entanto, o mecanismo pelo qual a galectina-3 medeia esse processo não é conhecido. Células que possuem fenótipo mais migratório apresentam um estado intermediário de adesão. Nós observamos que a galectina-3 se encontra nos complexos focais. Na presença de galectina-3 observamos diminuição de FAK fosforilado e recrutamento da fosfatase SHP-2 para os complexos focais. A diminuição de FAK fosforilado no lamelipódio leva ao turnover dos complexos focais e ao aumento da migração celular. Analisamos também a via de sinalização e observamos que a galectina-3 não ativa PAK. Contudo, o inibidor de PI3quinase, wortmanina, inibiu o efeito pró-migratório de galectina-3. Esses dados reforçam a noção do papel de galectina-3 na modulação do processo de migração de fibroblastos transformados, funcionando como uma molécula / Altered expression of cell surface N-linked oligosaccharides are often associated with malignant transformation of cells. One of the most common forms of aberrant glycosylation in transformed cells and human tumors is the highly elevated ?1,6 branching of N-linked oligosaccharides caused by increased expression of N-acetylglucosaminytransferase V (Mgat5). Galectin-3, a ?-galactoside binding protein, binds preferentially to poly-N-acetyllactosamines, which are the products of Mgat5. In order to exploit this hallmark of cancer cells, we have developed a tool for in situ identification of these tumors associated glycoconjugates. Human galectin-3 was fused to bacterial alkaline phosphatase, generating a hybrid molecule displaying both the carbohydrate binding properties of galectin-3 and enzymatic activity of alkaline phosphatase (Gal-3/FA). Gal-3/FA has the same fine of galectin-3 which was confirmed in direct binding assays. The tool presented herein was therefore useful for several immunoenzymatic assays, and will allow to establish whether the expression pattern of galectin-3 ligands have any physiological or clinical significance. We have identified ?1 integrin as a galectin-3 ligand. ?1 integrins are the actual effector of cell adhesion and migration. We have established cell lines from methylcholantrene-induced sarcomas from both wild type and galectin-3 null mice. In this system, galectin-3 null cells were less migratory than control cells in laminin-1. When galectin-3 was transiently expressed in galectin-3 null sarcoma cells, it inhibited cell adhesion to laminin-1 and stimulate the migratory response to laminin-1. The addition of exogenous galectin-3 also enhanced the migratory capacity of ?12 cells in a carbohydrate dependent way. Galectin-3 was found in focal contacts of ?12 cells where it may interact with many glycoproteins containing polyllactosamines on the cell surface. Here we showed that ?1 integrins are among them. Exogenously added galectin-3 led to a decrease in phosphorylated-FAK in lamellipodia and increased the recruitment of Shp-2 phosphatase of migrating cells. The effect of galectin-3 in migration was not dependent on the activation of the p21-activated kinase (PAK). Wortmannin inhibited the increased migration elicited by galectin-3, suggesting the involvement of the PI3-kinase signaling in the galectin-3 pathway. We propose that extracellular galectin-3 bound ?1integrins and disrupted the focal adhesion plaque, thus favoring cell migration.

Assim como tecidos normais, tumores possuem uma demanda de nutrientes e oxigênio, suprida através da vasculatura a eles associada que resulta do processo de angiogênese. Fatores pró-angiogênicos são capazes de atrair monócitos, os quais se diferenciam em macrófagos associados a tumores (TAMs). TAMs comumente apresentam fenótipo M2, cujas características são consideradas pró-tumorais, como a promoção da angiogênese e a degradação de matriz extracelular. Estudos indicam que galectina-3 (gal-3), uma proteína pleiotrópica que se liga a ?-galactosídeos, participa do controle da angiogênese e da infiltração de macrófagos M2 na massa tumoral, mas pouco se sabe sobre os mecanismos envolvidos. No presente estudo, utilizamos um modelo de sarcoma induzido por carcinógeno em camundongos selvagens (WT) e knockout para gal-3 (Gal- 3 KO). Comparando os tumores de animais WT e Gal-3 KO, não observamos diferenças no padrão de crescimento tumoral, na área necrótica relativa, na proliferação celular e na quantificação de fibras de colágeno. Demonstramos que, embora ambos os grupos desenvolvam tumores, a angiogênese foi inibida em um microambiente desprovido de gal-3. Entretanto, não houve diferença na produção do fator de crescimento endotelial vascular (VEGF). As imagens obtidas in vivo indicaram que gal- 3 também influencia na formação estrutural de vasos adjacentes ao tumor. Além de mediar aspectos morfológicos relacionados à angiogênese, demonstramos que gal-3 também contribuiu para a funcionalidade vascular, pois houve uma redução na velocidade de fluxo sanguíneo nos vasos intratumorais de animais Gal-3 KO. Nossos dados sugeriram que há menos macrófagos no tumor que não expressa gal-3 e, dentre os TAMs, há mais M2 em comparação ao tumor gal-3-positivo. A análise do tecido onde o tumor se desenvolve, na fase inicial da tumorigênese, indicou que a ausência de gal-3 está relacionada a uma maior densidade de macrófagos M2. Considerando que a presença maior de macrófagos M2 nos sarcomas gal-3-negativos não resultou em maior produção de VEGF, mas sim na inibição da angiogênese, nossos resultados apontam para uma participação significativa de gal-3 na mediação da angiogênese pelos macrófagos / As well as normal tissues, tumors require nutrients and oxygen, which are supplied by the associated vasculature that results from the process of angiogenesis. Pro-angiogenic factors are able to attract monocytes and they differentiate into tumor-associated macrophages (TAMs). TAMs commonly exhibit M2 phenotype, which has characteristics considered pro-tumoral, such as angiogenesis promotion and degradation of extracellular matrix. Studies show that galectin-3 (gal-3), a pleiotropic ?-galactosidebinding protein, participates in angiogenesis control and M2 macrophage infiltration into the tumor mass, but little is known about the mechanisms involved. In this work, we established a model of carcinogen-induced sarcoma in wild-type (WT) and gal-3 knockout (Gal-3 KO) mice. Comparing tumors from WT and Gal-3 KO animals, there were no differences in the pattern of tumor growth, relative necrotic area, cell proliferation and collagenous fibers. We demonstrated that, although both groups develop tumors, angiogenesis was inhibited in a microenvironment devoid of gal-3. However, there was no difference in the production of vascular endothelial growth factor (VEGF). The images obtained in vivo indicated that gal-3 also influenced the structural formation of vessels adjacent to the tumor. In addition to mediating morphological aspects related to angiogenesis, we demonstrated that gal-3 also contributes to vascular functionality, since there was a reduction in blood flow velocity in intratumoral vessels from Gal-3 KO animals. Our data suggested that there are fewer macrophages in tumors without gal-3 and, among TAMs, there are more M2 compared to gal-3-positive tumors. Analysis of the tissue where the tumor develops, in early stages of tumorigenesis, indicated that the lack of gal-3 is related to an increased density of M2 macrophages. Since the greater number of M2 macrophages in gal-3-negative fibrosarcomas did not result in increased VEGF production, but inhibited angiogenesis, our results suggest a significant role of gal-3 in regulation of angiogenesis by macrophages

Galectina-3 é uma lectina animal que apresenta afinidade por b- galactosídeos e que tem sido associada à progressão tumoral e metástase. A expressão de galectina-3 encontra-se alterada durante a progressão tumoral de diferentes neoplasias. Em tumores como carcinoma de tiróide e bexiga a expressão de galectina-3 encontra-se aumentada, enquanto que em tumores como carcinoma de mama e ovário a expressão desta lectina encontra-se diminuída. Neste trabalho nós utilizamos um modelo de progressão tumoral de melanoma murino para investigar os mecanismos envolvidos na perda de expressão de galectina-3. Este modelo é composto por uma linhagem de melanócitos imortalizados (melan-a) e duas linhagens de melanoma de crescimento vertical (Tm1 e Tm5) estabelecidas após submeter a linhagem melan-a a inúmeros ciclos de de-adesão. Enquanto melan-a acumula grandes quantidades de galectina-3, as linhagens Tm1 e Tm5 deixaram de expressar o gene de galectina-3. Análise da região 5\' do gene de galectina-3 demonstrou que esta região apresentava grande conteúdo de dinucleotídeos CpG e vários sítios SP1. O seqüenciamento desta região após tratamento do DNA com bissulfito de sódio mostrou que esta região estava totalmente metilada nas linhagens Tm1 e Tm5 e desmetilada na linhagem melan-a. O tratamento da linhagem Tm1 com 5-Aza-2\'-deoxicitidina (5-Aza-CdR), um inibidor da DNA metiltransferase, provocou um decréscimo significativo nos níveis de metilação da região 5\' do gene de galectina-3 que por sua vez levou a re-expressão do RNAm e da proteína. O tratamento de Tm1 com os inibidores de histono deacetilases tricostatina A e 4-ácido-fenilbutírico em combinação com 5-Aza-CdR não aumentou os níveis de expressão do gene de galectina-3 e curiosamente, reverteu o efeito induzido por 5-Aza-CdR. Em adição, a expressão da enzima DNMT1 apresentou um discreto aumento nas linhagens Tm1 e Tm5 em relação a melan-a. Em conjunto esses resultados sugerem que mecanismos epigenéticos como a metilação estão envolvidos no controle de expressão do gene de galectina- 3 ao longo da progressão tumoral de melanoma murino. / Galectin-3 is a b-galactoside-binding animal lectin, shown to be involved in tumor progression and metastasis. Galectin-3 expression has been found altered along tumor progression of different tumors. In some types of cancers such as thyroid carcinoma and bladder carcinoma, galectin-3 expression has been found increased, whereas in tumors such as breast carcinoma and ovary carcinoma the expression of this lectin has been found decreased along tumor progression. In this study, we have used a murine melanoma model to investigate the mechanisms responsible for the loss of galectin-3 gene expression. This model consists of a cell line of immortalized melanocytes (melan-a) and two cell lines of vertical growth phase melanoma (Tm1 and Tm5) established after submitting melan-a cells to several deadhesion cycles. While melan-a expressed high amounts of galectin-3, both Tm1 and Tm5 cells lost galectin-3 gene expression. Analysis of the 5\' upstream region of the galectin-3 gene demonstrated the presence of a high CpG content and several SP1 binding sites. Bisulfite sequencing of this region showed that it was fully methylated in Tm1 and Tm5 cells and unmethylated in melan-a cells. Treatment of Tm1 cells with 5-aza-2\'-deoxycytidine (5-Aza-CdR), a DNA methyltransferase inhibitor, led to a marked decrease in the methylation levels of the 5\' upstream region of the galectin-3 gene, which led to transcription of the galectin-3 gene. Treatment of Tm1 cells with the histone-deacetylase inhibitors trichostatin A and 4- acid-phenilbutyrate in combination with 5-Aza-CdR did not increase the levels of galectin-3 gene expression and intriguingly, reverted the effect of 5-Aza-CdR alone. In addition, the expression of DNMT1 showed a modest, but significant increase in Tm1 and Tm5 cells as compared with melan-a cells. Altogether these results indicate that epigenetic mechanisms such as methylation play a role in the regulation of the galectin-3 gene expression along murine melanoma progression.

A heterogeneidade intratumoral é um fenômeno extremamente importante para entender a progressão tumoral e a resposta à intervenção terapêutica. A galectina-3 pertence à família das lectinas, possuem a função de reconhecimento e ligação à ?-galactosídeos ramificados de glicolipídeos e glicoproteínas, e está envolvida em processos fisiológicos e patológicos como o câncer. Nesse trabalho, a heterogeneidade intratumoral em relação à expressão de galectina-3 foi observada em amostras de diferentes lesões melanocíticas de pacientes. Além disso, o inóculo de células de melanoma murino negativas para galectina-3 em animais gal3-/- gerou tumores constituídos por uma fração de células tumorais que passaram a expressar de novo galectina-3, sugerindo que pressões do microambiente tumoral modulam a expressão dessa lectina em melanomas. A acidose extracelular atuou como regulador negativo de galectina-3 in vitro, diminuindo a expressão dessa lectina tanto em células de melanoma murino e humano quanto em melanócito murino. Entretanto, a hipóxia, seja pela exposição aguda ou intermitente, não alterou a expressão in vitro de galectina-3 em células de melanoma humano. Por fim, tumores originados pelo inóculo de células tumorais positivas e negativas para galectina-3 (mimetizando tumores heterogêneos) obtiveram a maior taxa de crescimento tumoral comparados aos tumores constituídos por uma única população de células, seja positiva ou negativa para galectina-3. Portanto, foram apresentadas evidências de que a heterogeneidade intratumoral em relação à galectina-3 parece estar envolvida com o sucesso evolutivo do melanoma e que a acidose é indicada como uma das pressões microambientais que contribuem para o estabelecimento e manutenção da fração de células tumorais negativas para galectina-3 dentro da massa tumoral / The intratumoral heterogeneity observed in human tumors is extremely important to understand tumor progression and its therapeutic response. Galectin-3 belongs to animal lectin family and it is a ?-galactosidase binding protein which is involved in physiological and pathological processes, including cancer. In this work, an intratumor heterogeneous galectin-3 expression was observed in tissue sessions containing different human melanocytic lesions. Moreover, negative galectin-3 murine cells injected into gal3-/- mice were able to generate tumors composed of a positive galectin-3 cell fraction, suggesting that selective forces in tumor microenvironment modulate galectin-3 expression in melanoma. Extracellular acidosis acts as a negative regulator to galectin-3 in vitro, decreasing its expression in murine and human melanoma cells and even in murine melanocytes. However, intermittent or acute hypoxia exposure did not alter galectin-3 expression in human melanoma cells in vitro. In addition, tumors originated from a mixture of positive and negative galectin-3 cells (mimicking heterogeneous tumors) showed higher growth rate compared to those derived from only galectin-3 positive or negative cells. Therefore, we showed evidences that galectin-3 intratumoral heterogeneity seems to be involved with the evolutionary success of melanoma and that acidosis may be the microenvironmental pressure responsible for the establishment and maintenance of galectin-3 negative cell fraction into the tumor bulk

Les lymphomes sont constitués de sous-groupes tumoraux définis par différents critères cliniques, morphologiques, immunophénotypiques et moléculaires. Néanmoins, même au sein d’un sous-groupe, il existe une grande hétérogénéité dans l’évolution clinique des patients, ce qui rend leur prise en charge difficile. Ceci souligne l’importance de découvrir de nouveaux biomarqueurs pronostiques qui amélioreraient l’approche thérapeutique personnalisée. <p>La revue de la littérature fait apparaitre les galectines-1 et -3 comme des protéines impliquées dans le développement des cancers ainsi que dans la biologie des lymphocytes. Pourtant, au moment où nous avons débuté ce travail, peu de chercheurs s’étaient intéressés à l’implication de ces galectines dans les lymphomes.<p>Nous avons donc décidé d’évaluer le rôle diagnostique ou pronostique de la galectine-1 et de la galectine-3 dans les lymphomes.<p><p>La première étude a consisté en la caractérisation de l’expression immunohistochimique de ces deux galectines au sein d’une série clinique de lymphomes systémiques humains en comparaison au tissu lymphoïde normal. <p>En ce qui concerne la galectine-1, les cellules lymphomateuses l’expriment peu, de façon similaire à ce qui est observé pour les cellules lymphoïdes normales. Au contraire, les cellules endothéliales associées aux lymphomes sont caractérisées par une augmentation de l’expression de galectine-1 en comparaison aux cellules endothéliales normales. Cette expression endothéliale ne montre pas de relation avec les variables cliniques de notre étude mais est corrélée à la densité microvasculaire des lymphomes. <p>En ce qui concerne la galectine-3, les cellules lymphomateuses des lymphomes B diffus à grandes cellules se caractérisent par une expression plus élevée comparativement aux autres sous-groupes de lymphomes et aux cellules lymphoïdes normales. Cette expression de galectine-3 n’est pas associée aux variables cliniques dans notre étude. Contrairement à la galectine-1, la galectine-3 est exprimée de façon similaire par les cellules endothéliales des vaisseaux des tissus lymphoïdes normaux et des lymphomes.<p>Ces données suggèrent un rôle angiogénique de la galectine-1 dans les lymphomes systémiques.<p><p>Dans la deuxième étude, nous avons caractérisé l’expression des galectines-1 et -3 au sein d’une série clinique de lymphomes primitifs du système nerveux central (LPSNC). <p>Nous avons observé que les profils d’expression des cellules lymphomateuses sont similaires pour les lymphomes systémiques et les LPSNC, caractérisés par une absence d’expression de galectine-1 et une expression variable de galectine-3. <p>A l’opposé, nous avons observé une expression endothéliale de ces galectines différente entre les lymphomes systémiques et les LPSNC. La galectine-1 n’est pas exprimée par les cellules endothéliales des vaisseaux des LPSNC. Par contre, la galectine-3 est exprimée par les cellules endothéliales de certains LPSNC. Cette expression est associée à un mauvais pronostic. <p>Nous avons également démontré que l’hyperplasie endothéliocapillaire est associée à une survie diminuée. <p>Cette hyperplasie endothéliocapillaire et/ou l’expression endothéliale de galectine-3 sont des facteurs pronostiques indépendants au sein de notre série de patients immunocompétents atteints de LPSNC et traités par chimiothérapie.<p><p>Au vu des résultats des deux premières études, nous pensons que les galectines-1 et -3 jouent un rôle dans l’angiogenèse des lymphomes. Ce rôle est très certainement complexe comme l’illustre la différence d’expression endothéliale de ces galectines entre les lymphomes systémiques et les LPSNC. Ces variations d’expressions selon l’organe hôte sont à intégrer avec la notion générale d’hétérogénéité des cellules endothéliales. Cette hétérogénéité s’observe également entre les cellules endothéliales des vaisseaux normaux et les cellules endothéliales des vaisseaux dans un contexte tumoral (TAECs - tumor-associated endothelial cells). <p>Suite à ces observations, nous avons décidé d’étudier l’implication des galectines-1 et -3 extracellulaires sur deux modèles in vitro de cellules endothéliales (cellules HUVEC et EA.hy926). La revue de la littérature nous a permis de poser l’hypothèse que la lignée EA.hy926 pourrait s’apparenter aux TAECs. La première partie de cette troisième étude a pour but de valider cette hypothèse. La comparaison morphologique des lignées EA.hy926 et HUVEC cultivées sur un substrat de matrigel a permis de montrer que les tubes formés par les cellules EA.hy926 sont plus hétérogènes comme ce que l’on peut observer dans les vaisseaux tumoraux. La comparaison des profils d’expression des gènes de ces deux lignées a mis en évidence que différents gènes surexprimés dans les TAECs le sont également dans les cellules EA.hy926. Les cellules EA.hy926 présentent un profil d’expression des récepteurs au VEGF (VEGFR- vascular endothelial growth factor receptor) caractérisé par un taux élevé de VEGFR1 et un faible taux de VEGFR2 en comparaison aux cellules HUVEC. L’analyse in vivo des VEGFRs au sein des cellules endothéliales de tissus humains normaux et tumoraux nous a permis de confirmer la surexpression de VEGFR1 par les TAECs. L’ensemble de ces résultats supportent donc notre hypothèse que la lignée EA.hy926 présente des caractéristiques plus proches des TAECs en comparaison à la lignée HUVEC.<p>Nous avons ensuite, dans la deuxième partie de cette troisième étude, analysé les effets respectifs de la galectine-1, de la galectine-3 et de leur combinaison sur ces deux lignées. Nous avons observé que la galectine-1 extracellulaire dans notre modèle de cellules endothéliales normales (HUVEC) augmente la croissance cellulaire ainsi que la formation de tubes sans mise en évidence de phosphorylation significative de VEGFR1 ni de VEGFR2. Dans notre modèle de TAECs (EA.hy926), la galectine-1 extracellulaire ne favorise pas la croissance cellulaire et est faiblement angiogénique via la phosphorylation du VEGFR2, sans phosphorylation du VEGFR1. <p>En ce qui concerne la galectine-3, nous avons observé dans nos deux modèles des résultats similaires, à savoir une absence d’effet sur la croissance cellulaire et un effet positif sur la formation de tubes s’accompagnant d’une phosphorylation de VEGFR2 sans phosphorylation de VEGFR1. <p>Le résultat le plus original consiste en la mise en évidence d’un effet synergique des deux galectines sur la croissance et la formation de tubes des cellules EA.hy926. Cet effet synergique s’accompagne d’une phosphorylation de VEGFR1 et de VEGFR2. Il est important de noter que l’activation de VEGFR1 n’a été observée que pour la lignée EA.hy926 et uniquement en présence des deux galectines. <p>Dans ces conditions, nous avons démontré que les voies de signalisation activées suite à l’ajout de ces galectines impliquent la voie de l’extracellular signal regulated kinase1/2 et de l’heat shock protein 27. <p><p>En conclusion, ce travail nous a donc permis, à partir de la caractérisation de l’expression de galectine-1 et -3 dans une série de lymphomes systémiques et cérébraux, de proposer un rôle angiogénique pour ces galectines. Les études in vitro ont souligné les rôles différents que joueraient ces galectines extracellulaires dans l’angiogenèse non tumorale et tumorale et l’implication du VEGFR1 dans l’angiogenèse tumorale. <p> / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

Modification by glycosylation gives proteins a range of diverse functions reflecting their structural variability. N-glycans regulate many biological outcomes in mammalian cells under both normal and pathological conditions. They play a major role in various pathologies such as cancer and lysosomal storage diseases. Interplay between N-glycans and other regulators, such as membrane lipid domains, in the control of signaling pathways remains poorly understood. My thesis therefore focuses on how N-glycans and membrane lipid domains oppose and/or work together at different cellular levels to regulate various processes such as receptor signaling and diffusion, endocytosis and lysosomal organelle biogenesis. Mgat5 encodes for ß1,6-N-acetylglucosaminyltransferase V that produces N-glycans, the preferred ligand for galectins. In tumor cells, galectins bind glycosylated receptors at the cell surface forming a lattice, that restricts receptor endocytosis and enhances its residency at the plasma membrane. In the first part of my thesis, I report that Galectin/receptor crosslinking opposes receptor sequestration by oligomerized caveolin-1 (Cav1) domains overriding its negative regulation of epidermal growth factor receptor (EGFR) signaling, cell surface diffusion and tumor growth. These results identify Cav1 as a conditional tumor suppressor. I also demonstrate that Cav1 is a negative regulator of lipid raft-mediated endocytosis. Cav1 indirectly regulates the internalization of cholera toxin b subunit to the Golgi apparatus independently of caveolae formation. That identifies a new role for caveolin-1 outside caveolae in the regulation of raft-dependent endocytosis Finally, Mgat5 overexpression in pneumocytes is associated with the expression of a lysosomal organelle, the multilamellar body (MLB), via autophagy. MLB expression is also a characteristic of various lysosomal storage diseases. I demonstrate that cholesterol accumulation can override the need for Mgat5 overexpression in MLB formation indicating that they may form via multiple mechanisms. However, I also demonstrate that a contribution of the autophagic pathway is a common determinant of biogenesis of MLB of various lipid compositions. In conclusion, Mgat5-dependent protein glycosylation and Cav1/raft domains therefore both function as regulators of plasma membrane interactions, endocytosis and lysosomal organelle biogenesis. Understanding of this interplay is crucial for the understanding of the mechanisms involve in various pathologies such as cancer and lysosomal storage diseases. / Medicine, Faculty of / Graduate

Les progéniteurs hématopoïétiques se différencient dans la moelle osseuse, au contact des cellules stromales, indispensables pour leur survie. Lors de la lymphopoïèse B, les progéniteurs se différencient suivant plusieurs étapes successives en cellules pro-B, pré-BI, pré-BII, puis B immatures, au cours desquelles se mettent en place les réarrangements des gènes codant pour les chaînes lourdes et légères des immunoglobulines. Les cellules pré-BII, qui expriment le récepteur pré-B (pré-BCR) à leur surface, constituent un point de contrôle essentiel lors du développement B. Le laboratoire a précédemment démontré que la galectine-1 (gal1), produite par les cellules stromales, fait un lien entre le pré-BCR et certaines intégrines afin de former une « synapse immunologique » entre les cellules pré-BII et les cellules stromales, conduisant ainsi à la relocalisation puis à l’activation du pré-BCR.Au cours de ma thèse, je me suis intéressée au rôle du stroma médullaire à l’étape des cellules pré-BII. Nous avons tout d’abord démontré l’importance de la formation de la synapse et des interactions entre les pré-BCR, les intégrines et la gal1 pour la prolifération et la différenciation des cellules pré-B in vivo. Enfin, nous avons caractérisé les cellules stromales de la moelle osseuse qui expriment la gal1. Ce travail a montré que la majorité des cellules pré-BII, au contraire des autres progéniteurs B, se situe au contact des cellules stromales gal1+, apportant pour la première fois la preuve de l’existence d’une niche stromale spécifique aux cellules pré-BII. / In the bone marrow, hematopoietic progenitors differentiate in close contact with stromal cells, essential for their survival. During B cell development, the progenitors differentiate through several sequential stages including pro-B cells, pre-BI cells, pre-BII cells and immature B cells, which allow setting up the rearrangements of genes encoding the immunoglobulin heavy and light chains. The expression of a functional pre-B cell receptor (pre-BCR) at the surface of the pre-BII cells represents a crucial checkpoint during B cell differentiation. The laboratory has previously demonstrated that the stromal cell-derived galectin-1 (gal1) is a link between the pre-BCR and certain integrins. This binding drives the pre-BCR in the pre-BII/stromal cell synapse, leading to the initiation of pre-BCR signaling.During my PhD, I have been interested in the role of stromal cells at the pre-BII cell stage. We first demonstrated the importance of synapse formation and the importance of the interactions between the pre-BCR, integrins and gal1 for the proliferation and differentiation of pre-BII cells in vivo. Finally, we characterized the bone marrow stromal cells which express gal1. This work showed that the majority of pre-BII cells, unlike other B cell progenitors, was in contact with gal1+ stromal cells, providing the first evidence of the existence of a specific stromal cell niche for the pre-BII cells.

A galectina-1 (Gal-1) é uma lectina que reconhece ?-galactosídeos e participa de vários processos biológicos, incluindo a modulação da resposta inflamatória. Dados da literatura mostram a participação desta lectina na indução da exposição de fosfatidilserina (FS - um marcador de apoptose), na geração de espécies reativas do oxigênio (EROs) e na modulação quimiotática de neutrófilos. Entretanto, ainda são escassos os dados relacionados ao impacto biológico da Gal-1, exógena e endógena, sobre a biologia destas células. Neste trabalho foram avaliados, in vitro, alguns aspectos funcionais da interação Gal-1/neutrófilo. Determinou-se o nível de expressão da Gal-1 (Western Blotting) e de seu mRNA (PCR real time), em leucócitos humanos obtidos do sangue periférico de doadores sadios e em células da linhagem promielocítica humana (HL-60). Leucócitos do sangue periférico e células HL-60 não expressam níveis detectáveis da proteína e também do mRNA para Gal-1. Por meio de ensaios de quimiluminescência (QL) foi possível analisar a capacidade da Gal-1 recombinante humana de induzir e modular a produção de EROs em neutrófilos humanos não ativados e ativados com fMLP (n-Formil-Methionyl-Leucyl-Phenylalanine). A Gal-1 induz a produção de EROs de modo dose-dependente em neutrófilos ativados com fMLP. Entretanto, em neutrófilos não ativados esta lectina não induz o metabolismo oxidativo e, além disso, é capaz de modular negativamente a produção de EROs em resposta ao fMLP. Tanto nas células não ativadas quanto ativadas com fMLP, os efeitos da Gal-1 na produção de EROs estão parcialmente associados a sua propriedade lectínica. Na literatura ainda não há relatos sobre a interferência da Gal-1 no metabolismo oxidativo em neutrófilos ativados com repetidas doses de fMLP. Sabe-se que o tratamento sucessivo com fMLP reduz os níveis de produção de EROs por neutrófilos, no entanto, a presença de Gal-1 não interferiu neste processo. Interessantemente, neutrófilos recuperados do peritônio de camundongos Gal-1-/- liberam mais EROs em resposta ao fMLP e a Gal-1 exógena quando comparado aos neutrófilos de animais selvagens. Com base nos achados in vitro e sabendo que na sepse polimicrobiana o neutrófilo desempenha um papel importante, o próximo passo foi utilizar o modelo de M-CLP (sepse moderada) em camundongos destituídos (Gal-1-/-) ou não (Gal-1+/+) do gene da Gal-1. Animais Gal-1-/-, apresentam menor taxa de sobrevivência. O influxo de neutrófilos e a carga bacteriana no peritônio são maiores nos animais Gal-1-/- apesar da menor quantidade de bactérias detectadas no sangue, em relação aos animais selvagens. No pulmão, o influxo de neutrófilos é semelhante para ambos os grupos. No entanto, após a injeção intraperitoneal de 107 Unidades Formadoras de Colônias (UFC) de bactérias, camundongos Gal-1-/- apresentam maior atividade bactericida no lavado peritoneal e sangue, em relação aos selvagens. A participação da Gal-1 na homeostase de neutrófilos foi demonstrada in vitro, por citometria de fluxo (anexina-V-FITC), onde a indução de FS nos neutrófilos tratados com Gal-1 favoreceu a fagocitose destas células por macrófagos. Portanto, este conjunto de resultados sugere que a Gal-1, exógena ou endógena, pode modular funções imunológicas de neutrófilos e participar da regulação do processo inflamatório/infeccioso sistêmico. / Galectin-1 (Gal-1) is a lectin that recognizes ?-galactosides and participates in biological processes, including modulation of the inflammatory response. Literature data show the involvement of this lectin to induce exposure of phosphatidylserine (PS - a marker of apoptosis), in the generation of reactive oxygen species (ROS) and in modulation of neutrophil chemotaxis. However, there are few data related to the biological impact of exogenous and endogenous Gal-1 on the biology of these cells. This study evaluated, in vitro, some functional aspects of the interaction of Gal-1 and neutrophil. It was determined the expression level of Gal-1 (Western blotting) and its mRNA (real time PCR) on human leukocytes obtained from peripheral blood of healthy donors and human promyelocytic cell line (HL-60). Peripheral blood leukocytes and HL-60 cells do not express detectable levels of this protein as well as the Gal-1 mRNA. Through chemiluminescence testing (CL) it was possible to analyze the ability of recombinant human Gal-1 to induce and modulate the production of ROS in naïve and activated (n-Formyl-Methionyl-Leucyl-Phenylalanine-fMLP) human neutrophils. Gal-1 induces ROS production in a dose-dependent way in fMLP activated neutrophils. However, in naive neutrophils this lectin does not induce oxidative stress and can negatively modulate ROS production in response to fMLP. The effects of Gal-1 on ROS production in both non-activated cells and activated cells are partially associated with their lectin property. In the literature there are no data about the interference of Gal-1 on ROS production in activated neutrophils with repeated doses of fMLP. It is known that the subsequent treatment with fMLP reduced levels of ROS production by neutrophils; however, the presence of Gal-1 did not affect this process. Interestingly, peritoneum neutrophils from Gal-1-/- mice release more ROS in response to fMLP and exogenous Gal-1 when compared to neutrophils from wild type animals. Based on the in vitro findings and considering that in polymicrobial sepsis, neutrophils play an important role, the next step was to use the M-CLP model (moderate sepsis) in mice lacking (Gal-1-/-) or not (Gal-1+/+) Gal-1 gene. Gal-1-/- animals present lower survival rate and fewer bacteria in the blood despite having higher bacterial load in infectious focus in relation to wild type mice. The rates of neutrophils influx into the peritoneum and lungs are similar for both groups. The participation of Gal-1 in the homeostasis of neutrophils was demonstrated in vitro by flow cytometry (annexin V-FITC), where induced PS by Gal-1 in the neutrophils enhanced the phagocytosis of these cells by macrophages. Therefore, this set of results suggests that Gal-1, exogenous or endogenous, can modulate immune functions of neutrophils and participate in the regulation of inflammatory/infectious disorders.

A galectina-1 (Gal-1) é uma proteína que reconhece ?-galactosídeos e participa de vários processos biológicos, incluindo a modulação da resposta imunológica. Vários relatos da literatura reportam o potencial uso terapêutico da Gal-1 para doenças auto-imunes, inflamatórias e infecciosas. Entretanto, são escassos os relatos sobre o envolvimento da Gal-1 na infecção causada por Tripanosoma cruzi. Neste trabalho foi avaliada a participação da Gal-1 endógena e a administração de Gal-1 exógena na evolução da infecção aguda induzida experimentalmente por T. cruzi. Foram realizados experimentos in vivo e/ou in vitro com o uso de Gal-1 recombinante humana, camundongos C57BL/6 deficientes do gene da Gal-1 (Gal-1 KO, knock-out) ou do gene Toll Like Receptor 4 (TLR-4 KO, knock-out) e selvagens (WT, wild type), além de seus macrófagos. A forma de T. cruzi utilizada foi a tripomastigota da cepa Y. Os parâmetros analisados na caracterização do processo de infecção foram: i) parasitemia e sobrevivência de animais; ii) histopatologia do tecido cardíaco; iii) imunofenotipagem de leucócitos; iv) dosagem de citocinas; v) determinação de taxas de invasão e liberação de parasitas a partir de células infectadas; vi) produção de óxido nítrico por macrófagos. A Gal-1 e/ou anticorpos anti-glicopeptídeos miméticos de mucina de T. cruzi ligam-se à glicanas da superfície deste parasita e impedem sua invasão em fibroblastos e sua captura por macrófagos. O tratamento de macrófagos infectados com Gal-1 reduz a liberação de parasitas e aumenta a produção de óxido nítrico (NO) por um mecanismo, aparentemente, independente da sinalização via TLR-4. Os camundongos KO Gal-1 e os WT tratados com Gal-1 exógena apresentaram as menores taxas de parasitemia, sendo que os primeiros são mais resistentes à infecção aguda por T. cruzi. A ausência da Gal-1 endógena nos animais infectados provocou vários efeitos como a redução no infiltrado inflamatório e carga parasitária no tecido cardíaco, níveis séricos elevados de citocinas (IL-2, IL-4, IL-6, IL-10 e IL-17A), menor porcentagem de linfócitos T CD4+ e maior de T CD8+ no coração dos animais, aumento do influxo de neutrófilos na cavidade peritoneal e no coração. Com base nesse conjunto de resultados sugerimos que a ausência de Gal-1 endógena ou o tratamento de animais com Gal- 1 exógena promoveram perfis imunológicos (resposta inata e adaptativa) favorecedores da resolução da infecção experimental aguda por T. cruzi / Galectin-1 (Gal-1) is a protein that recognizes ?-galactosides and participates in many biological processes, including the modulation of the immune response. Several reports in the literature show the potential therapeutic use of Gal-1 to autoimmune, inflammatory and infectious diseases. However, there are few reports on the involvement of Gal-1 on infection caused by Trypanosoma cruzi. Here, we evaluated the involvement of endogenous Gal-1 and Gal-1 administration of exogenous in the development of acute experimental infection by T. cruzi. Recombinant human Gal-1, C57BL/6 mice deficient for Gal-1 gene (Gal-1 KO, knockout) or for Toll like receptor 4 gene (TLR-4 KO, knock-out) or C57BL/6 wild type mice (WT), and macrophages from these animals were used in experiments in vivo and / or in vitro. The form of T. cruzi used in this work was trypomastigotes from Y strain. The analyzed parameters characterizing the process of infection were: i) parasitemia and survival of animals; ii) histopathology of cardiac tissue; iii) leucocyte immunophenotyping; iv) cytokine assay; v) determination of invasion and release rates of parasites from infected cells; vi) nitric oxide production by macrophages. The Gal-1 and / or antibodies anti- glycopeptides that mimics T. cruzi mucin bind to glycans on the surface of this parasite and prevent invasion of the parasite in fibroblasts and its capture by macrophages. Treatment of infected macrophages with Gal-1 promotes a lower release of parasites and increased production of nitric oxide (NO) by these phagocytes, and this production of NO is independent of TLR-4 signaling pathway. The Gal-1 KO mice and WT mice treated with exogenous Gal-1 had the lowest rates of parasitemia and the first group is more resistant to acute infection with T. cruzi. The absence of endogenous Gal-1 in infected animals caused various effects such as a reduction in the inflammatory infiltrate and the parasite load in the cardiac tissue, elevated serum levels of cytokines (IL-2, IL-4, IL-6, IL-10 and IL- 17A), a lower percentage of T CD4+ and increased T CD8+ the hearts of animals, increased influx of neutrophils into the peritoneal cavity and heart tissue. Based on this set of results we suggest that the absence of endogenous Gal-1 or treatment of animals with exogenous Gal-1 promoted immunological profiles (innate and adaptive response) favoring the resolution of acute experimental T. cruzi infection.

La cystinose est une maladie lysosomale héréditaire due à une mutation du gène CTNS codant pour un transporteur de cystine, cystinosin, et est caractérisée par l’accumulation de cystine dans les organes causant leur dégénération.Si le rein est le premier organe affecté par la maladie, sa pathogénèse n’est pas encore totalement comprise. La recherche de nouveaux partenaires de la cystinosine a révélé une interaction avec un membre de la famille des galactines, galectine-3 (Gal3). L’étude de cette interaction a mis en évidence un nouveau rôle de la cystinosine dans l’inflammation chronique impliquée dans la pathogénèse rénale de la cystinose. Dans les reins du modèle murin de la cystinose (Ctns-/-), l’expression de Gal3 est augmentée et de nombreux infiltrats de cellules inflammatoires sont observés. De plus, l’expression de la cytokine pro-inflammatoire Monocyte Chemoattractant Protein-1 (MCP1) est augmentée dans le sérum des souris Ctns-/-.Au contraire, peu d’infiltrat et un taux normal de MCP1 sont observés dans les souris Ctns-/-Gal3-/-, ainsi qu’une meilleure structure et fonction rénale.Ce travail pourrait permettre la découverte de nouvelles cibles thérapeutiques pour la cystinose.Des études antérieures ont montré que la transplantation de Cellules Souches Hématopoïétiques (CSH) saines peut efficacement traiter la cystinose dans les souris Ctns-/-. Toutefois, dû aux risques liés à une transplantation allogénique, une transplantation autologue de CSH génétiquement modifiées ex vivo pour exprimer une copie fonctionnelle du gène CTNS a été développée.Nous résumons ici les études pharmacologiques et toxicologiques ainsi que le développement du protocole de transduction des cellules humaines qui seront inclus dans une application intitulée« Investigational New Drug » qui sera soumise à la FDA afin de débuter un essai clinique de phase I/II. / Cystinosis is an inherited lysosomal storage disorder caused by mutations in the gene CTNS encoding the cystine transporter cystinosin, and is characterized by accumulation of cystine in the tissues leading to multiorgan degeneration.The kidney is the first organ impacted by cystinosis but the pathogenesis is still not fully understood. The study of new partners of cystinosin revealed an interaction with galectin-3, a member of the galectin’s family. The investigation of this interaction unraveled a new role for cystinosin in chronic inflammation associated with kidney pathology in cystinosis. Indeed, the cystinosis mouse model, Ctns-/ mice, had increased expression of Gal3 and abundant pro-inflammatory infiltrates in their kidney, as well as increased expression of Monocyte Chemoattractant Protein-1 (MCP1), a proinflammatory cytokine, in their serum.In contrast, few infiltrates and normal MCP1 levels were observed in the Ctns-/- Gal3-/- mice, which also demonstrated better kidney structure and function.This study may lead to the discovery of new drug targets for cystinosis treatment.Previous studies showed that wild-type Hematopoietic Stem Cells (HSCs) transplantation had the potential to rescue cystinosis in Ctns-/- mice.However, due to the risks associated with allogeneic transplant, an autologous transplantation of HSCs genetically modified ex vivo to express a functional CTNS gene has been developed in the laboratory. In this work, we summarized the pharmacology and toxicology studies and manufacturing development that will be included in an Investigational New Drug application to be submitted to the FDA to start a phase I/II clinical trial for cystinosis.