Le cytosquelette d'actine est une structure dynamique capitale pour la cellule, qui intervient dans les processus de signalisation et génère des forces mécaniques pour compléter des fonctions aussi diverses que l'adhésion, la migration, la division ou la différenciation. Les protéines qui régulent cette structure sont capables de moduler ces fonctions. J'ai identifié une nouvelle famille de protéines régulatrices de l'actine, les protéines Refilines (RefilineA et RefilineB), dont l'expression est corrélée avec l'engagement des cellules dans des programmes de différenciation. La RefilineA est induite lors de la différenciation des cellules précurseurs neurales multipotentes en cellules progénitrices gliales. La RefilineB est stabilisée dans les cellules épithéliales lors de la transition épithélio-mésenchymateuse (TEM) induite par le TGF-β. Dans ces cellules, les Refilines agissent en se complexant à la FilamineA, une protéine qui se lie aux filaments d'actine et forme le maillage. Des syndromes génétiques de mutations sur le gène de la FilamineA entrainent d'importants défauts développementaux, cependant la fonction précise de la protéine reste à ce jour obscure. Le complexe Refiline/FilamineA induit la formation de câbles d'actine et génère également une nouvelle structure d'actine périnucléaire appelée coiffe d'actine (« actin cap ») ou « ligne TAN» qui s'ancre à l'enveloppe nucléaire pour réguler les mouvements et la morphologie du noyau. Les Refilines sont les seules protéines identifiées à ce jour capables de catalyser la formation de structures périnucléaires d'actine. Ces résultats ouvrent donc de nouvelles perspectives pour appréhender les fonctions de la FilamineA ainsi que la biologie et les fonctions des structures périnucléaires d'actine. / The actin cytoskeleton is a highly dynamic structure involved in cell signaling and that creates mechanical force for the completion of diverse functions such as adhesion, migration, division or differentiation. Proteins that regulate this structure can modulate its function. We identified a new protein family that regulates the actin cytoskeleton, Refilin proteins (RefilinA and RefilinB), and whose expression correlates with differentiation switches. RefilinA is induced during differentiation of neural multipotent precursors into glial progenitors, while RefilinB is stabilized in epithelial cells during epithelial-mesenchymal transition (EMT) induced by TGF-β. In cells, Refilins interact with FilaminA, a protein that binds actin filaments to organize them into a network. Genetic syndromes where the FilaminA gene is mutated lead to important developmental defects, The Refilin/FilaminA complex generates actin cables as well as a new perinuclear structure called « actin cap » or «TAN line» that interacts with the nuclear envelope to regulate nuclear movement and shape. Refilin proteins are the only proteins identified so far that induce the formation of perinuclear actin structures. These results open up new perspective for the understanding of FilaminA's function as well as for the biology and functions of perinuclear actin structures.

Epithelial-mesenchymal transition (EMT) regulates the cellular processes of migration, growth, and proliferation - as well as the collective cellular process of tissue remodeling - in response to mechanical and chemical stimuli in the cellular microenvironment. Cells of the epithelium form cell-cell junctions with adjacent cells to function as a barrier between the body and its environment. By distributing localized stress throughout the tissue, this mechanical coupling between cells maintains tensional homeostasis in epithelial tissue structures and provides positional information for regulating cellular processes. Whereas in vitro and in vivo models fail to capture the complex interconnectedness of EMT-associated signaling networks, previous computational models have succinctly reproduced components of the EMT program. In this work, we have developed a computational framework to evaluate the mechanochemical signaling dynamics of EMT at the molecular, cellular, and tissue scale. First, we established a model of cell-matrix and cell-cell feedback for predicting mechanical force distributions within an epithelial monolayer. These findings suggest that tensional homeostasis is the result of cytoskeletal stress distribution across cell-cell junctions, which organizes otherwise migratory cells into a stable epithelial monolayer. However, differences in phenotype-specific cell characteristics led to discrepancies in the experimental and computational observations. To better understand the role of mechanical cell-cell feedback in regulating EMT-dependent cellular processes, we introduce an EMT gene regulatory network of key epithelial and mesenchymal markers, E-cadherin and N-cadherin, coupled to a mechanically-sensitive intracellular signaling cascade. Together these signaling networks integrate mechanical cell-cell feedback with EMT-associated gene regulation. Using this approach, we demonstrate that the phenotype-specific properties collectively account for discrepancies in the computational and experimental observations. Additionally, mechanical cell-cell feedback suppresses the EMT program, which is reflected in the gene expression of the heterogeneous cell population. Together, these findings advance our understanding of the complex interplay in cell-cell and cell-matrix feedback during EMT of both normal physiological processes as well as disease progression.

Sporadic breast cancers are mainly attributable to long-term exposure to environmental factors, via a multi-year, multi-step, and multi-path process of tumorigenesis involving cumulative genetic and epigenetic alterations in the chronic carcinogenesis of breast cells from a non-cancerous stage to precancerous and cancerous stages. Epidemiologic and experimental studies have suggested that various dietary compounds like green tea and grape seed may be used as preventive agents for breast cancer control. In this research, I have developed a cellular model that mimics breast cell carcinogenesis chronically induced by cumulative exposures to low doses of environmental carcinogens. I used the chronic carcinogenesis model as a target system to investigate the activity of dietary compounds at non-cytotoxic levels in intervention of cellular carcinogenesis induced by cumulative exposures to pico-molar 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (B[a]P). I used various cancer-associated properties like, reduced dependence on growth factors, anchorage-independent growth, increased cell mobility, and acinar-conformational disruption as measurable endpoints of carcinogenesis. The first part (Part-I) of this dissertation focuses on the understanding the breast cancer progression, importance of environmental carcinogens, role of diet in cancer prevention and importance of epithelial to mesenchymal transition and stem-like cells in chronic carcinogenesis. The next three parts (Part II-IV) focus on understanding the role and mechanisms of dietary compounds in prevention of carcinogenesis and stem-like cell properties. Results in part II revealed the green tea extract at bio-achievable concentration can suppress carcinogen-induced cancerous properties. In Part-III, I compared the four major catechins in green tea extract in suppressing chronic carcinogenesis and the results revealed that epicatechin gallate to be most effective. I also identified that short-term exposure to NNK and B[a]P resulted in elevation of reactive oxygen species, ERK pathway activation and induction of cell proliferation and DNA damage, which can be blocked by green tea catechins. Results in Part-IV describe the roles of properties and markers associated with stem-like cells and the epithelial to mesenchymal transition induced by chronic carcinogenesis and their suppression by green tea catechins and grape seed proanthocyanidin extract. The last section (Part-V) summarizes the findings with their importance and discusses future directions.

A terapia fotodinâmica (TFD) é uma técnica não invasiva usada no tratamento de lesões de pele, como câncer basocelular, queratose actínica, e doença de Bowen, dentre outros. Basicamente, a combinação da administração de um fotossensibilizador (FS), com a irradiação de luz adequada e o oxigênio celular, gera uma série de reações oxidativas que provocam a morte do tecido. Contudo, o principal efeito colateral desta terapia é a fotossensibilidade prolongada ocasionada pela administração de fotossensibilizadores sistêmicos. Por outro lado, a via tópica não apresenta esta limitação, pois o tratamento é realizado no local da lesão através de pró-drogas. O ácido aminolevulínico, ALA, está entre as pró-drogas mais utilizadas para indução do acúmulo do agente fotossensível na pele, a protoporfirina IX, ou PpIX. Contudo, a via tópica não permite penetração suficiente e homogênea do creme para o tratamento de lesões espessas. Visando a melhoria da TFD, foram realizados estudos in vivo e in vitro. Nos estudos in vivo, técnicas mecânicas - rolos de microagulhas, tape stripping e injeção livre de agulhas foram estudadas buscando encontrar a mais eficiente nos quesitos de: promoção da penetração da pró-droga no tecido, distribuição homogênea e de indução do acúmulo de PpIX. Para isto, foi o utilizado o modelo porcino, in vivo, conhecido como o modelo que possui a pele mais similar à pele humana. Os resultados in vivo mostram que as técnicas têm resultados similares na produção de PpIX e na distribuição de porfirina mais homogênea na superfície. Além disso, todas as técnicas estudadas in vivo têm se destacado em promover uma entrega mais homogênea de ALA também na profundidade da pele quando comparadas ao grupo controle. Nos estudos in vitro, foram examinadas possíveis diferenças na capacidade de formação da PpIX e/ou de resistência de células ao tratamento por TFD entre células expressando diferentes características de transição epitélio-mesenquimal. Os resultados in vitro indicam que as células com características epitélio-mesenquimal mais acentuadas produzem mais PpIX e são mais responsivas à TFD. Estes resultados indicam que a TFD tem maior efetividade no tratamento de células mesenquimais, e os estudos in vivo mostram que no tecido normal há maior seletividade de produção na camada da epiderme e apêndices da pele sugerindo que a terapia pode ser utilizada com maior eficiência em lesões superficiais e, até mesmo diminuir as taxas de recorrência devido a heterogeneidade de distribuição do creme na pele quando umas das técnicas mecânicas são utilizadas. / Photodynamic therapy (PDT) is a noninvasive technique used to treat skin lesions, such as basal cell cancer, actinic keratosis and Bowen\'s disease. Basically, the administration of a photosensitizer (PS), combined with the illumination of adequate light and the cellular oxygen, generate a series of oxidative reactions that cause tissue death. However, the major side effect of the treatment is prolonged photosensitivity caused by the systemic administration of photosensitizers. On the other hand, the topical therapy does not show this limitation, and it is performed at the lesion site via prodrugs. The aminolevulinic acid, ALA, is the most popular pro-drug in topical PDT. This prodrug induces PpIX production that is a photosensitive porphyrin. However, when ALA is used topically, the cream does not provide enough or homogeneous penetration for the treatment of deep lesions. Therefore, with the aim of improving PDT therapy, studies in vivo and in vitro were performed. In the in vivo analysis, mechanical techniques - microneedle roller, tape stripping, and needle-free injection- were studied looking for the most effective regarding to improve the following purposes: promoting penetration of the prodrug into the tissue, homogeneous distribution, and at inducing PpIX accumulation. The evaluations were made by fluorescence spectroscopy, biopsy of skin, and fluorescence images, using the porcine model, in vivo, known as the most similar of human skin tissue. The in vivo results showed that all techniques have similar results in the production of PpIX, and perform a more homogeneous porphyrin distribution in the skin surface. Moreover, all the techniques have excelled in promoting a homogeneous distribution of PpIX in the deep of the skin when compared to the control group. In addition to the skin penetration, studies of PpIX production were performed in vitro in cells expressing different levels of epithelial-mesenchymal transition characteristics. The studies were made in regard to a possible difference in PpIX formation capacity and / or a resistance to the PDT treatment. The in vitro results showed that cells with more epithelial-mesenchymal characteristics produce more PpIX and are more responsive to the PDT therapy. These results indicate that PDT therapy may have a better effectiveness in the treatment of mesenchymal cells and also the results in vivo showed that the ALA-induced PpIX in normal tissue seems to be selective to epidermal and skin appendages, indicating that the topical therapy may be used with a higher efficiency in superficial injuries providing lower recurrence rates when they combine with one of the techniques studied.

Como em diversos tumores sólidos, no câncer de cabeça e pescoço (HNSCC) a presença de metástases é um importante fator de mal prognóstico. Até o momento, estudos indicam que, no microambiente inflamatório tumoral, a estimulação com o Fator de Necrose Tumoral Alpha (TNF-?) leva à ativação de diferentes vias moleculares, como a via do Fator Nuclear Kappa-B (NF-kB) e PI3K/AKT, que inibem GSK3? e consequentemente, promovem a estabilização e translocação nuclear de SNAIL e betacatenina. De um modo geral, em diversos tipos de câncer, enquanto beta-catenina tem ação em promover a proliferação celular, membros da família SNAIL são capazes de induzir o processo de transição epitélio-mesenquimal (EMT). Sabe-se que os eventos de EMT estão envolvidos tanto na iniciação de metástases quanto na geração de célulastronco tumorais (CSCs), que por sua vez estão associadas à falha terapêutica e recidiva, devido à características que lhe conferem resistência aos tratamentos convencionais. Ao silenciar a expressão gênica de modo pós-transcricional, microRNAs (miRs) tem sido associados à regulação tanto da EMT quanto da geração de CSCs. Com uso da abordagem de High-Content Screening (HCS, análise celular multiparamétrica quantitativa por microscopia automatizada), buscamos investigar a capacidade de um grupo de 30 miRs humanos, muitos deles envolvidos em vias inflamatórias e na pluripotência, em modular aspectos relacionados a sobrevivência celular e EMT, em uma linhagem celular derivada de HNSCC (FADU) sob estímulo inflamatório. Inicialmente, avaliamos o potencial do TNF-? em modular parâmetros morfométricos, bem como a presença/localização de proteínas relacionadas com a EMT e capacidade migratória. Posteriormente, avaliamos o efeito de moléculas miméticas dos miRs em suprimir ou potencializar a sobrevivência celular e EMT em células estimuladas com TNF-?, seguido da identificação de transcritos alvos preditos (bem como das vias de sinalização enriquecidas para estes alvos) comumente alvejados por grupos de miRs que levaram a alterações multiparamétricas similares. De modo geral, miRs que alvejaram RELA e AKT2/AKT3 foram responsáveis pela redução na proliferação celular e EMT, enquanto o oposto foi observado em miRs que alvejaram GSK3B e ARHGAP5 (inibidor de RhoA). O silenciamento por siRNAs específicos contra RELA e CTNNB1, causou à redução na sobrevivência celular, enquanto que o silenciamento de AKT1 e CTNNB1 levou à redução na expressão proteica de SNAIL/SLUG. Finalmente, o silenciamento de RELA, AKT1, GSK3B e CTNNB1 levou a redução na sobrevivência celular e indução a apoptose mesmo na ausência de estimulação com TNF-?. Como um todo, nós demonstramos que a abordagem de HCS permitiu a identificação de miRs com efeitos fenotípicos similares (no contexto de proliferação e EMT) e que, a predição de alvos compartilhados por estes miRs, levou à identificação de alvos e vias de sinalização relevantes do ponto de vista terapêutico. / Like many solid cancers, in head and neck cancer (HNC) the presence of metastases is an important factor of poor prognosis. To date, studies indicate that, in the tumor inflammatory microenvironment, stimulation with Tumor Necrosis Factor Alpha (TNF-?) leads to the activation of different molecular pathways, such as the Nuclear Factor-Kappa B (NF-kB) and PI3K/AKT pathway, inhibiting GSK3? and the degradation of SNAIL and beta-catenin, stabilizing them promoting their nuclear translocation. In general, in several types of cancer, while beta-catenin acts to promote cell proliferation, members of the SNAIL family induce the epithelial-mesenchymal (EMT) transition process. It is known that EMT events are involved both in the initiation of metastases and generation of cancer stem cells (CSCs), which in turn are associated with therapeutic failure and relapse, due to its properties that confer resistance to conventional treatments. By silencing gene expression in a post-transcriptional fashion, microRNAs (miRs) have been associated with the regulation of both EMT and CSCs generation. Using the HighContent Screening (HCS) approach, we sought to investigate the ability of a group of 30 human miRs, many of them involved in inflammatory pathways and pluripotency, to modulate aspects related to cell survival and EMT, in a HNSCC-derived cell line (FADU) under inflammatory stimuli. Initially, we evaluated the potential of TNF-? in modulating morphometric parameters, as well as the presence/location of EMT-related proteins and migratory capacity. Subsequently, we evaluated the effect of miRs mimetic molecules on suppressing or potentiating cell survival and EMT in TNF-?-stimulated cells, followed by the identification of predicted target transcripts (as well as signaling pathways enriched for these targets) commonly targeted by groups of miRs that led to similar multiparametric changes. Overall, miRs that targeted RELA and AKT2/AKT3 were responsible for the reduction in cell proliferation and EMT, while the opposite was observed in miRs that targeted GSK3B and ARHGAP5 (RhoA inhibitor). Gene silencing by specific siRNAs against RELA and CTNNB1 caused a reduction in cell survival, while silencing of AKT1 and CTNNB1 led to reduced protein expression of SNAIL/SLUG. Finally, the silencing of RELA, AKT1, GSK3B and CTNNB1 led to a reduction in cell survival and induction of apoptosis even in the absence of TNF-? stimulation. As a whole, we demonstrated that the HCS approach allowed the identification of miRs with similar phenotypic effects (in the context of proliferation and EMT) and that the prediction of targets shared by these miRs led to the identification of relevant targets and signaling pathways from the therapeutic point of view.

Sabemos hoje que os tumores sólidos apresentam uma composição celular heterogênia e que apenas uma pequena parcela dessas células apresenta capacidade de se proliferar e gerar novos tumores. Estudos prévios sobre a formação do câncer de mama têm sido realizados com base na combinação dos marcadores de superfície celular CD44 e CD24. Já foi demonstrado que uma subpopulação de células do câncer de mama com alta expressão de CD44 e baixa expressão de CD24 (CD44+/CD24-) tem maior capacidade de gerar tumores, quando comparadas com a subpopulação de células CD44-/CD24+. O objetivo do estudo foi identificar a taxa de células com fenótipo CD44+/CD24- presentes nos tumores mamários e relaciona-la com a taxa de comprometimento dos linfonodos axilares ipsilaterais por neoplasia, além de avaliar também sua relação com outros fatores sabidamente relacionados com mal prognóstico da paciente. Pacientes e métodos: avaliamos prospectivamente 53 amostras cirúrgicas provenientes de 42 pacientes com diagnóstico histopatológico de carcinoma de mama, quantificando as células CD44+/CD24- por citometria de fluxo. Relacionamos a porcentagem destas células encontrada em cada amostra com o comprometimento axilar, os receptores hormonais e Her-2, a idade da paciente, o grau histológico do tumor, o diâmetro patológico do tumor e o tipo histológico. Resultados: verificamos um significante aumento da população de células CD44+/CD24- no grupo de carcinomas ductais invasivos em pacientes que apresentavam metástase axilar [mediana 8,53% (3,6 71,2%)] em relação ao grupo de pacientes sem linfonodos comprometidos pela neoplasia [mediana 1,49% (0,3 17,1%)] (p=0,0002). Conclusão: concluímos então que quando estudamos vários tumores mamários invasivos de mesma classificação histológica, podemos notar que existe uma variação na quantidade de células CD44+/CD24- entre eles. Nosso estudo mostrou que essa variação está relacionada à agressividade tumoral e à sua capacidade de gerar metástases já que, tumores com maior quantidade de células CD44+/CD24- apresentam maior taxa de comprometimento dos linfonodos axilares. / It is known that solid tumors are composed by a heterogeneous combination of cells and only a small portion of these cells has the capacity to proliferate and generate new tumors. Previous studies about the breast cancer initiation have been based on a combination of CD44 and CD24cell surface markers. It has been shown that this subpopulation of breast cancer cells with high expression of CD44 and low expression of CD24 (CD44+/CD24-) has a greater capacity to generate tumors when compared with the subpopulation of cells CD44- /CD24+. The study objective was to identify whether the rate of cells with CD44+/CD24- phenotype present in breast tumors is related with the rate of ipsilateral lymph node metastasis, in addition to evaluate its relationship with other risk factors known to be related with worst prognosis. Patients and methods: we prospectively evaluated 53 surgical specimens from 42 patients with histological diagnosis of breast cancer, quantifying CD44+/CD24- cells through flow cytometry. We list the percentage of these cells found in each sample with axillary lymph node status, hormone receptors and Her-2, patient age, histological grade, pathological tumor diameter and histological tumorclassification. Results: we find a significant increase of CD44+/CD24- population in the invasive ductal carcinomas, in patients with axillary metastasis [median 8.53% (3.6 - 71.2%)] than in the group of patients without lymph nodes metastasis [median 1.49% (0.3 - 17.1%)] (p = 0.0002). Conclusion: when we studied several invasive breast tumors of same histological classification, we note that there is variation in the number of CD44+/CD24- cells. Our study showed that this variation is related to tumor aggressiveness and their ability to generate metastasis, because tumors with high rate of CD44+/CD24- cells have a higher rate of lymph node metastasis.

Orientadores: José Antonio Rocha Gontijo, Flávia Fernandes Mesquita Vieira / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T00:59:05Z (GMT). No. of bitstreams: 1 Block_DanielBueno_M.pdf: 3824410 bytes, checksum: 3f680ece69f85c5b515f67c4ee1f67b4 (MD5) Previous issue date: 2013 / Resumo: O ambiente em que vivemos tem grande influência no desenvolvimento e na vida adulta do feto, sendo que a alimentação ou o tabagismo vêem como hábitos e estilo de vida que estão diretamente relacionados a modificações na organogênese fetal. O tabagismo é um dos fatores de maior preocupação das autoridades em saúde pública, devido aos graves problemas à saúde causados pelo cigarro, os custos sociais e econômicos decorrentes destas afecções e, atualmente às possíveis implicações epigenéticas dada incidência do tabagismo em gestantes que pode repercutir sobre gerações futuras. Vários estudos tratam dos efeitos danosos e das repercussões do cigarro no organismo de gestantes e no desenvolvimento do feto, tais como hipertensão arterial, doenças cardiovasculares, maior prevalência de aborto espontâneo, morbidade intrauterina , retarde no crescimento fetal, entre outros. A inalação de monóxido de carbono (CO) pelas gestantes, através do cigarro, causa no feto um estado de hipóxia que pode ser, muitas vezes, fatal. Em resposta a este déficit de oxigênio alguns mecanismos fisiológicos podem ser observados, como: o aumento da expressão do hormônio endógeno eritropoietina (EPO) que regula a eritropoiese e consequentemente, os níveis de hemoglobina e a hematose dos tecidos. O fator induzível por hipóxia (HIF-1) atua na regulação da expressão EPO, sobre a angiogênese, e na viabilidade e proliferação celular vascular entre outras funções. Nesta emaranhada rede de estímulos, está intimamente envolvido o fator de crescimento do endotélio vascular (VEGF) que tem na hipóxia um dos principais estímulos a sua expressão. Este fator é o mais importante mediador do desenvolvimento vascular renal, principalmente do processo de diferenciação do corpúsculo glomerular. Observamos que a inalação de tabaco não modificou significativamente a evolução da massa corporal das mães durante a prenhes (figura 6). No entanto, a prole de animais submetidos ao fumo apresentou uma expressiva redução da massa corporal ao nascer - Ct 7,2± 0,05DPM g vs. Fm 6,3± 0,24DPM g (figura 7), e da nona e décima semana de vida - Ct 322± 20,5DPM g vs. Fm 286± 32,3DPM g e Ct 329± 20,4DPM g vs. Fm 294± 32,6DPM g, respectivamente (figura 8). Os resultados referente à função renal na prole Ct e Fm na 5ª semana de vida não mostraram diferenças significativas na filtração glomerular (CCr) tão pouco na reabsorção proximal de sódio. Contudo, a prole Fm apresentou um aumento significativo na excreção de sódio (FENa+ 24,5%, FEK 13,8%, FEPPNa+ 25,3% e CENa+ 20%) quando comparado ao Ct (figura 13). Por outro lado, na 10ª semana de vida, observamos um aumento significativo (p=0,01) no CCr - 13,9% e na CENa+ - 17,7%, na prole Fm vs. Ct. Nestes animais não houve diferença na reabsorção de Na+ no túbulo proximal e pós-proximal, consequentemente não observamos diferenças significativas na FENa+ e FEK (figura 14). Nos animais Fm de 13 semanas de vida nenhum dos parâmetros das provas funcionais renais se alteraram (figura 15). Contudo, estritamente nesta idade podemos observar um elevação na pressão arterial (p=0,02) entre os grupos Ct e Fm - 134± 9,79DPM mmHg e 146± 11,07DPM mmHg, respectivamente (figuras 11). Não observamos modificações significativas, através da estereologia renal, no volume renal (Ct 0,12 ±0,01 vs. Fm 0,11 ±0,004), na massa renal (Ct 0,43 ±0,03 vs. Fm 0,37 ±0,01) nos animais com 12 dias de vida. Embora, não estatisticamente significativo, a prole Fm apresentou uma redução de 10% no volume glomerular (Ct 16420 ±2411 vs. Fm 15860 ±1078) e 8,2% menos glomérulos (Ct 10450 ±2030 vs. Fm 8628 ±900) quando comparados ao Ct (figuras 16 a 19). Os resultados quantitativos das proteínas envolvidas na angiogênese e eritropoiese - VEGF e EPO, dados pelo ensaio de western blotting, não apresentaram diferenças significativas entre os grupos (figuras 20 a 22). Contudo, os resultados semi-quantitativos por imunolocalização mostrou uma elevada intensidade fluorocrômica do VEGF nos animais Fm e de HIF1? nos animais Ct no período embrionário - E17 (figuras 23 a 27). Observamos, também, uma expressiva modificação na estrutura da matriz extracelular por deposição de proteínas no sitio intersticial e perivascular renal nos animais Fm de 16 semanas vida comparadas ao Ct dadas pela histoquímica de picrossíruis e imunofluorêscencia de fibronectina (figura 29 a 32). Assim, podemos concluir que, a exposição intrauterina ao fumo e seus componentes, podem levar a uma modificação morfofuncional renal na vida adulta que reflete diretamente na manutenção da pressão arterial / Abstract: Prior study about developmental plasticity hypothesis suggests that various adverse intra-uterine exposures lead to persistent fetal developmental adaptations. Maternal smoking is a very important modifiable adverse fetal exposure in western countries and leads to a decrease in the offspring's birth weight. Thus far, the specific adverse fetal smoking exposures and mechanisms underlying these associations on renal development and functional disorder are unclear. The present study investigates, in adult male rats, the effect of smoking exposure (Sk) in utero on blood pressure (BP), and its association with nephron structure and function changes. In the current study, showed in 13-week old Sk offspring enhanced arterial blood pressure, reduced nephron number are associated with higher TGF-?1 glomerular expression. Sk glomeruli also presented an upregulated collagen and fibronectin deposition intrinsically related to fibrotic process as compared to age-matched control group. From our present knowledge, these are the first data showing renal morphological and functional modifications in the gestational smoking model of fetal programming. The fetal-programmed adult rats showed structural kidney disorders associated with a striking stage of fibrosis, which led us to state that the glomerular overflow and subsequently TGF-?1 activity inducing fibrotic protein expression that may cause glomerular EMT / Mestrado / Fisiopatologia Médica / Mestre em Ciências

Une transition épithélio-mésenchymateuse (TEM) et une expression élevée de la sphingosine kinase 1 (SPHK1) sont souvent observées dans les cancers. Notre étude du génome et du transcriptome d'adénocarcinomes pulmonaires (AP) montre que l'expression élevée de SPHK1 est en rapport, d'une part, avec des gains de la région incluant le locus SPHK1 et, d'autre part, avec une signature d'expression génique de TEM dans des tumeurs invasives. L'expression de SPHK1 est restreinte aux cellules tumorales. La surexpression de SPHK1 dans des cellules d'AP et l'exposition à son produit, la sphingosine-1-phosphate (S1P), entraînent une TEM, de manière réversible pour la S1P. La surexpression de SPHK1 active aussi NF-kB. La surexpression du facteur anti-apoptotique FLIP active NF-kB, induit une TEM et augmente l'expression de SPHK1, suggérant une boucle d'amplification entre NF-kB et SPHK1. Une TEM et la surexpression de FLIP ont été impliquées dans la résistance primaire aux inhibiteurs pharmacologiques de l'EGFR (EGFR TKI). Nous montrons que la surexpression de SPHK1 dans des cellules A549 diminue modestement la sensibilité au gefitinib, alors que l'inhibition de SPHK1 ou la déplétion du sérum en S1P l'augmentent modestement. L'invalidation de SPHK1 entraîne l'apoptose d'A549 y compris quand FLIP est surexprimé. L'activation et le maintien d'une TEM sont généralement attribués à des signaux contextuels du stroma. Cette thèse montre que les cellules tumorales elles-mêmes favorisent la surexpression de SPHK1 qui peut induire une TEM de façon autonome. De plus, la surexpression de FLIP impliquée dans la résistance aux EGFR TKI, n'empêche pas l'apoptose induite par l'invalidation de SPHK1. / Epithelial-mesenchymal transition (EMT) and sphingosine kinase 1 (SPHK1) high expression are often seen in cancers. Our study of genomic and gene expression data in pulmonary adenocarcinomas (AP) shows that SPHK1 high expression correlates with both gains in the region encompassing the SPHK1 locus, and an EMT gene expression signature in invasive tumors. SPHK1 expression is restricted to tumors cells. SPHK1 overexpression in AP cells, as well as exposure to its productsphingosine-1-phosphate (S1P),induce an EMT -in a reversible manner for S1P. SPHK1 overexpression also activates NF-kB. Overexpression of FLIP – an antiapoptotic factor - activates NF-kB, induces an EMT, and increases SPHK1 expression, suggesting an amplification loop between NF-kB and SPHK1. EMT and FLIP overexpression are known to favor primary resistance to EGFR pharmacological inhibitors (EGFR TKI). We show that SPHK1 overexpression in A549 cells slightly decreases cell sensitivity to gefitinib, while pharmacologic inhibition of SPHK1 or serum S1P depletionincrease it. Downregulation of SPHK1 expression induces apoptosis of A549 cells even when FLIP is overexpressed. Activation and maintenance of EMT are generally attributed to contextual signals from the stroma. Here, we show that tumor cells themselves favor SPHK1 overexpression, which can led to EMT in cell-autonomous manner. In addition, FLIP overexpression which is implicated in EGFR TKI resistance, cannot prevent apoptosis that is induced by SPHK1 invalidation.

Le mélanome métastatique représente le plus mortel des cancers cutanés par sa forte résistance aux thérapies conventionnelles. Les cellules initiatrices de tumeurs (CIT) sont présentes dans de nombreux cancers dont le mélanome. Ces cellules, capables de s’autorenouveller, sont à l’origine de la récidive tumorale et des métastases représentant une cible pour le développement de nouveaux traitements. Les CIT sont confinées dans un microenvironnement tumoral dans lequel des facteurs sécrétés tels que l'Insulin-Like Growth Factor-1 (IGF-1) et le Transforming growth factor (TGF-β) favorisent la transition épithéliomésenchymateuse (TEM), un processus clef lié à l’émergence des CIT. En utilisant des cellules de mélanome métastatique, nous avons montré qu’une inhibition d’IGF-1 induit une diminution de la tumorigénicité des cellules en diminuant la capacité des B16-F10 à former des métastases pulmonaires. Outre son action sur la prolifération cellulaire, IGF-1 est impliqué dans le processus de TEM favorisant les propriétés migratoires et invasives des cellules B16-F10. Par ailleurs, IGF-1 joue un rôle majeur dans le maintien des CIT expliquant la forte résistance des mélanomes aux thérapies conventionnelles. Des expériences préliminaires suggèrent que ces activités induites par IGF-1 pourraient être médiées en partie par le facteur TGF-β, un facteur clef de la TEM. D'autres résultats confortent cette hypothèse en montrant une implication directe du TGF-β dans le caractère souche des cellules B16-F10. Ces travaux montrent que l’inhibition de la voie IGF-1/IGF-1R dans le microenvironnement tumoral pourrait être une bonne stratégie pour le développement de traitements anti-tumoraux contre le mélanome. / Metastatic melanoma is arguably the most virulent among human cancers, owing to its propensity to metastasize, and its resistance to conventional therapies. Like in many other cancers, tumor stem cells or tumor initiating cells (TIC), have been identified in melanoma. These cells have the unique ability to self-sustain and renew the tumor and thus represent an interesting target for the development of new therapeutic strategies. TIC are nested in a confined microenvironment where secreted-factors such as Insulin-Like Growth Factor- 1 (IGF-1) and transforming growth factor (TGF-β) promote epithelialmesenchymal transition (EMT), a key process in stemness features acquisition. In this context, we investigated the effects IGF-1 on TIC behavior. Using B16-F10 metastatic melanoma cell line, we show that IGF-1 downregulation curbs lung metastasis suggesting that IGF-1 plays a direct role in the intrinsic tumorigenic potential of these cells.markers associated with an increased expression of the epithelial marker E-cadherin and of the major regulator of melanocyte differentiation MITF. Most importantly, IGF-1 inhibition sharply decreased stemness features, reducing the expression of key stem markers and functional characteristics of MIC. This was associated with an important sensitivity to mitoxantrone treatment. Interestingly, our preliminary data suggest the EMT key component, TGF-β, conveys IGF-1-mediated effects. Indeed, TGF-β directly affects B16-F10 stemness phenotype and markers. In summary, we show that the IGF-1/IGF-1R nexus represents an interesting target for the development of novel therapeutic strategies against metastatic melanoma.

Les canaux anioniques ont toujours été sous-estimés par rapport aux canaux cationiques principalement en raison des difficultés à caractériser leurs identités moléculaires et par l'absence d'inhibiteurs sélectifs puissants. C’est une classe comportant une grande hétérogénéité et une importante complexité comprenant entre autres le CFTR activé par l’AMP cyclique, les anoctamines activées par le calcium intracellulaire et les canaux VRAC activés lors d’un gonflement cellulaire. L’identité moléculaire d’une protéine essentielle aux courants ioniques déclenchés par le gonflement cellulaire a été découverte en 2014, permettant de pouvoir enfin investiguer le fonctionnement et le rôle de ces canaux. Dans une première partie, nous avons démontré l’absence de spécificité des inhibiteurs des principaux canaux chlorures. Cette étude met en exergue le besoin de développer de nouveaux inhibiteurs permettant de discriminer les différents canaux chlorures, mais aussi la nécessité de coupler l’approche pharmacologique à une approche génétique. Dans une deuxième partie, nous avons mis en évidence la perméabilité des canaux anioniques sensibles au gonflement cellulaire au glutathion, principal antioxydant cellulaire. Nos résultats suggèrent donc que les canaux VRAC pourraient être impliqués dans la régulation du stress oxydatif. Dans la troisième partie de ma thèse, nous avons montré que le facteur de croissance TGFβ1 stimule l’ouverture des canaux anioniques sensibles au gonflement cellulaire. Cette activation s’accompagne d’une perte de glutathion et d’une accumulation intracellulaire d’espèces réactives de l’oxygène. Cette dernière est à l’origine d’une transition épithélio-mésenchymateuse des cellules rénales. L’inhibition pharmacologique ainsi que la répression génétique des canaux VRAC réduit significativement les effets du TGFβ1. Enfin dans la dernière partie, nous avons caractérisé le rôle des canaux anioniques sensible au gonflement cellulaire dans la sécrétion de facteurs pro-inflammatoires par les macrophages stimulés par un choc hypotonique. En collaboration, nous avons montré que le blocage de la régulation du volume cellulaire, sous le contrôle de ces canaux, entraine une inhibition de l’activation de l’inflammasome et donc de la sécrétion des facteurs pro-inflammatoires. Bien que les mécanismes de signalisation cellulaire restent à déterminer, ces travaux mettent en lumière l’importance des canaux anioniques sensibles au gonflement cellulaire dans la transition épithéliomésenchymateuse et l’inflammasome. Il apparait que ces canaux pourraient être des cibles thérapeutiques prometteuses dans un certain nombre de pathologies et qu’il est primordial de poursuivre les investigations dans ce domaine. / Anion channels have always been underestimated compared to cationic channels mainly because of the difficulty in characterizing their molecular identities and the absence of potent selective inhibitors. It is a class with great heterogeneity and complexity, including CFTR activated by cAMP, anoctamines activated by intracellulaire calcium and VRAC channels activated by cell swelling. The molecular identity of an essential protein to the ionic currents triggered by cell swelling was discovered in 2014, allowing the scientist community to investigate the role of these channels. In a first part, we highlighted the lack of specificity of the inhibitors of different chloride channels. This study shed lights on the need to develop new inhibitors to discriminate the different chloride channels, but also the need to couple the pharmacological approach to a genetic approach. In a second part, we have demonstrated the glutathione permeability of Volume Regulated Anion Channels. Our results therefore suggest that VRAC channels might be involved in the regulation of oxidative stress as glutathione is the main antioxidant within the cell. In the third part of my thesis, we have shown that TGFβ1 growth factor stimulates the opening of Volume Regulated Anion Channels which is correlated with both a loss of glutathione and an intracellular accumulation of reactive oxygen species. The latter is at the origin of an epitheliomesenchymal transition of renal cells. The pharmacological inhibition as well as the genetic repression of the VRAC channels significantly reduces the effects of TGFβ1. Finally, in the last part, we characterized the role of Volume Regulated Anion Channels in the secretion of pro-inflammatory factors by macrophages stimulated by hypotonic shock. In collaboration, we have shown that blocking the regulation of cell volume, under the control of these channels, leads to an inhibition of the inflammasome and therefore of the secretion of pro-inflammatory factors. Although cell signaling mechanisms remain to be determined, this work highlights the importance of VRAC in the epithelial-mesenchymal transition and inflammasome. It appears that these channels could be promising therapeutic targets in a certain number of pathologies and that it is essential to continue the investigations in this field.