Submitted by LUCAS POSSEBON null (lucas_possebon@hotmail.com) on 2017-03-07T01:37:03Z No. of bitstreams: 1 Dissertação Mestrado Lucas Possebon.pdf: 3575174 bytes, checksum: bd6f3f0fb08741b8fd22772ba8dded60 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2017-03-10T17:07:31Z (GMT) No. of bitstreams: 1 possebon_l_me_sjrp.pdf: 3575174 bytes, checksum: bd6f3f0fb08741b8fd22772ba8dded60 (MD5) / Made available in DSpace on 2017-03-10T17:07:31Z (GMT). No. of bitstreams: 1 possebon_l_me_sjrp.pdf: 3575174 bytes, checksum: bd6f3f0fb08741b8fd22772ba8dded60 (MD5) Previous issue date: 2017-02-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O processo inflamatório causado pelo tabagismo está relacionado a diferentes tipos de doenças como enfisema pulmonar, doença pulmonar obstrutiva crônica (DPOC) e aterosclerose. Neste cenário, a proteína anti-inflamatória AnxA1 pode representar uma alternativa terapêutica. Por essas razões, o objetivo da pesquisa foi avaliar os efeitos do peptídeo mimético Ac2-26 da proteína AnxA1, em modelo de tabagismo. Ratas Wistar foram divididas em 3 grupos (n=10/grupo): expostos ao fumo não tratados (F) e tratados com o peptídeo (F+Ac2-26) e controles (C). Os grupos de animais expostos ao fumo foram colocados em um equipamento específico da Unidade Didática e de Pesquisa Experimental (UDPE) das Faculdades Integradas Padre Albino (FIPA), e expostos à queima de 10 cigarros comerciais, um após o outro, 2x/dia, por 5 semanas. O grupo C foi mantido no mesmo regime, porém na ausência da fumaça do cigarro e tratamento. Para avaliar a eficácia do Ac2-26, animais F+Ac2-26 foram administrados intraperitonealmente com o peptídeo (1mg/kg), 1x/dia, antes da primeira exposição ao cigarro. As ratas foram pesadas e tiveram a pressão arterial aferida no início e final do experimento. No período final, a ventilação pulmonar foi verificada por meio da pletismografia e também foram realizadas imagens de raios-X. Os animais foram eutanasiados e coletados o lavado bronco alveolar (LBA) para quantificações de células inflamatórias e citocinas, o sangue para dosagens bioquímicas de citocinas e hemoglobina e os órgãos, pulmão e traqueia, para os estudos histopatológicos e imuno-histoquímicos. As análises fisiológicas mostraram perda de peso, aumento da pressão arterial, reduções da frequência e ventilação pulmonares, bem como alterações macroscópicas das dimensões pulmonares por imagens de raio-X no grupo F. Enquanto, nos F+Ac2-26, esses valores foram semelhantes aos controles. As análises histopatológicas mostraram maiores espaços intra-alveolares e aumento do tecido linfoide no pulmão e perda dos cílios no epitélio da traqueia no grupo F comparado as F+Ac2-26 e C. Nas análises do LBA, foi observado aumento na quantidade de linfócitos e macrófagos em F, com redução significante dessas células promovida após o tratamento. Nas quantificações de células inflamatórias nos tecidos, os macrófagos e mastócitos foram observados aumentados no grupo F comparado aos C e F+Ac2-26. As análises imuno-histoquímicas do pulmão e da traqueia mostraram menor expressão de AnxA1, COX-2 e MMP-9 nos animais C e F+Ac2-26. As dosagens de citocinas e quimiocina indicaram aumento no sobrenadante do macerado do pulmão, plasma sanguíneo e LBA no grupo F e redução nos níveis desses mediadores em C e F+Ac2-26. Ainda, as dosagens bioquímicas do sangue mostraram que o tratamento com o peptídeo ocasionou aumento da concentração de hemoglobina e glicose e redução do colesterol total e transaminase glutâmico oxalacética (TGO) comparados aos animais não tratados. Nossos resultados evidenciaram a ação protetora do peptídeo mimético Ac2-26 no modelo de DPOC, atenuando o processo inflamatório causado pela exposição à fumaça do cigarro e abre novas perspectivas para o tratamento das doenças relacionadas ao tabagismo. / The inflammatory process caused by smoking is related to different kinds of diseases such as pulmonary emphysema, chronic obstructive pulmonary disease (COPD) and atherosclerosis. In this scenario, an anti-inflammatory protein AnxA1 may represent a therapeutic alternative. For these reasons, the objective of this research was to analyze the effects of the mimetic peptide Ac2-26 of the AnxA1 protein, in a smoking model. Wistar rats were divided into 3 groups (n = 10/group). The groups of animals exposed to smoke were placed in a specific equipment of the Didactic and Experimental Research Unit (UDPE) of the Integrated Colleges Padre Albino (FIPA), and exposed to the burning of 10 cigarettes, one after another, 2x / day for 5 weeks. To evaluate the efficacy of Ac2-26, CS+Ac2-26 animals were administered intraperitoneally with peptide (1mg / kg), 1x / day, prior to first exposure to the cigarette. Group C was maintained in the same regimen, but in the absence of cigarette smoke or treatment. The rats were weighed and had blood pressure measured at the beginning and ending of the experiment. In the final period, pulmonary ventilation was verified through plethysmography and also performed ray-X images. The animals were euthanized and collected the alveolar bronchus (BAL) for quantification of inflammatory cells and cytokines, the blood for biochemical measurements of cytokines and hemoglobin and organs, lung and trachea, for histopathological and immunohistochemical studies. The physiological analyzes showed weight loss, increased blood pressure, reductions and in the pulmonary frequency and ventilation, as well as macroscopic alternative in the lung dimensions by X-ray images in group CS. While, in CS+Ac2-26, these values were similar to controls. The histopathological analyzes showed enlargement of the intra-alveolar spaces and increased lymphoid tissue (BALT), and loss of the cilia in the epithelium of the trachea in the CS group, compared to CS+Ac2-26 and C. Numerous lymphocytes and macrophages were observed in the BAL in CS, with significant reduction of the cells after treatment. In the quantifications of inflammatory cells in the tissues, macrophages and mast cells were increased in the CS+Ac2-26 group. The immunohistochemical analyzes of the lung and trachea showed lower expression of AnxA1, COX-2 and MMP-9 in C and CS+Ac2-26 animals. The dosages of cytokines and chemokine indicated incr ease in the supernatant of lung macerate, blood plasma and BAL in the F group and reduction of these mediators levels in C and CS+Ac2-26 groups. Also, the biochemical blood measurements showed that treatment with the peptide caused an increase in hemoglobin and glucose concentrations and reduction of total cholesterol and Glutamic oxaloacetic transaminase (GOT) compared to untreated animals. Our results evidenced a protective action of the Ac2-26 mimetic peptide in the COPD model, by attenuating the inflammatory process caused by exposure to cigarette smoke, which opens new perspectives for the treatment of smoking-related diseases.

A inflamação no sistema nervoso central (SNC) está envolvida na gênese de uma série de doenças neurodegenerativas, sendo assim, compreender o processo inflamatório nessas circunstâncias se torna essencial para propor novas abordagens terapêuticas. Sabemos que a Anexina A1 (ANXA1) e o receptor TSPO são dois moduladores importantes da neuroinflamação. Enquanto se sabe que a ANXA1 possui propriedades antiinflamatórias, o papel do TSPO ainda não está esclarecido. Desta forma, este projeto avaliou a atuação da ANXA1 sobre a expressão do TSPO em linhagem de células da microglia (BV2), e sua conexão com o receptor Toll-like receptor-4 (TLR4) em BV2 ativada pelo lipopolisacarídeo de E.coli (LPS). Os resultados obtidos mostram que o tratamento de BV2 com LPS induz a expressão de TSPO, dependente de ativação de TLR4, através das vias da molécula adaptadora do fator de diferenciação mielóide 88 (MyD88) e do fator nuclear κB (NFκB). O tratamento com ANXA1 recombinante induz um perfil antiinflamatório em células BV2 estimuladas com LPS, por reduzir a secreção de citocinas proinflamatórias e, ao mesmo tempo, aumentar secreção de citocinas antiinflamatórias. A exposição com ANXA1 ainda impede o aumento da expressão de TSPO induzida pelo LPS. Mostramos também que esta ação da ANXA1 é dependente da interação com o receptor de peptídeo formilado (FPR2). Adicionalmente, o silenciamento de TSPO em células BV2 predispõe essas células a um perfil ativado exacerbando a secreção do fator de necrose tumoral (TNFα) em resposta ao LPS, o que não pode ser revertido pelo tratamento com ANXA1 recombinante. Em conjunto, os resultados expõe a relação existente entre ANXA1 e TSPO em micróglia ativada pelo LPS, mostrando que a ANXA1 9 modula negativamente a expressão do TSPO. Ademais, o silenciamento de TSPO inibiu a fagocitose de neurônios apoptóticos, o que ainda sugere a participação do TSPO na eferocitose. / Inflammation in the Central Nervous System (CNS) is involved in the genesis of a number of neurodegenerative diseases, so understanding the inflammatory process in these circumstances is essential to proposal new therapeutic approaches. We know that Annexin A1 (ANXA1) and the TSPO receptor are two important modulators of neuroinflammation. While it is known that ANXA1 has anti-inflammatory properties, the role of TSPO has not yet been clarified. Thus, this project evaluated the interference of ANXA1 on the expression of TSPO in microglia cell line (BV2), and its connection with the Toll-like receptor-4 receptor (TLR4) in BV2 activated by E. coli lipopolysaccharide LPS). The results show that the treatment of BV2 with LPS induces the expression of TSPO, dependent on activation of TLR4, through the pathways of the adapter molecule of myeloid differentiation factor 88 (MyD88) and nuclear factor κB (NFκB). Treatment with recombinant ANXA1 induces an anti-inflammatory profile in LPS-stimulated BV2 cells, by reducing the secretion of proinflammatory cytokines and, at the same time, increasing secretion of anti-inflammatory cytokines. Exposure with ANXA1 still prevents the increase of LPS-induced TSPO expression. We also show that this action of ANXA1 is dependent on the interaction with the formylated peptide receptor (FPR2). In addition, TSPO silencing in BV2 cells predisposes these cells to an activated profile exacerbating secretion of tumor necrosis factor (TNFα) in response to LPS, which can not be reversed by treatment with recombinant ANXA1. Together, the results show the relationship between ANXA1 and TSPO in LPS activated microglia, showing that ANXA1 negatively modulates TSPO 11 expression. In addition, TSPO silencing inhibited the phagocytosis of apoptotic neurons, which still suggests the participation of TSPO in eferocytosis.

Le mélanome cutané est le plus agressif des cancers de la peau. Une fois métastasé, les options thérapeutiques sont limitées et peu efficaces. Dans le but de trouver de nouveaux marqueurs d'agressivité du mélanome cutané, une étude protéomique comparative entre deux lignées de mélanome murin, génétiquement semblables mais avec des agressivités différentes, a permis d'identifier l'annexine A1 (ANXA1) comme une protéine pro-invasive. Le but de ce travail de thèse a été d'évaluer la valeur pronostique d'ANXA1 et de déterminer son rôle et son mode d'action dans les processus d'invasion des mélanomes humains. Lors d'une étude immunohistologique rétrospective sur deux centres (Clermont-Ferrand et Saint-Etienne), il a été observé, indépendamment de l'indice de Breslow, une corrélation inverse entre le taux d'ANXA1 dans les tumeurs primitives de 61 patients et le délai d’apparition des métastases. Cette association est due à l’implication d’ANXA1 dans les processus d’invasion. En effet,nous avons démontré dans différentes lignées de mélanome qu'ANXA1 extracellulaire stimule les récepteurs aux peptides formylés (FPRs) exprimés par ces cellules. Cette fixation aux FPRs induit les voies des MAPK et STAT3 qui entraînent l'activation des métalloprotéases (MMP2). L'induction des MMP2 par ANXA1 augmente significativement le pouvoir invasif des lignées de mélanome. Nous avons aussi démontré qu’ANXA1 est transloquée coté externe de la membrane cytoplasmique là où elle subit un clivage par une sérine protéase qui pourrait être la nicaline. Ce clivage qui se produit après la sérine 28 n'a pas été décrit et jouerait un rôle dans la capacité invasive des mélanomes en libérant un ou des peptide(s) proinvasif(s). A long terme, ce travail vise à utiliser ANXA1 comme marqueur pronostique et/ou cible thérapeutique du mélanome cutané. / Cutaneous melanoma is the most aggressive skin cancer. Treatment options are limited and inefficient when melanoma has metastasized. In order to identify new markers of melanoma dissemination, protein profiles of two genetically similar murine melanoma cell lines have been compared. Both B16F10 and B16Bl6 cells induced primary tumours after subcutaneous graft, however, only B16Bl6 melanomas develop metastases. Among proteins differentially expressed, annexin A1 (ANXA1) is overexpressed in the aggressive B16Bl6 melanoma.The aim of the present work was to assess ANXA1 prognostic value in human melanoma and todecipher its implication in the invasion process. We report that, regardless of Breslow index, ANXA1 expression in primary tumours of 61 patients is inversely correlated with time to metastasis. This correlation is due to ANXA1 involvement in the invasion processes. Indeed, we show that in different human melanoma cell lines, extra cellular ANXA1 stimulates Formylated Peptide Receptors (FPRs). FPRs/ANXA1 interaction induces MMP2 activity through MAP Kinase and STAT3 pathways. ANXA1-stimulated MMP2 induces a significant increase of cell invasion ability. We also show that ANXA1 is externalized and localized on the cell surface where it is cleaved by a serine protease, which could be nicalin. ANXA1 cleavage occurs after Serine 28, a so far not described site. These data suggest that ANXA1 cleavage might be associated with invasion ability of melanoma cells by release of proinvasive peptide(s).These findings identify ANXA1 as a melanoma proinvasive protein that might be a promising prognosis marker and therapeutic target.

A inflamação no sistema nervoso central (SNC) está envolvida na gênese de uma série de doenças neurodegenerativas, sendo assim, compreender o processo inflamatório nessas circunstâncias se torna essencial para propor novas abordagens terapêuticas. Sabemos que a Anexina A1 (ANXA1) e o receptor TSPO são dois moduladores importantes da neuroinflamação. Enquanto se sabe que a ANXA1 possui propriedades antiinflamatórias, o papel do TSPO ainda não está esclarecido. Desta forma, este projeto avaliou a atuação da ANXA1 sobre a expressão do TSPO em linhagem de células da microglia (BV2), e sua conexão com o receptor Toll-like receptor-4 (TLR4) em BV2 ativada pelo lipopolisacarídeo de E.coli (LPS). Os resultados obtidos mostram que o tratamento de BV2 com LPS induz a expressão de TSPO, dependente de ativação de TLR4, através das vias da molécula adaptadora do fator de diferenciação mielóide 88 (MyD88) e do fator nuclear κB (NFκB). O tratamento com ANXA1 recombinante induz um perfil antiinflamatório em células BV2 estimuladas com LPS, por reduzir a secreção de citocinas proinflamatórias e, ao mesmo tempo, aumentar secreção de citocinas antiinflamatórias. A exposição com ANXA1 ainda impede o aumento da expressão de TSPO induzida pelo LPS. Mostramos também que esta ação da ANXA1 é dependente da interação com o receptor de peptídeo formilado (FPR2). Adicionalmente, o silenciamento de TSPO em células BV2 predispõe essas células a um perfil ativado exacerbando a secreção do fator de necrose tumoral (TNFα) em resposta ao LPS, o que não pode ser revertido pelo tratamento com ANXA1 recombinante. Em conjunto, os resultados expõe a relação existente entre ANXA1 e TSPO em micróglia ativada pelo LPS, mostrando que a ANXA1 9 modula negativamente a expressão do TSPO. Ademais, o silenciamento de TSPO inibiu a fagocitose de neurônios apoptóticos, o que ainda sugere a participação do TSPO na eferocitose. / Inflammation in the Central Nervous System (CNS) is involved in the genesis of a number of neurodegenerative diseases, so understanding the inflammatory process in these circumstances is essential to proposal new therapeutic approaches. We know that Annexin A1 (ANXA1) and the TSPO receptor are two important modulators of neuroinflammation. While it is known that ANXA1 has anti-inflammatory properties, the role of TSPO has not yet been clarified. Thus, this project evaluated the interference of ANXA1 on the expression of TSPO in microglia cell line (BV2), and its connection with the Toll-like receptor-4 receptor (TLR4) in BV2 activated by E. coli lipopolysaccharide LPS). The results show that the treatment of BV2 with LPS induces the expression of TSPO, dependent on activation of TLR4, through the pathways of the adapter molecule of myeloid differentiation factor 88 (MyD88) and nuclear factor κB (NFκB). Treatment with recombinant ANXA1 induces an anti-inflammatory profile in LPS-stimulated BV2 cells, by reducing the secretion of proinflammatory cytokines and, at the same time, increasing secretion of anti-inflammatory cytokines. Exposure with ANXA1 still prevents the increase of LPS-induced TSPO expression. We also show that this action of ANXA1 is dependent on the interaction with the formylated peptide receptor (FPR2). In addition, TSPO silencing in BV2 cells predisposes these cells to an activated profile exacerbating secretion of tumor necrosis factor (TNFα) in response to LPS, which can not be reversed by treatment with recombinant ANXA1. Together, the results show the relationship between ANXA1 and TSPO in LPS activated microglia, showing that ANXA1 negatively modulates TSPO 11 expression. In addition, TSPO silencing inhibited the phagocytosis of apoptotic neurons, which still suggests the participation of TSPO in eferocytosis.

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.