Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O diagnóstico da hanseníase neural pura baseia-se em dados clínicos e laboratoriais do paciente, incluindo a histopatologia de espécimes de biópsia de nervo e detecção de DNA de Mycobacterium leprae (M. leprae) pelo PCR. Como o exame histopatológico e a técnica PCR podem não ser suficientes para confirmar o diagnóstico, a imunomarcação de lipoarabinomanana (LAM) e/ou Glicolipídio fenólico 1 (PGL1) - componentes de parede celular de M. leprae foi utilizada na primeira etapa deste estudo, na tentativa de detectar qualquer presença vestigial do M. leprae em amostras de nervo sem bacilos. Além disso, sabe-se que a lesão do nervo na hanseníase pode diretamente ser induzida pelo M. leprae nos estágios iniciais da infecção, no entanto, os mecanismos imunomediados adicionam severidade ao comprometimento da função neural em períodos sintomáticos da doença. Este estudo investigou também a expressão imuno-histoquímica de marcadores envolvidos nos mecanismos de patogenicidade do dano ao nervo na hanseníase. Os imunomarcadores selecionados foram: quimiocinas CXCL10, CCL2, CD3, CD4, CD8, CD45RA, CD45RO, CD68, HLA-DR, e metaloproteinases 2 e 9. O estudo foi desenvolvido em espécimes de biópsias congeladas de nervo coletados de pacientes com HNP (n=23 / 6 BAAR+ e 17 BAAR - PCR +) e pacientes diagnosticados com outras neuropatias (n=5) utilizados como controle. Todas as amostras foram criosseccionadas e submetidas à imunoperoxidase. Os resultados iniciais demonstraram que as 6 amostras de nervos BAAR+ são LAM+/PGL1+. Já entre as 17 amostras de nervos BAAR-, 8 são LAM+ e/ou PGL1+. Nas 17 amostras de nervos BAAR-PCR+, apenas 7 tiveram resultados LAM+ e/ou PGL1+. A detecção de imunorreatividade para LAM e PGL1 nas amostras de nervo do grupo HNP contribuiu para a maior eficiência diagnóstica na ausência recursos a diagnósticos moleculares. Os resultados da segunda parte deste estudo mostraram que foram encontradas imunoreatividade para CXCL10, CCL2, MMP2 e MMP9 nos nervos da hanseníase, mas não em amostras de nervos com outras neuropatias. Além disso, essa imunomarcação foi encontrada predominantemente em células de Schwann e em macrófagos da população celular inflamatória nos nervos HNP. Os outros marcadores de ativação imunológica foram encontrados em leucócitos (linfócitos T e macrófagos) do infiltrado inflamatório encontrados nos nervos. A expressão de todos os marcadores, exceto CXCL10, apresentou associação com a fibrose, no entanto, apenas a CCL2, independentemente dos outros imunomarcadores, estava associada a esse excessivo depósito de matriz extracelular. Nenhuma diferença na frequência da imunomarcação foi detectada entre os subgrupos BAAR+ e BAAR-, exceção feita apenas às células CD68+ e HLA-DR+, que apresentaram discreta diferença entre os grupos BAAR + e BAAR- com granuloma epitelioide. A expressão de MMP9 associada com fibrose é consistente com os resultados anteriores do grupo de pesquisa. Estes resultados indicam que as quimiocinas CCL2 e CXCL10 não são determinantes para o estabelecimento das lesões com ou sem bacilos nos em nervo em estágios avançados da doença, entretanto, a CCL2 está associada com o recrutamento de macrófagos e com o desenvolvimento da fibrose do nervo na lesão neural da hanseníase. / The diagnosis of pure neural leprosy (PNL) is based on clinical and laboratory data, including the histopathology of nerve biopsy specimens and detection of M. leprae DNA by polymerase chain reaction (PCR). Given that histopathological examination and PCR methods may not be sufficient to confirm diagnosis, immunolabeling of lipoarabinomanan (LAM) and/or phenolic glycolipid 1 (PGL1) M. leprae wall components were utilized in the first step of this investigation in an attempt to detect any vestigial presence of M. leprae in AFB- nerve samples. Furthermore, its well known that nerve damage in leprosy can be directly induced by Mycobacterium leprae in the early stages of infection; however, immunomediated mechanisms add gravity to the impairment of neural function in symptomatic periods of the disease. Therefore, this study also investigated the immunohistochemical expression of immunomarkers involved in the pathogenic mechanisms of leprosy nerve damage. These markers selected were CXCL10, CCL2 chemokines and CD3, CD4, CD8, CD45RA, CD45RO, CD68, HLA-DR, metalloproteinases 2 and 9 in nerve biopsy specimens collected from leprosy (23) and nonleprosy patients (5) suffering peripheral neuropathy. Twenty-three PNL nerve samples (6 AFB+ and 17 AFB-PCR+) were cryosectioned and submitted to LAM and PGL1 immunohistochemical staining by immunoperoxidase; 5 nonleprosy nerve samples were used as controls. The 6 AFB-positive samples showed LAM/PGL1 immunoreactivity. Among the 17 AFB- samples, only 8 revealed LAM and/or PGL1 immunoreactivity. In 17 AFB-PCR+ patients, just 7 had LAM and/or PGL1-positive nerve results. In the PNL cases, the detection of immunolabeled LAM and PGL1 in the nerve samples would have contributed to enhanced diagnostic efficiency in the absence of molecular diagnostic facilities. The results of the second part of this study showed that CXCL10-, CCL2-, MMP2- and MMP9-immunoreactivities were found in the leprosy nerves but not in nonleprosy samples. Immunolabeling was predominantly found in recruited macrophages and Schwann cells composing the inflammatory cellular population in the leprosy-affected nerves. The immunohistochemical expression of all the markers, but CXCL10, was associated with fibrosis; however, only CCL2 was, independently from the other markers, associated with this excessive deposit of extracellular matrix. No difference in the frequency of the immunolabeling was detected between the AFB+ and AFB- leprosy subgroups of nerves, exception made to some statistical tendency to difference in regard to CD68+ and HLA-DR+ cells in the AFB- nerves exhibiting epithelioid granuloma. MMP9 expression associated with fibrosis is consistent with previous results of this research group. The findings conveys the idea that CCL2 and CXCL10 chemokines at least in advanced stages of leprosy nerve lesions are not determinant for the establishment of AFB+ or AFB- leprosy lesions, however, CCL2 is associated with macrophage recruitment and fibrosis.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O diagnóstico da hanseníase neural pura baseia-se em dados clínicos e laboratoriais do paciente, incluindo a histopatologia de espécimes de biópsia de nervo e detecção de DNA de Mycobacterium leprae (M. leprae) pelo PCR. Como o exame histopatológico e a técnica PCR podem não ser suficientes para confirmar o diagnóstico, a imunomarcação de lipoarabinomanana (LAM) e/ou Glicolipídio fenólico 1 (PGL1) - componentes de parede celular de M. leprae foi utilizada na primeira etapa deste estudo, na tentativa de detectar qualquer presença vestigial do M. leprae em amostras de nervo sem bacilos. Além disso, sabe-se que a lesão do nervo na hanseníase pode diretamente ser induzida pelo M. leprae nos estágios iniciais da infecção, no entanto, os mecanismos imunomediados adicionam severidade ao comprometimento da função neural em períodos sintomáticos da doença. Este estudo investigou também a expressão imuno-histoquímica de marcadores envolvidos nos mecanismos de patogenicidade do dano ao nervo na hanseníase. Os imunomarcadores selecionados foram: quimiocinas CXCL10, CCL2, CD3, CD4, CD8, CD45RA, CD45RO, CD68, HLA-DR, e metaloproteinases 2 e 9. O estudo foi desenvolvido em espécimes de biópsias congeladas de nervo coletados de pacientes com HNP (n=23 / 6 BAAR+ e 17 BAAR - PCR +) e pacientes diagnosticados com outras neuropatias (n=5) utilizados como controle. Todas as amostras foram criosseccionadas e submetidas à imunoperoxidase. Os resultados iniciais demonstraram que as 6 amostras de nervos BAAR+ são LAM+/PGL1+. Já entre as 17 amostras de nervos BAAR-, 8 são LAM+ e/ou PGL1+. Nas 17 amostras de nervos BAAR-PCR+, apenas 7 tiveram resultados LAM+ e/ou PGL1+. A detecção de imunorreatividade para LAM e PGL1 nas amostras de nervo do grupo HNP contribuiu para a maior eficiência diagnóstica na ausência recursos a diagnósticos moleculares. Os resultados da segunda parte deste estudo mostraram que foram encontradas imunoreatividade para CXCL10, CCL2, MMP2 e MMP9 nos nervos da hanseníase, mas não em amostras de nervos com outras neuropatias. Além disso, essa imunomarcação foi encontrada predominantemente em células de Schwann e em macrófagos da população celular inflamatória nos nervos HNP. Os outros marcadores de ativação imunológica foram encontrados em leucócitos (linfócitos T e macrófagos) do infiltrado inflamatório encontrados nos nervos. A expressão de todos os marcadores, exceto CXCL10, apresentou associação com a fibrose, no entanto, apenas a CCL2, independentemente dos outros imunomarcadores, estava associada a esse excessivo depósito de matriz extracelular. Nenhuma diferença na frequência da imunomarcação foi detectada entre os subgrupos BAAR+ e BAAR-, exceção feita apenas às células CD68+ e HLA-DR+, que apresentaram discreta diferença entre os grupos BAAR + e BAAR- com granuloma epitelioide. A expressão de MMP9 associada com fibrose é consistente com os resultados anteriores do grupo de pesquisa. Estes resultados indicam que as quimiocinas CCL2 e CXCL10 não são determinantes para o estabelecimento das lesões com ou sem bacilos nos em nervo em estágios avançados da doença, entretanto, a CCL2 está associada com o recrutamento de macrófagos e com o desenvolvimento da fibrose do nervo na lesão neural da hanseníase. / The diagnosis of pure neural leprosy (PNL) is based on clinical and laboratory data, including the histopathology of nerve biopsy specimens and detection of M. leprae DNA by polymerase chain reaction (PCR). Given that histopathological examination and PCR methods may not be sufficient to confirm diagnosis, immunolabeling of lipoarabinomanan (LAM) and/or phenolic glycolipid 1 (PGL1) M. leprae wall components were utilized in the first step of this investigation in an attempt to detect any vestigial presence of M. leprae in AFB- nerve samples. Furthermore, its well known that nerve damage in leprosy can be directly induced by Mycobacterium leprae in the early stages of infection; however, immunomediated mechanisms add gravity to the impairment of neural function in symptomatic periods of the disease. Therefore, this study also investigated the immunohistochemical expression of immunomarkers involved in the pathogenic mechanisms of leprosy nerve damage. These markers selected were CXCL10, CCL2 chemokines and CD3, CD4, CD8, CD45RA, CD45RO, CD68, HLA-DR, metalloproteinases 2 and 9 in nerve biopsy specimens collected from leprosy (23) and nonleprosy patients (5) suffering peripheral neuropathy. Twenty-three PNL nerve samples (6 AFB+ and 17 AFB-PCR+) were cryosectioned and submitted to LAM and PGL1 immunohistochemical staining by immunoperoxidase; 5 nonleprosy nerve samples were used as controls. The 6 AFB-positive samples showed LAM/PGL1 immunoreactivity. Among the 17 AFB- samples, only 8 revealed LAM and/or PGL1 immunoreactivity. In 17 AFB-PCR+ patients, just 7 had LAM and/or PGL1-positive nerve results. In the PNL cases, the detection of immunolabeled LAM and PGL1 in the nerve samples would have contributed to enhanced diagnostic efficiency in the absence of molecular diagnostic facilities. The results of the second part of this study showed that CXCL10-, CCL2-, MMP2- and MMP9-immunoreactivities were found in the leprosy nerves but not in nonleprosy samples. Immunolabeling was predominantly found in recruited macrophages and Schwann cells composing the inflammatory cellular population in the leprosy-affected nerves. The immunohistochemical expression of all the markers, but CXCL10, was associated with fibrosis; however, only CCL2 was, independently from the other markers, associated with this excessive deposit of extracellular matrix. No difference in the frequency of the immunolabeling was detected between the AFB+ and AFB- leprosy subgroups of nerves, exception made to some statistical tendency to difference in regard to CD68+ and HLA-DR+ cells in the AFB- nerves exhibiting epithelioid granuloma. MMP9 expression associated with fibrosis is consistent with previous results of this research group. The findings conveys the idea that CCL2 and CXCL10 chemokines at least in advanced stages of leprosy nerve lesions are not determinant for the establishment of AFB+ or AFB- leprosy lesions, however, CCL2 is associated with macrophage recruitment and fibrosis.

Diversas doenças estão associadas com a formação de novos vasos a parti vasos pré-existentes, ou angiogênese. Dentre elas está a aterosclerose (Griffioen & Molema, 2000). Pesquisas recentes demonstram que a hipercolesterolemia, que têm um papel importante na fisiologia da aterosclerose, também pode prejudicar a ação de fatores angiogênicos (Jang et.al., 2000). A hipercolesterolemia que é decorrente de aumento de LDL no plasma ocasiona um aumento no tempo de permanência desta partícula na circulação (Yasunobu, 2001). Contudo, a LDL pode sofrer modificação na circulação, dando origem a uma subfração mais eletronegativa da LDL, a LDL (-). A LDL (-) pode prejudicar cada etapa da angiogênese, desregulando a função endotelial (Tai et. al., 2006). Em nosso estudo, vimos que apesar da LDL (-) ter estimulado a miga celular, esta partícula inibiu a formação de túbulos in vitro. A LDL (-) não foi capa afetar a angiogênese in vivo. / A large number of diseases is associated with formation of new blood vessels out of pre-existing capillaries, or angiogenesis. These diseases include the atherosclerosis (Griffioen & Molema, 2000). Resents researches demonstrate that the hypercholesterolemia, that have a important role in the physiology of the atherosclerosis, can impaired the angiogenesis (Jang et. al., 2000) . The hypercholesterolemia that is decurrente of high levels of LDL in the plasma causes an increase in the time of permanence this particle in the circulation (Yasunobu, 2001). However, the LDL can to suffer modification in the circulation, giving rise to a subfration more eletronegative from LDL, the LDL (-). The LDL (-) could impair each one of the steps of the angiogenesis, thereby dysregulating endothelial function (Tai et. al., 2006). In our study, see that despite the LDL (-) have stimulated the cell migration, this particle inhibited the Tube formation in vitro. The LDL (-) didn\'t affect the angiogenesis in vivo.

L’altération de la barrière hématoencéphalique (BHE) par les cellules tumorales et les cellules immunes circulantes peut mener à la neuroinflammation. Les cellules leucémiques promyélocytaires HL-60 sont un excellent modèle pour étudier et comprendre les mécanismes de signalisation moléculaires qui caractérisent le développement tumoral et métastatique. La cancérogenèse peut s’accompagner de modulations de l’expression de biomarqueurs tels que la cyclooxygénase-2 et la métalloprotéase-9. Les recherches décrites dans ce mémoire relatent l’analyse des biomarqueurs inflammatoires et invasifs régulés lors de la différenciation induite par le PMA des cellules HL-60 en macrophages. Le statut de différenciation cellulaire pourrait avoir un impact sur les gènes cibles de la voie NF-κB. Nous émettons l’hypothèse que le PMA active la voie NF-κB et que cette signalisation peut être renversée par l’(-)-épigallocatéchine-gallate (EGCG). En effet, une régulation à la hausse de l’expression de plusieurs gènes combinée à la diminution de l’expression d’IκB mettent en évidence l’implication de la voie NF-κB dans l’activation des mécanismes pro-inflammatoires et pro-invasifs. Les mêmes observations sont faites dans les cellules différenciées appelées «macrophages-like». L’EGCG, un polyphénol dérivé du thé vert, a un potentiel chimiopréventif. Il est capable d’inhiber la signalisation moléculaire passant par la voie NF-κB dans les cellules HL-60 traitées simultanément par l’EGCG et le PMA, mais pas dans les cellules «macrophages-like». Cette différence peut s’expliquer par une modulation de l’expression du récepteur de surface cellulaire de l’EGCG, le récepteur à la laminine de 67 kDa, et de son précurseur de 37 kDa. Collectivement, nos résultats montrent que le statut de différenciation des cellules promyélocytaires HL-60 concorde avec l’activation des mécanismes favorisant le développement d’un cancer et des métastases. Cet effet peut être prévenu par l’utilisation d’agents naturels tel l’EGCG. Le ciblage de biomarqueurs liés au statut de différenciation des cellules tumorales impliquées dans la perturbation de la barrière hématoencéphalique qui cause la neuroinflammation permettrait l’avancement des connaissances dans la prévention de la cancérogenèse. / Blood-brain barrier (BBB) disruption by circulating tumor and immune cells leads to secondary inflammatory infections. Promyelocytic HL-60 cells represent an excellent model to study and to get a better understanding of the molecular signaling mechanisms involved in carcinogenesis and metastasis. The research described in this thesis shows the analysis of several inflammatory and invasive biomarkers regulated during PMA-induced differentiation of promyelocytic HL-60 cells into macrophages. Carcinogenesis involves some modifications in the expression of biomarkers such as cyclooxygenase-2 and matrix metalloprotease-9. The differentiation status could have an impact on the NF-κB signaling pathway that regulates the target genes, given that these target genes expression varies during cell differentiation. We hypothesize that the activation of the NF-κB pathway by PMA can be reverse by (-)-epigallocatechin-gallate (EGCG). Indeed, the up-regulation of downstream genes combined with the down-regulation of IκB expression showed the significant implication of the NF-κB signaling pathway to activate pro-inflammatory and pro-invasive mechanisms linked to carcinogenesis. The same evidence exhibits in the differentiated cells called «macrophages-like». Moreover, the green tea polyphenol, EGCG, shows chemopreventive property since it better inhibited NF-κB signaling in cells treated simultaneously with EGCG and PMA compared to the «macrophages-like». This difference could be due, in part, to the down-regulation of the 67 kDa laminin receptor, known to be the non-integrin membrane receptor for EGCG. All together, our results suggest that the differentiation status of promyelocytic cells is linked to the activation of mechanisms involved in carcinogenesis and metastasis. These phenomena can be prevented by using natural agents such as EGCG. Targeting the specific biomarkers linked to the differentiation status of tumor cells and involved in the disruption of the BBB may help reduce secondary neuroinflammation and enable the advancement of knowledge towards carcinogenesis prevention.

En 2015, la récidive tumorale et les métastases du cancer du sein demeurent une cause importante de décès à travers le monde. Toutefois, ces cancers sont souvent hétérogènes car en dépit d’un phénotype similaire, l’évolution clinique et la réponse au traitement peuvent varier considérablement. Il y a donc un intérêt évident à identifier et à caractériser de nouveaux biomarqueurs pour permettre classer les tumeurs mammaires dans des sous-groupes plus homogènes. Notre hypothèse est que chaque cancer mammaire possède des caractéristiques distinctes au plan des altérations du génome et des profils d’expression géniques et que ces changements se traduisent cliniquement par une prédisposition à former des métastases ou à répondre ou non à la chimiothérapie et aux thérapies ciblées. Dans le cadre de nos travaux, nous nous sommes intéressés aux sous-types agressifs de tumeurs mammaires et notamment les cancers de type triple négatif. Nous avons aussi tenté d’identifier des marqueurs capables de distinguer l’une de l’autre les tumeurs de type luminal A et luminal B. Pour ce faire, nous avons d’abord utilisé une stratégie in silico à partir de données publiques (micro-puces d’ADN et séquençage de l’ARN). Nous avons ensuite construit sept micro-matrices tissulaires (TMA) provenant de tissus mammaires normaux et tumoraux fixés à la formaline et enrobés en paraffine. Ces outils nous ont permis d’évaluer par immunohistochimie les niveaux d’expression différentielle des marqueurs suivants : ANXA1, MMP-9, DP103 et MCM2. Ceux-ci ont été comparés aux marqueurs usuels du cancer du sein (ER, PR, HER2, CK5/6 et FOXA1) et corrélés aux données cliniques (survie globale et métastase). Nos résultats indiquent que ces nouveaux marqueurs jouent un rôle important dans l’évolution clinique défavorable des tumeurs de haut grade. Dans un premier article nous avons montré que l’expression d’ANXA1 est dérégulée dans les cancers de type triple-négatif et aussi, dans une certaine mesure, dans les tumeurs HER2+. Nous croyons qu’ANXA1 permet de mieux comprendre le processus d’hétérogénéité tumorale et facilite l’identification des tumeurs de haut grade. Nous proposons également qu’ d’ANXA1 stimule la transition épithélio-mésenchymateuse (EMT) et la formation des métastases. Dans un second temps, nous avons montré que les niveaux d’expression de MMP-9 reflètent la différenciation cellulaire et corrèlent avec les sous-types de cancers mammaires ayant un mauvais pronostic. Nous estimons que MMP-9 permet de mieux comprendre et d’identifier les tumeurs mammaires à haut risque. De fait, la surexpression de MMP-9 est associée à une augmentation des métastases, une récidive précoce et une diminution de la survie globale. Dans le cadre d’un troisième article, nous avons montré que la surexpression du marqueur de prolifération MCM2 s’observe dans les cancers triple-négatifs, HER2+ et Luminal B par comparaison aux cancers luminal A (p< 0.0001). Nos résultats suggèrent qu’en utilisant un seuil de 40% de noyaux marqués, nous pourrions distinguer l’une de l’autre les tumeurs de type luminal A et luminal B. Cela dit, avant de pouvoir envisager l’utilisation de ce marqueur en clinique, une étude de validation sur une nouvelle cohorte de patientes s’impose. En somme, les résultats de nos travaux suggèrent qu’ANXA1, MMP-9 et MCM2 sont des marqueurs intéressants pour mieux comprendre les mécanismes physiopathologiques impliqués dans la progression tumorale et le développement des métastases. À terme, ces nouveaux marqueurs pourraient être utilisés seuls ou en combinaison avec d’autres gènes candidats pour permettre le développement de trousses « multigènes » ou d’essais protéomiques multiplex pour prédire l’évolution clinique des cancers mammaires. / In 2015, breast cancer remains a leading cause of death among women worldwide due to relapse and metastases. However, mammary tumors are known to be heterogeneous in terms of their clinical course and response to treatment, despite a seemingly similar phenotype. There is therefore an obvious need to identify and characterize new biomarkers of progression in breast cancers so that each tumor can be properly classified. Our hypothesis is that each breast cancer has its own set of genomic abnormalities or altered pattern of gene expression that can explain the aggressiveness of each tumor, its ability to metastasize and its response to chemotherapeutic agents or other forms of targeted therapies. In this study, our aim is to identify and characterize new biomarkers with prognostic value in aggressive subsets of breast cancer focusing primarily on triple-negative tumors and luminal B breast cancer. To achieve those aims, we conducted an in silico search from public databases of DNA microchip and RNA sequencing data. We next constructed seven tissue microarrays (TMA) using paraffin blocks from human breast cancer along with normal breast to examine the differential expression of new putative markers: ANXA1, MMP-9, DP103 and MCM2. Expression levels measured by immunohistochemistry were then compared to other conventional markers of breast cancer (ER, PR, HER2, Ki-67, CK 5/6, FOXA1) and correlated with clinical data (overall survival and metastasis). By comparing the relative expression of these markers in human breast tumors we were able to pinpoint the important role of ANXA1, MMP-9, DP103, and MCM2 in aggressive tumor subtypes recognized for their poor clinical course. Firstly, we have shown that ANXA1 expression is severely deregulated in high-grade breast cancers including triple-negative and, to some extent, HER2-positive breast cancers. In addition, our results also indicated a possible role of ANXA1 in regulating EMT and breast cancer cell metastasis. Secondly, expression of MMP-9 was found to mirror the degree of tumor differentiation and to correlate with breast cancers of unfavorable outcome. This implies that MMP-9 can help better characterize the biology of breast carcinoma and to identify subgroups of high-risk breast tumors. In fact, we found that high levels of MMP-9 in tumors were associated with increased metastatic dissemination, early relapse and reduced survival. Thirdly, we demonstrated that MCM2 is overexpressed in triple-negative, HER2 positive and luminal B breast cancer in comparison to luminal A breast cancer (p-value < 0.0001). Our findings support the notion that MCM2 can be used to distinguish luminal A from luminal B breast cancer based on a 40% index cut-point. However, an independent validation cohort is needed to confirm the clinical utility of MCM2. Lastly, our results suggest that ANXA1, MMP-9 and MCM2 are valuable genes/proteins candidate that can help better understand the mechanisms involved in tumor progression and metastasis. One may also envisage their use, alone or in combination with other genes, in the development of a multi-gene panel or multiplex proteomic assay to predict clinical outcome and guide therapeutic decisions.

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L’altération de la barrière hématoencéphalique (BHE) par les cellules tumorales et les cellules immunes circulantes peut mener à la neuroinflammation. Les cellules leucémiques promyélocytaires HL-60 sont un excellent modèle pour étudier et comprendre les mécanismes de signalisation moléculaires qui caractérisent le développement tumoral et métastatique. La cancérogenèse peut s’accompagner de modulations de l’expression de biomarqueurs tels que la cyclooxygénase-2 et la métalloprotéase-9. Les recherches décrites dans ce mémoire relatent l’analyse des biomarqueurs inflammatoires et invasifs régulés lors de la différenciation induite par le PMA des cellules HL-60 en macrophages. Le statut de différenciation cellulaire pourrait avoir un impact sur les gènes cibles de la voie NF-κB. Nous émettons l’hypothèse que le PMA active la voie NF-κB et que cette signalisation peut être renversée par l’(-)-épigallocatéchine-gallate (EGCG). En effet, une régulation à la hausse de l’expression de plusieurs gènes combinée à la diminution de l’expression d’IκB mettent en évidence l’implication de la voie NF-κB dans l’activation des mécanismes pro-inflammatoires et pro-invasifs. Les mêmes observations sont faites dans les cellules différenciées appelées «macrophages-like». L’EGCG, un polyphénol dérivé du thé vert, a un potentiel chimiopréventif. Il est capable d’inhiber la signalisation moléculaire passant par la voie NF-κB dans les cellules HL-60 traitées simultanément par l’EGCG et le PMA, mais pas dans les cellules «macrophages-like». Cette différence peut s’expliquer par une modulation de l’expression du récepteur de surface cellulaire de l’EGCG, le récepteur à la laminine de 67 kDa, et de son précurseur de 37 kDa. Collectivement, nos résultats montrent que le statut de différenciation des cellules promyélocytaires HL-60 concorde avec l’activation des mécanismes favorisant le développement d’un cancer et des métastases. Cet effet peut être prévenu par l’utilisation d’agents naturels tel l’EGCG. Le ciblage de biomarqueurs liés au statut de différenciation des cellules tumorales impliquées dans la perturbation de la barrière hématoencéphalique qui cause la neuroinflammation permettrait l’avancement des connaissances dans la prévention de la cancérogenèse. / Blood-brain barrier (BBB) disruption by circulating tumor and immune cells leads to secondary inflammatory infections. Promyelocytic HL-60 cells represent an excellent model to study and to get a better understanding of the molecular signaling mechanisms involved in carcinogenesis and metastasis. The research described in this thesis shows the analysis of several inflammatory and invasive biomarkers regulated during PMA-induced differentiation of promyelocytic HL-60 cells into macrophages. Carcinogenesis involves some modifications in the expression of biomarkers such as cyclooxygenase-2 and matrix metalloprotease-9. The differentiation status could have an impact on the NF-κB signaling pathway that regulates the target genes, given that these target genes expression varies during cell differentiation. We hypothesize that the activation of the NF-κB pathway by PMA can be reverse by (-)-epigallocatechin-gallate (EGCG). Indeed, the up-regulation of downstream genes combined with the down-regulation of IκB expression showed the significant implication of the NF-κB signaling pathway to activate pro-inflammatory and pro-invasive mechanisms linked to carcinogenesis. The same evidence exhibits in the differentiated cells called «macrophages-like». Moreover, the green tea polyphenol, EGCG, shows chemopreventive property since it better inhibited NF-κB signaling in cells treated simultaneously with EGCG and PMA compared to the «macrophages-like». This difference could be due, in part, to the down-regulation of the 67 kDa laminin receptor, known to be the non-integrin membrane receptor for EGCG. All together, our results suggest that the differentiation status of promyelocytic cells is linked to the activation of mechanisms involved in carcinogenesis and metastasis. These phenomena can be prevented by using natural agents such as EGCG. Targeting the specific biomarkers linked to the differentiation status of tumor cells and involved in the disruption of the BBB may help reduce secondary neuroinflammation and enable the advancement of knowledge towards carcinogenesis prevention.

Dans les tumeurs, les cellules cancéreuses invasives induisent l'expression de la métalloprotéase matricielle 11 (MMP-11) dans les adipocytes adjacents (cancer-associated adipocytes) entrainant leur " dédifférenciation " en fibroblastes, la MMP-11 régulant négativement l'adipogénèse. Au cours de ma thèse, j'ai étudié la fonction de la MMP-11 sur le développement mammaire postnatal. La structure de la glande ainsi que la production de lait sont altérées dans les souris déficientes pour la MMP-11. De plus, la MMP-11 régule l'homéostase du collagène. In vivo, des transplantations montrent une fonction locale paracrine de la MMP-11 essentielle à la morphogenèse mammaire. In vitro, la MMP-11 adipocytaire favorise le branchement d'organoïdes primaires. Ainsi, la MMP-11 est un facteur paracrine majeur pour le développement de la glande mammaire. Enfin, pour poursuivre ces travaux, j'ai établi des souris transgéniques conditionnelles ciblant l'expression de la MMP-11 dans les adipocytes.

Made available in DSpace on 2014-08-20T14:30:10Z (GMT). No. of bitstreams: 1 tese_anelise_fernandes_montagner.pdf: 2069040 bytes, checksum: f8827dc228fd6085f72280a7c6d30b98 (MD5) Previous issue date: 2013-08-29 / The aim of this study was to evaluate the influence of chlorhexidine digluconate and sodium hypoclorite application on dentin after acid ecthing on the performance of adhesive restorations subjected to aging of the adhesive interface. For this, three studies were performed (in vitro, in situ and a systematic review). The in vitro study aimed to evaluate the effect of chlorhexidine digluconate (CHX) and sodium hypochlorite (NaOCl) on the microtensile bond strength values (μTBS) in dentin after cariogenic challenge and aging. Thirty sound human molars were selected and randomly assigned into 3 dentin pre-treatments (control, 2% chlorhexidine and 10% NaOCl) and 4 different types of aging (control -C-, biofilm without cariogenic challenge -NCC-, biofilm with cariogenic challenge -CC- and 18-month water storage -WS-). The same etch-and-rinse adhesive system (Adper Single Bond 2, 3M ESPE) and composite resin (Z250, 3M ESPE) were used for all the groups (n=30). For NCC and CC groups, dental biofilm in microcosm derived from saliva of a healthy donor was added on the samples. The biofilm grew up in a defined medium enriched with mucin (DMM), with or without 10% sucrose, according to the group. After the experimental period, all the samples were cleaned up and microtensile test was carried out. The data, in MPa, were subjected to ANOVA and a post-hoc Tukey Test (p < 0.05). The pre-treatment did not influence the μTBS, for all the aging conditions (p = 0.188); however the type of aging statistically affected the bond strength (p = 0.000). The bond strength values showed the following trend: C = NCC < CC = WS. Cariogenic challenge and water storage aging affected the bond strength stability and the former seems to be a suitable short-term methodology to assess the degradation of the resin-dentin. The in vivo study aimed to evaluate the effect of chlorhexidine (CHX) application in etched dentin on the 6-month retention of restorations of noncarious cervical lesions (NCCLs). A randomized controlled splitmouth and blind trial was carried out. Patients (n = 42) with at least two noncarious cervical lesions were included. NCCLs were randomly assigned according to the type of tooth into two groups: control (application of a placebo solution) or experimental group (application of 2% CHX for 60s after acid etching). Class V restorations (n = 169) were performed with an etch-and-rinse adhesive system (Adper Single Bond 2) and a composite resin (Filtek Z350) according to the manufacturer instructions, by 10 previously trained operators. The restorations were evaluated by a calibrated examiner at 1 week (baseline) and 6-month using the FDI criteria. The outcome was retention of the restoration. The analysis of factors associated to the failure of restoration was carried out through Fisher s Exact Test (α = 0.05). After 6 months of follow-up, 3.4% (CI 95% 1.3 - 7.3) of the Class V restorations failed. There was no significant difference between control and CHX (p = 0.920). Regarding the cavity s variables, deeper (p = 0.024) and wider cavities (p = 0.004) showed more failures, and wedge-shaped cavities failed more than saucer-shaped ones (p = 0.033). Regarding the patient s variables, restorations in patients with lower salivation 8 showed a higher percentage of failure (p = 0.034). Both treatments provided acceptable clinical performance of the restorations. The use of CHX as a coadjuvant in dentin adhesion did not influence the retention of Class V restorations after 6 months of follow-up. The systematic review aimed to systematically review the literature for in vitro and ex vivo studies that evaluated the resin-dentin bond strength immediately and longitudinal with the use of MMP inhibitor on the adhesive procedure. The systematic review was conducted sourcing the following databases (Pubmed, TRIP, LILACS, Scielo, Cochrane and ISI web Science) with no publication year or language limit. From 1.336 potentially eligible studies, 48 were selected for full text analysis and 30 were included for review. Two independently reviewers selected the studies, abstracted information and assessed the risk of bias on standardize scales. The results showed high heterogeneity in some comparisons and showed no difference for CHX 2% and control at baseline, however after 6-month of aging the CHX 2% and 0.2% presented a trend showing higher bond strength values than control groups (p < 0.05). The literature review was possible to observe the trend towards the decreased of the bond strength through time most for the control groups than for the experimental one. From of the 30 studies included, only 5 studies (16.6%) presented medium risk of bias while 25 (83.4%) showed high risk of bias and none showed low risk of bias. The use of MMPs inhibitor did not negatively affect the immediate bond strength, and it influenced the bond strength values long-time regarding the dentin adhesion stability. / O objetivo deste estudo foi avaliar a influência da aplicação do digluconato de clorexidina (CRX) e hipoclorito de sódio (NaOCl) em dentina, após o condicionamento ácido, na performance da interface adesiva submetida a envelhecimento da interface adesiva. Para isto, foram realizados 3 estudos (in vitro, in vivo e revisão sistemática). O estudo in vitro objetivou avaliar o efeito do prétratamento da dentina nos valores de resistência de união (RU) após diferentes tipos de envelhecimentos da interface adesiva. A dentina de todos os espécimes foi submetida ao condicionamento com ácido fosfórico por 15s e em seguida a 3 tipos de pré-tratamento: água destilada (controle - soluçao placebo - 60s), digluconato de clorexidina 2% - 60s, ou hipoclorito de sódio 10% - 60s. O sistema desivo (Single Bond 2 3M/ESPE) foi aplicado na superfície dentinária conforme indicação do fabricante. Palitos resina-dentina foram obtidos e submetidos a 4 níveis de envelhecimento: controle (CO), biofilme sem desafio cariogênico (BSD), biofilme com desafio cariogênico (BCD) e armazenamento em água por 18 meses (AGU). Nos grupos com biofilme, modelo de microcosmo foi utilizado e o biofilme foi crescido em meio definido enriquecido com mucina, com (BCD) ou sem (BSD) sacarose por 14 dias. Após o período experimental os espécimes foram limpos e preparados para o teste de microtração e o padrão de fratura foi analisado. Os valores de RU, em MPa, foram submetidos a ANOVA e teste post hoc Tukey (p < 0,05). O pré-tratamento não influenciou nos valores de RU, para todos os envelhecimentos avaliados (p = 0,188), entretanto o tipo de envelhecimento estatisticamente influenciou os valores de RU (p = 0,000). O envelhecimento CO foi similar ao BSD, com valores RU maiores que o BCD e AGU. A interface adesiva foi negativamente afetada pelo desafio cariogênico e pela degradação em água. No estudo in vivo, com objetivo de avaliar a retenção de restaurações classe V em dentina tratada ou não com CRX, um ensaio clínico controlado randomizado, boca dividida e triplo-cego foi conduzido. Pacientes (n = 42) com no mínimo 2 lesões cervicais não cariosas (LCNC) foram selecionados. As LCNC foram randomizadas em 2 grupos: tratamento controle e tratamento experimental (CRX 2% - 60s). Restaurações (n = 169) foram realizadas com Single Bond 2 (3M EPSE) e Filtek Z350 por 10 operadores previamente treinados. As restaurações foram avaliadas por um examinador calibrado em 1 semana e 6 meses após, usando os critérios da FDI. A análise dos fatores associados às falhas nas restaurações foi conduzida através do teste exato de Fisher (α = 0,05). Após 6 meses de acompanhamento, 3,4% (IC95% 1,3 - 7,3) das restaurações falharam devido a retenção. Não houve diferença significante entre os tratamentos controle e CRX para retenção (p = 0,920) e para manchamento marginal (p = 0,734). Em relação às variáveis relativas à cavidade, cavidades com maior profundidade (p = 0,024) e com maior altura (p = 0,004) 6 apresentaram maior ocorrência de falhas. Ainda, cavidades em formato de V apresentaram mais falhas que aquelas em formato de U (p = 0,033). Quanto as variáveis relativas aos pacientes, restaurações em pacientes que apresentavam boca seca falharam em maior porcentagem (p = 0,034). O uso de clorexidina como um coadjuvante na adesão a dentina não influenciou na retenção das restaurações após 6 meses de acompanhamento. E a revisão sistemática teve o objetivo de revisar a literatura acerca de estudos in vitro que avaliaram a RU resina-dentina imediata e após envelhecimento com o uso de inibidores de MMPs no procedimento adesivo. A revisão sistemática foi conduzida em 6 databases (Pubmed, TRIP, LILACS, Scielo, Cochrane and ISI web Science) sem limite de ano ou língua. De 1.336 estudos potencialmente elegíveis, 48 foram selecionados para análise de todo texto e 30 estudos foram incluiídos na revisāo. Dois revisores selecionaram os estudos, extraíram as informações e dados e verificaram o risco de viés em escalas padronizadas. Os resultados demostraram uma grande heterogeneidade em algumas comparações e nāo mostraram diferenças entre clorexidina 2% e o grupo controle sem envelhecimento, porém após 6 meses de envelhecimento observou-se uma tendência a favor dos grupos controle (p < 0,05). Todavia, foi possível observar na revisāo de literatura descritiva a tendência da diminuiçāo dos valores de RU (porcentagem) ao longo do tempo, mais para o grupo controle do que para o grupo experimental. De 30 estudos incluídos, apenas 5 estudos (16,6%) apresentaram médio risco de viés, enquanto 25 estudos (83,4%) mostraram alto risco de vieés e nenhum mostrou baixo risco. Os inibidores de MMPs nāo afetaram negativamente a RU da resina a dentina sem envelhecimento, e nāo influenciaram os valores de RU após envelheciemento.

The control of reproductive cycles in higher primates is largely dependent on negative and positive feedback mechanisms by both steroidal and non-steroidal substances of the ovaries which regulate the function of hypothalamo-pituitary system. To gain insights into the role of INH A, the non steroidal ovarian hormone in the feedback control of pituitary FSH secretion, studies were conducted to examine the interrelationships of hormones throughout the menstrual cycle of the bonnet macaque. The findings of chapter II provide a detailed description of endocrine hormone profile during the menstrual cycle of the bonnet macaques with special attention to the feedback role of INH A on pituitary FSH secretion. To characterize the endocrine profile of different hormones, both ovarian (E2, P4, INH A) and pituitary (FSH, LH) hormones were measured daily for more than 40 days. To further examine the site of secretion of INH A and its relationship with pituitary FSH dynamics, surgical lutectomy and pharmacological induction of luteolysis employing the third generation GnRH R antagonist, Cetrorelix (CET) studies were carried out in the subsequent experiments. The results obtained from these studies suggest that INH A and P4 secreted from the CL during luteal phase regulate pituitary FSH secretion. The selective rise in FSH observed during the late menstrual cycle and during menstruation (referred to as luteo-follicular transition), as has been reported previously in higher primates, considered necessary for initiation of follicular growth and recruitment of follicles for ensuing menstrual cycle was characterized in the monkey. Surgical lutectomy and induction of luteolysis by CET experiments suggested that increased GnRH secretion is essential for this selective rise in FSH following withdrawal of inhibition by P4 and INH A. In clinical cases of reproductive ageing, the shortened follicular phase in human females has been identified to be the result of occurrence of early onset of FSH rise during the luteal-follicular transition period. The cause(s) of declining fertility with age in women who still have regular menstrual cycles are not clear, but issues of relationship between dysregulation of selective FSH rise in the late luteal phase and associated infertility could be examined using bonnet monkey as a model system. INH A is secreted in significant quantities by CL in higher primates and the feto placental unit suggesting its importance during fertility and pregnancy. Apart from the negative feedback regulation of pituitary FSH secretion, the complete repertoire of actions of this hormone during pregnancy is yet to be fully understood. The data presented in this thesis is the first comprehensive report showing the endocrine hormone profile of gonadotropins and ovarian hormones including INH A throughout the menstrual cycle of the bonnet macaque. The characterization of INH A profile in bonnet monkey will be of significant value for studies examining the role of INH A in higher primates. Dimeric inhibin has been suggested to be important for regulation of fertility and reproductive functions. Also, inhibin-α (one of the subunits of the dimeric protein) knock out mice model has provided convincing evidence that it acts as a tumour suppressor. A great deal of new information has been generated in recent years regarding the potential clinical usefulness of monitoring inhibin levels in blood and biological fluids in gynaecological diseases, pathological pregnancies and other disorders. Emerging clinical roles of inhibin have made INH A an important candidate molecule to study its molecular regulation. The results presented in chapter II suggested that LH regulates luteal INH A secretion (induction of luteolysis by CET administration experiment). As a first step towards understanding molecular regulation of inhibin-α expression in the macaque CL, in silico promoter analysis of macaque inhibin-α was performed and it revealed several transcriptional factor binding sites that were conserved across species. In rats FSH up regulates while preovulatory LH surge suppresses inhibin-α mRNA expression in the ovary and this suppression has been suggested to be necessary for occurrence of secondary FSH surge during metestrus. To address differential regulation of inhibin-α by LH and FSH in rat ovary during the periovulatory period, studies employing immature rats were carried out and the results are presented in chapter III. The results suggest that immature rat ovaries respond to exogenous gonadotropins in terms of LH signaling (cAMP production), luteinization (P4 production) and as well induction of ICER expression required for repression of inhibin-α subunit expression. PDE4 inhibitor (rolipram) treatment enhanced the ovarian cAMP concentrations suggesting that PDE4 play a major role in controlling intraovarian cAMP concentrations in rat ovaries. However increased cAMP concentrations did not appear to up regulate the ICER expression at the time point examined in this study. In higher primates time course of second FSH surge and continued synthesis and secretion of INH A in the CL are different from non primate species. In the monkey, the second FSH rise occurs during the late luteal phase and experiments have been carried out to examine the regulation of inhibin-α subunit expression by ICER. Expressions of ICER (mRNA/protein) and INH A were examined during different stages of CL and the results indicated no clear inverse relationship between the ICER and inhibin-α mRNAs. With no conclusive role for the ICER in regulating luteal inhibin-α observed in the study, the role of transcriptional activators in the regulation of inhibin-α like GATA4, SF-1, β-catenin were further examined. Since luteal INH A secretion was dependent on pituitary LH as determined earlier in chapter II, expressions of transcriptional activators were examined in CL of different stages and also during induced luteolysis and the results are described in chapter IV. In conclusion, our results indicate cross talk between WNT, cAMP and P38 MAP kinase signaling pathways in the regulation of luteal INH A secretion. The pituitary gonadotropin, LH, is the primary luteotropin in primate species acting to maintain the structure and function of the CL during the menstrual cycle. However whether the actions of LH are direct or mediated by local factors such as P4 remain unknown. Moreover, P4 secretion which is dominant during luteal phase has any role in regulating CL structure and function is not clearly defined. To address these and issues concerning P4 actions, initially, experiments were performed in the rat model to study the importance of P4 in the regulation of ovarian functions. An antiprogestin, RU486, was employed as a tool to uncover the PR regulated pathways during ovulation in rats and the findings are presented in the chapter V. The results indicated that blockade of PR action by RU486 during gonadotropin-induced superovulation resulted in inhibition of follicular rupture and ovulation in immature rats. Further to understand the downstream effectors of PR action, and to identify the candidate target genes of PR activation, semi-quantitative RT-PCR and western blot analyses were performed. The results obtained indicated that betacellulin, a member of EGF family and MMP-9 a proteolytic enzyme, were markedly repressed in response to RU486 treatment in rat ovaries. Also, the down stream pathway of EGF signaling leading to activation of ERK was markedly repressed in RU486 treated ovaries. It was next examined what role the P4/PR system has in the regulation of CL structure and function. Surprisingly, PR expression is absent in CL of rats, while it is present in higher primates. Experiments were carried out to examine intracrine actions of P4 in the regulation of CL structure and function in monkeys. The recently reported model system of induced luteolysis yet capable of responsive to trophic support from the laboratory provided an ideal opportunity to examine direct effects of P4 on structure and function of CL in the monkey. A series of pilot experiments were carried out in monkeys experiencing summer amenorrhea, to determine dose and mode of administration of exogeneous P4 to simulate mid luteal phase circulating P4 concentrations in monkeys subjected to induced luteolysis. Based on the results of pilot experiments, implantation of Alzet pumps containing 97.5mg of P4 was selected for maintaining mid luteal phase P4 concentrations. The microarray data of induced luteolysis previously deposited by the laboratory in NGBI’s gene expression omnibus were mined for identification and validation of differentially expressed genes of PR and its target genes following LH depletion and LH replacement experiments. Expressions of PR, PR cofactors and expressions of PR downstream target genes through out the luteal phase and in CL from day1 of menses were also examined. Analysis of expressions of genes revealed that of the 45 genes identified to be regulated by LH treatment, 4 genes were found to be responsive to P4, and 14 were identified to be responsive to both P4 and LH. Morphology of CL tissue sections revealed that P4 treatment appeared to have reversed the induced-luteolysis changes. In another experiment, implantation of P4 during late luteal phase (i.e., the period of declining P4 concentrations) for 24h caused changes in expressions of genes associated with tissue remodeling and morphology of luteal cells. Taken together, the results suggest that induced luteolysis plus P4 replacement model is suitable for assessing the effects of P4 on CL function. The results also suggest that CL could serve as target tissue for examining the genomic and non genomic actions of P4. In summary, studies carried out in the present thesis provides a comprehensive endocrine hormone profile throughout the menstrual cycle of the bonnet monkey with special emphasis on time course of INH A and FSH secretion which is very useful for future investigations. Studies have been carried out in rats and monkeys with different experimental model systems to address molecular mechanisms underlying inhibin-α regulation in the ovary in general and CL in particular. Experimental findings in monkeys could help elucidate the underlying molecular nature of CL functionality and extrapolate to understand luteal insufficiency and infertility producing conditions in humans. Also different model systems have been validated to examine the actions of P4/PR system in rats and monkeys and more importantly to address the direct effects of P4 upon monkey CL structure and function were established. Future investigations based on findings of these studies should help clarify relative roles for LH and P4 during maintenance of CL function and luteolysis.