Estudos mostram que a exposição ao meio hiperglicêmico ou diabetes protege o coração contra insultos patológicos, incluindo isquemia. A ativação de fatores anti-apoptóticos e proliferativos parece estar envolvida com esta cardioproteção. Este estudo foi desenhado para investigar a modulação da viabilidade de fibroblastos cardíacos submetidos à hipóxia tratados previamente com meio hiperglicêmico, e os efeitos de 15 dias de infarto do miocárdio (IM) na função ventricular em ratos diabéticos sobre os fatores de morte e sobrevida celular. Foram utilizados ratos Wistar machos e as análises foram realizadas no ventrículo esquerdo. Para as análises in vitro, os fibroblastos cardíacos foram obtidos por digestão enzimática (tripsina/colagenase), e cultivados em dois meios: baixa Glicose (5mM) e alta Glicose (25mM análogo ao plasma diabético). As células confluentes (80%) foram submetidas à hipóxia por incubadora modular com gás (5,6% de CO2 e 94,4% de N2); após as células foram mantidas em estufa a 37ºC por 6; 12; 24; 48 e 72hs. Experimentos de citometria de fluxo foram realizados para análise da viabilidade celular e fragmentação de DNA, ambas para verificação de apoptose e confirmadas pelas medidas de AnexinaV/FITC/IP. A expressão do fator de crescimento endotelial vascular (VEGF) foi medida no sobrenadante dos meios por ELISA. Para as análises in vivo, os ratos foram divididos (N=8/grupo) em grupos: controle (C), diabético (D), infartado (I) e diabético infartado (DI). Quinze dias após a indução do diabetes ou não, com injeção de estroptozotocina (50mg/Kg) ou tampão citrato, os grupos I e DI foram submetidos à ligadura da artéria coronária. Após 15 dias de IM e/ou 30 de diabetes, a função cardíaca (sistólica e diastólica) foi analisada por ecocardiograma, além da avaliação do tamanho do IM em 24 horas e 15 dias. As citocinas inflamatórias foram medidas por ELISA, a expressão gênica dos fatores de morte e sobrevida celular foram avaliados por PCR em tempo real, a atividade da caspase 3 foi medida por espectrofluorimetria, e a expressão protéica dos transportadores de glicose (GLUT) por Western blotting. A densidade capilar e a fibrose do coração foram quantificadas por análise histológica. A viabilidade celular mostrou homogenia em todos os tempos de hipóxia e em ambos os tratamentos de glicose. Em 6 horas verificamos fragmentação do DNA com marcação dos fosfolípides de membrana (apoptose) e % elevada de morte em ambos os tratamentos até 12hs. Após 24hs, ocorreu adaptação, havendo aumento de VEGF e diminuição da fragmentação do DNA com 48hs; após 72hs, observou-se apenas que o VEGF continuava aumentado. No meio hiperglicêmico, os valores de VEGF estavam maiores e a fragmentação menor quando comparados ao meio hipoglicêmico. O grupo DI apresentou melhora na função sistólica (fração de ejeção e de encurtamento), acompanhada de manutenção da função diastólica (tempo de relaxamento isovolumétrico e tempo de desaceleração do pico E), redução de 36% do tamanho do IM, redução das citocinas pró-inflamatórias (TNF- e IL-1), com ativação da apoptose (expressão aumentada de Fas, p53, Bax e atividade de caspase 3) e aumento dos fatores de sobrevivência celular (Bcl-2, HIF-1, VEGFa e IL8r). Além disso, o grupo DI apresentou aumento do GLUT-1 com diminuição do escore de fibrose e aumento da densidade capilar; resultados comparados ao grupo I. Este cenário é, provavelmente, um mecanismo de compensação, associado ao saldo positivo entre genes regulatórios relacionados com a sobrevivência celular programada, redução de citocinas inflamatórias, aumento da utilização de glicose como substrato energético, redução da fibrose e angiogênese. Em conjunto, estes achados sugerem uma maior plasticidade e resistência celular de fibroblastos cardíacos frente à hipóxia sendo esta favorecida pela hiperglicemia crônica em ratos diabéticos / Studies showed that exposure to hyperglycemia or diabetes protects the heart against pathological insults, including ischemia. The activation of anti-apoptotic factors and proliferation seem to be involved in this cardioprotection. This study was designed to investigate the feasibility of modulation of hyperglycemic-environment pretreated-cardiac fibroblasts subjected to hypoxia, and the effects of 15 days of myocardial infarction on ventricular function in diabetic rats and on the causes of death and cell survival. Male Wistar rats were used and the analyses were performed in the left ventricle. For in vitro experiments, cardiac fibroblasts were obtained by enzymatic digestion (trypsin/collagenase) and they were cultivated in two different mediums: Low Glucose (5mM) and High Glucose (25mM similar to diabetic plasma). Confluent cells (80%) were subjected to hypoxia by modular incubator with gas (5.6% CO2 and 94.4% N2).at 37 ° C for 6, 12, 24, 48 and 72h. Flow cytometry was employed to the analysis of cell viability and DNA fragmentation, both for verification of apoptosis process and confirmed by AnexinaV / FITC / IP. The expression of vascular growth factor (VEGF) was measured in the supernatant medium by ELISA. For in vivo experiments, the rats were divided (N = 8/group) in control (C), diabetic (D), infarcted (I) and diabetic infarcted (DI).groups After 15 days of induction of diabetes by estreptozotocin (50mg/kg) or citrate buffer, the I and DI groups underwent coronary artery ligation. After 15 days of myocardium infarction (MI) and/or 30 of diabetes, cardiac function (systolic and diastolic) was analyzed by echocardiogram, as well as the size of infarction at both, 24 hours and 15 days. The inflammatory cytokines were measured by ELISA, the gene expression of factors for death and cell survival by real-time PCR, the activity of caspase 3 by spectrofluorimetry, the protein expression of glucose transporters (GLUT) by Western- blotting and, lastly, capillary density and fibrosis by histological analysis. Cell viability showed homogeneous at all times of hypoxia and in both glucose treatments. In 6 hours we found DNA fragmentation in the main phospholipid membrane (apoptosis) and high % of death in both treatments until 12 hours. After 24 hours, the cells entered into adaptation and after 48 hours it was verified an increase in VEGF expression and a decrease in DNA fragmentation. At 72 hours, no significant differences in the fragmentation were observed; however, there was an increase in VEGF expression. In the hyperglycemic medium we observed higher values of VEGF expression and less DNA fragmentation when compared to the hypoglycemic medium. In the DI group systolic function (% ejection fraction and fractional shortening) was better than in the I group and it was accompanied by the maintenance of diastolic function (IVRT and deceleration time of peak E). There was also a 36% reduction in infarction size in the DI group and reduced pro-inflammatory cytokines (TNF- and IL-1) with activation of apoptosis (increased expression of Fas, p53, Bax and caspase 3 activity) and increased cell survival factors (Bcl-2, HIF-1, VEGF and IL8r). Moreover, DI rats also showed an increase in GLUT-1 with decreased scores of fibrosis and increased capillary density when compared to the I group. This scenario is probably a compensatory mechanism associated with the positive balance of regulatory genes related to programmed cell survival, reduction of inflammatory cytokines, increased use of glucose as energy substrate, reduction of fibrosis and angiogenesis. Altogether, these findings suggest a greater plasticity and cellular resistance of cardiac fibroblasts to hypoxia and this is favored by chronic hyperglycemia in diabetic rats.

Pentoxifilina (PTF), um derivado da metilxantina, tem ação terapêutica como agente antifibrótico. In vitro, a PTF causa inibição na produção de colágeno, glicosaminoglicanos e fibronectina, bem como promove acentuada redução na proliferação dos próprios fibroblastos de quelóides. A PTF na concentração de 1.000 g/mL foi seletiva no controle da inibição da síntese protéica pelos fibroblastos. O objetivo deste estudo foi verificar o comportamento in vitro de fibroblastos oriundos de cicatrizes hipertróficas (HSHF) e de pele não-cicatricial (NHF) na presença e ausência de PTF (1.000 g/mL), quanto à: proliferação celular, produção de colágeno e capacidade dos fibroblastos gerarem contração em modelo experimental de matriz de colágeno. Para tanto, foram utilizados fibroblastos cultivados a partir de amostras de cicatrizes hipertróficas e pele não-cicatricial doadas, com finalidade de pesquisa, pelo banco de Tecidos do Instituto Central do Hospital das Clínicas da FMUSP. Culturas celulares expostas a PTF apresentaram diminuição na proliferação celular em HSHF (46,35%) e em NHF (37,73%) (p<0,0001). Na presença de PTF, foi observada seletividade de inibição na síntese de colágenos, havendo inibição mais expressiva de colágeno tipo III em HSHF e de colágeno tipo I em NHF (p<0,0001). O modelo experimental de matriz de colágeno povoada por fibroblastos de cicatriz hipertrófica apresentou contração menor (12%) na presença de PTF (p<0,0001), em relação à sua ausência / Fibroblasts are thought to be partially responsible for the persisting contractile forces that result in burn contractures. Using a monolayer and fibroblast populated collagen lattice (FPCL) three-dimensional (3D) model we subjected hypertrophic scar and non-cicatricial fibroblasts to the antifibrogenic agent pentoxifylline (PTF) 1000g/mL attempting to reduce proliferation, collagen type I and III synthesis and contraction in this 3D model. Fibroblasts were isolated from post burn hypertrophic scars (HSHF) and non-scarred skin (NHF). Cells were grown in monolayer or incorporated into FPCL\'s and exposed to PTF. In monolayer, cell number proliferation was reduced (46.35% in HSHF group and 37.73% in NHF group) p<0,0001. The PTF also demonstrated to be selective on collagen type I and III synthesis inhibition suggesting higher inhibition of collagen type III on HSHF group and more evident inhibition of type I on NHF group. FPCL\'s containing PTF had surface areas reduced in about 12% p<0,0001. PTF showed inhibition effects on cell proliferation and reduced contraction in both HSHF and NHF

Made available in DSpace on 2014-08-20T14:30:05Z (GMT). No. of bitstreams: 1 dissertacao_Rodrigo_Varella_de_Carvalho.pdf: 598355 bytes, checksum: 322409e41faaa4fcdb65cd8f7b7e200d (MD5) Previous issue date: 2006-05-29 / The aim of the present study was to evaluate the gelatinase activity (MMP-2 and 9), and proliferation of human pulp fibroblasts in vitro, after irradiation with a low level diode laser (780nm). The activity was evaluated with zymography and the proliferation rate was obtained through growth curves made in SigmaPlot (8.0), after the counting in Neubauer chamber.in light microscope. Cells used to zimography were cultived in DMEM, 10% fetal bovine serum and incubated at 37ºC in atmosphere of 5% CO2 and 95% air, in 100% humidity. Cells used to proliferation rate were cultivated in nutritional deficit (5% SFB). Groups to zymography and cell proliferation were determined: zymography - Z1 = not irradiated, Z2 = 3J/cm2 e Z3 = 6J/cm2, cell proliferation: L1 = not irradiated, L2 = 3J/cm2 e L3 = 6J/cm2. The counting was made after 2, 4 and 6 days. Assays were performed in triplicate. The zymography showed that the MMP-2 activity was major with MMP-9, and, Z2 e Z3 produced a higher activity than MMP-2, when compared to Z1. Results of the zymography and proliferation test were compared by either ANOVA complemented by Tukey´s test. The level of significance was 5% (p<0,05). Statistical analysis demonstrate difference between the groups in different days. The L3 obtained higher proliferation in the finish of the six days. L2 demonstrated higher growth than L1. Irradiated fibroblasts with the low power laser expressed a higher amount of MMP-2 activity and 6J/cm2 produced a higher proliferation rate in the initial days / O objetivo do presente estudo foi avaliar a atividade das gelatinases (MMP-2 e MMP-9), e a proliferação de fibroblastos pulpares humanos in vitro, após a irradiação com um diodo laser de baixa de baixa potência (780nm). A atividade foi avaliada com zimografia e as taxas de proliferação e contagem celular foram obtidas através de curvas de crescimento feitas com o software SigmaPlot (8.0), após a contagem em câmara de Neubauer ao microscópio óptico. As células usadas para a zimografia foram cultivvadas em DMEM, com SFB a 10% e incubadas a 37ºC em atmosfera de 5% CO2 e 95% de ar, em 100% de umidade. Já, as células usadas para a taxa de proliferação e contagem celular foram cultivadas em déficit nutricional (5% SFB). Os grupos para zimmografia e para a contagem e taxa de proliferação foram determinados: a) zimografia - Z1 = sem irradiação, Z2 = 3J/cm2 e Z3 = 6J/cm2 e b) proliferação e contagem celular - L1 = sem irradiação, L2 = 3J/cm2 e L3 = 6J/cm2. A contagem celular foi realizada em após 2, 4 e 6 dias. Os ensaios foram realizados em triplicata. A zimografia mostrou que a ativiidade da MMP-2 foi maior que a MMP-9, e Z2 e Z3 produziram uma grande atividade, quando comparados ao rupo Z1. Os resultados da zimografia e dos testes de contagem e proliferação celular foram comparados por análise de variância (ANOVA) e complementado pelo teste Tukey. O nível de significância utilizado foi de % (p<0,05). A análise estatística demonstrou diferença entre os grupos nos diferentes dias para a proliferação e contagem celular. O grupo L3 obteve a maior contagem celular ao final dos seis dias avaliados. O grupo L2 demonstrou maior proliferação que o grupo L1. Os fibroblastos irradiados com o laser de baixa potência expressaram uma alta atividade de MMP-2 e a dose 6J/cm2 produziu uma alta taxa de proliferação nos primeiros dias

Made available in DSpace on 2014-08-20T14:30:16Z (GMT). No. of bitstreams: 1 Dissertacao_Laura_Siqueira_Pintado.pdf: 1321397 bytes, checksum: a54cf33fb1a88d6f8f068fcdad1d0588 (MD5) Previous issue date: 2011-08-26 / Aiming at the success of conservative pulp treatment, the MTA (mineral trioxide aggregate) has been considered the first choice product for performing direct pulp capping. It has features such as biocompatibility, ability to stimulate the formation of dentin and cellular proliferation, among others. However, drawbacks such as difficulty of handling after the manipulation and extended setting times stimulated new researches in an attempt to improve such characteristics. Thus, a light-cured cement-based MTA was developed at the CDC-BIO in the Faculty of Dentistry of the Federal University of Pelotas. It is suggested that the presence of resin monomers may have a deleterious effects on the dental pulp. Based on this fact, the study evaluated the cytotoxicity and genotoxicity of the experimental MTA compared with the conventional commercially available as well as their physical and mechanical properties. Primary cultures of human pulp fibroblasts (HPF) and one strain of mouse fibroblasts 3T3/NIH were employed. Pulp of extracted sound human third molars was used for the pulpal fibroblast cell culture. Specimens were made from the two materials and embedded in 1 mL of DMEM for obtaining the test eluate. For cytotoxicity, cells were incubated for 24 or 48 hours with the eluate and evaluated by the MTT assay [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide]. To assess the genotoxicity of the products through micronucleus development the Feulgen staining technique was employed after cell incubation for 24 hours with the eluate. For the evaluation of physical and mechanical properties of the experimental MTA diametral tensile strength and sorption and solubility tests were performed. The results were analyzed by ANOVA and Kruskal Wallis tests considering p <0.05. The light-curing MTA showed cytotoxic effects similar to conventional MTA in the two cell lineages. The percentage of micronuclei was different between the materials, but both were similar to control. The diametral tensile strength was lower for the experimental MTA. The conventional MTA was more soluble and both had the same behavior in relation to water sorption. The results suggest that the experimental light-cured MTA had similar behavior and biocompatibility comparable to the commercially available MTA and is a promising material for pulp capping / Visando o sucesso do tratamento conservador da polpa, o MTA (agregado trióxido mineral) tem sido considerado o produto de primeira escolha para a realização de capeamento pulpar direto. Apresenta características como biocompatibilidade, capacidade de estimular a formação de dentina e proliferação celular, entre outros. No entanto, os inconvenientes como a dificuldade de manuseio após a manipulação e longo tempo de presa tem estimulado novas pesquisas na tentativa de melhorar essas características. Assim, um novo cimento fotopolimerizável baseado em MTA (MTA F) foi desenvolvido na Faculdade de Odontologia da Universidade Federal de Pelotas. Sugere-se que a presença de monômeros resinosos pode ter um efeito deletério sobre a polpa dentária. Baseado neste fato, o estudo avaliou a citotoxicidade e genotoxicidade do MTA F em comparação ao convencional disponível no mercado bem como suas propriedades físico-mecânicas. Foram utilizadas culturas primárias de fibroblastos de polpa humana (FPH) e uma linhagem de fibroblastos de camundongo 3T3/NIH. Polpa extraída de terceiros molares humanos íntegros foi utilizada para a cultura de fibroblastos pulpares. Os corpos de prova de ambos os materiais foram embebidos em 1 mL de DMEM para a obtenção do eludato teste. Para citotoxicidade, as células foram incubadas por 24 ou 48 horas com o eludato e avaliado pelo ensaio de MTT [3 - (4,5-dimetil-2-il) -2,5-brometo difeniltertrazolim]. Para avaliar a genotoxicidade dos produtos através da técnica de micronúcleos, foi utilizada a técnica de coloração de Feulgen após a incubação de células por 24 horas com o eludato. Para a avaliação das características físico-mecânicas do MTA experimental foram realizados os testes de resistência a tração diametral e sorção e solubilidade. Os resultados foram analisados pelos testes ANOVA e Kruskal Wallis considerando p<0,05. O MTA fotopolimerizável apresentou efeitos citotóxicos semelhantes ao MTA convencional nas duas linhagens celulares. O percentual de micronúcleos foi diferente entre os materiais, porém ambos foram semelhantes ao controle. A resistência a tração diametral do MTA experimental foi inferior ao MTA comercial. O MTA convencional foi mais solúvel e ambos tiveram o mesmo comportamento em relação à sorção de água. Os resultados sugerem que o MTA experimental fotopolimerizável teve comportamento e biocompatibilidade semelhante ao MTA disponível comercialmente, sendo um material promissor para o capeamento pulpar

Nous nous intéressons aux mécanismes moléculaires impliqués dans la physiopathologie de la fibrose pulmonaire idiopathique. La fibrose pulmonaire estcaractérisée par l’accumulation de protéines de la matrice extracellulaire et de fibroblastes dans les espaces aériens distaux. La désorganisation et la destructionalvéolaires qui résultent de la fibrose aboutissent à une altération des propriétés mécaniques du poumon et à une incapacité à réaliser les échanges gazeux responsables d’une insuffisance respiratoire parfois mortelle. Le pronostic de la fibrose pulmonaire idiopathique (FPI) est particulièrement mauvais puisque la médiane de survie est de 3 à 5 ans. Il n’existe actuellement aucune thérapeutique efficace dans la fibrose pulmonaire. Ainsi, il est crucial d’explorer de nouvelles hypothèses physiopathologiques dans cette maladie afin d’ouvrir de nouvelles voies thérapeutiques. L'apoptose joue un rôle clé dans le développement de nombreux organes et dans l'homéostasie tissulaire chez l'adulte. Les protéines de la famille BCL-2 sont des éléments essentiels de la machinerie apoptotique. Ces protéines agissent comme des régulateurs anti- ou pro-apoptotiques. Parmi les membres de la famille BCL-2, le facteur pro-apoptotique BAX contrôle la voie mitochondriale de l’apoptose. Des perturbations de l’apoptose ont été mises en cause dans des maladies pulmonaires comme la fibrose pulmonaire idiopathique. Des études récentes suggèrent fortement que les protéines de la famille BCL-2 sont également impliquées dans d’autres fonctions cellulaires que le contrôle de l'apoptose. De plus, la protéine BAX est aussi localisée dans le noyau de nombreux types cellulaires. Même si le rôle de la fraction cytoplasmique de BAX au cours de l’apoptose est assez bien caractérisé, les fonctions nucléaires de BAX ne sont pas connues. Ce travail de thèse a pour but de mieux comprendre le rôle de la forme nucléaire de BAX dans différents processus cellulaires fondamentaux impliqués dans la fibrogenèse. Notre étude montre que la protéine BAX est présente dans le noyau à proximité de l’euchromatine dans différentes lignées d’origine pulmonaire in vitro. Ensuite, nous montrons que la forme nucléaire de BAX est impliquée dans la progression du cycle cellulaire et dans le contrôle de l’état de différenciation myofibroblastique en condition basale. Enfin, nous avons détecté la forme nucléaire de BAX dans l’épithélium hyperplasique et les foyers de fibrose dans le poumon de FPI. / Summary not transmitted

Les tumeurs sont constituées de deux groupes cellulaires :les cellules tumorales et les cellules dites « hôtes ». Au sein de ce dernier groupe, on retrouve les cellules endothéliales, les cellules inflammatoires mais également les fibroblastes associés au cancer (FAC). De plus en plus de données dans la littérature suggèrent l’importance des FAC dans l’invasion tumorale et le développement métastatique. De nombreuses études immunohistochimiques ont démontré le rôle pronostique des FAC dans différents cancers. Les traitements néoadjuvants modifient l’environnement tumoral mais l’impact de ces traitements sur les FAC est assez méconnu.Notre travail porte, tout d’abord, sur une revue de littérature à propos de l’impact pronostique négatif des FAC dans différents cancers. Ensuite, nous avons étudié le ratio des FAC SMA+/surface épithéliale tumorale dans une cohorte de patients ayant été opérés d’un adénocarcinome rectal ayant bénéficié ou pas d’une thérapie néoadjuvante. La comparaison du ratio entre les groupes traités et non traités a montré un ratio plus élevé chez les patients ayant bénéficié d’une thérapie néoadjuvante. Par ailleurs, les tumeurs présentant un ratio élevé présentent également un indice de prolifération faible suggérant que la thérapie néoadjuvante modifie l’environnement tumoral par une majoration des FAC aux dépens des cellules tumorales et par l’acquisition d’un phénotype quiescent des cellules tumorales. Finalement, chez les patients traités par une thérapie néoadjuvante, un ratio supérieur à un est associé à une survie sans récidive mauvaise.Nous nous sommes ensuite intéressés à la protéine FHL2 qui est exprimée par les FAC mais également par les cellules tumorales. L’étude de l’expression immunohistochimique de la protéine FHL2 sur un tissue microarray de cancers coliques a démontré que celle-ci est exprimée fréquemment dans les FAC de cancers coliques alors que l’expression dans les cellules tumorales est plus variable. Nous avons démontré que les tumeurs coliques exprimant fortement FHL2 sont associées à un plus mauvais pronostic (survie et survie sans récidive). Ce résultat est probablement lié à l’implication de cette protéine dans la transition épithélio-mésenchymateuse.Finalement, nous avons étudié la possibilité d’une interaction entre FHL2 et ADAM-17. Nous avons démontré que cette interaction est plus fréquente dans le cancer colique comparé au tissu colique normal. Ce dernier résultat ouvre la voie vers un partenaire potentiel de FHL2 qui pourrait notamment interférer avec la voie de signalisation de l’EGF. / Tumours are composed of two cellular groups: the tumoural cells and the host cells. In this latter, we find endothelial cells, inflammatory cells but also the cancer-associated fibroblasts (CAFs). More and more litterature data suggest a key role of CAFs in the tumoural invasion and in the metastatic development. Several immunohistochemical studies have highlighted the prognostic role of CAFs in different cancers. Neoadjuvant treatments modulate the tumoural environment but the impact of such treatment on the CAFs is relatively unknown.In the first part of this thesis we revise the current knowledge on the adverse prognostic impact of CAFs in different cancers. We studied by immunohistochemistry the ratio CAFs SMA+/tumoural epithelial area within a patient cohort operated for rectal adenocarcinoma receiving or not a neoadjuvant treatment. The comparison of the ratio between the treated group and the non-treated group showed that this ratio is higher in patients treated with neoadjuvant therapy. Moreover, rectal cancer with a high ratio displayed a lower proliferation rate suggesting that the neoadjuvant treatment modifies the tumoural environment by an increase of CAFs and by acquisition of a quiescent phenotype of the tumoural cells. In the group of patients treated with neoadjuvant treatment, a ratio >1 was associated with an adverse impact on recurrence free survival.Secondly, we studied Four-and-a-half LIM Domain protein 2 (FHL2) which is a protein expressed by CAFs but also by the tumoural cells. Immunohistochemical study of FHL2 expression on a colon cancer tissue microarray demonstrated that FHL2 is frequently expressed in colon cancer CAFs while its expression is variable in the tumoural cells. We demonstrated that high FHL2 expression in colon cancer is related to poor prognosis (overall survival and metastasis free survival). Aggressive behaviour in such tumours might be related to the implication of FHL2 in epithelial-to-mesenchymal transition.Finally, we studied a possible interaction between FHL2 and ADAM-17. We demonstrated that this interaction is more frequent in colon cancer compared to normal colonic tissue, suggesting a role for it in colon cancer development. This interaction possibly interferes with the EGF pathway / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

Bipolar disorder and schizophrenia are two severe psychiatric disorders characterized by a complex genetic basis, coupled to the influence of environmental factors. In this thesis, functional genomic analysis tools were used for the study of the underlying pathophysiology of these disorders, focusing on gene expression and function on a global scale with the application of high-throughput methods. Datasets from public databases regarding transcriptomic data of postmortem brain and skin fibroblast cells of patients with either schizophrenia or bipolar disorder were analyzed in order to identify differentially expressed genes. In addition, fibroblast cells of bipolar disorder patients obtained from the Biobank of the Neuropsychiatric Research Laboratory of Örebro University were cultured, RNA was extracted and used for microarray analysis. In order to gain deeper insight into the biological mechanisms related to the studied psychiatric disorders, the differentially expressed gene lists were subjected to pathway and target prioritization analysis, using proprietary tools developed by the group of Metabolic Engineering and Bioinformatics, of the National Hellenic Research Foundation, thus indicating various cellular processes as significantly altered. Many of the molecular processes derived from the analysis of the postmortem brain data of schizophrenia and bipolar disorder were also identified in the skin fibroblast cells. Additionally, through the use of machine learning methods, gene expression data from patients with schizophrenia were exploited for the identification of a subset of genes with discriminative ability between schizophrenia and healthy control subjects. Interestingly, a set of genes with high separating efficiency was derived from fibroblast gene expression profiling. This thesis suggests the suitability of skin fibroblasts as a reliable model for the diagnostic evaluation of psychiatric disorders and schizophrenia in particular, through the construction of promising machine-learning based classification models, exploiting gene expression data from peripheral tissues.

Abstract Tissue repair and cancer progression involve similar mechanisms, including degradation of extracellular matrix in which matrix metalloproteinases (MMPs) play essential roles. The action of MMPs is important in normal physiological processes but MMPs also contribute to various pathological conditions. MMP-8 belongs to a family of collagenases with a diverse set of substrates. MMP-8 action is involved in skin wound healing and in various human cancers. The function of MMP-8 in cancer appears to be highly complex and varies depending on the cancer type and location. Little is known about the involvement of MMP-8 in oral physiology and pathology. The aim of this study was to clarify the role of MMP-8 in oral tissue repair and oral tongue squamous cell carcinoma (OTSCC). Studies with MMP-8 deficient mice revealed that the function of MMP-8 in tissue repair is highly dependent on the spatial aspects. In alveolar bone, MMP-8 increased inflammation and affected collagen metabolism. In tongue wounds, MMP-8 impaired early healing and reduced transforming growth factor (TGF) -β1 levels. This study also revealed the protective role of MMP-8 in OTSCC patients, in agreement with previous studies indicating positive features of MMP-8 in cancer. Low MMP-8 level and high vascular endothelial growth factor (VEGF) -C levels in tumors correlated with worse prognosis in these patients. In mouse tongue fibroblast cell cultures, MMP-8 reduced TGF-β1 signaling molecule phosphorylated Smad2 levels and impaired the collagen contraction ability. TGF-β1, apoptosis factor Fas-ligand (Fas-L) and estrogen receptors (ERs) were identified as novel MMP-8 substrates. In OTSCC cell cultures, MMP-8 impaired cell migration and invasion. Diminished TGF-β1 levels were involved in the defective migration of MMP-8 overexpressing cells. Moreover, MMP-8 affected the expression of MMP-9, MMP-1, cathepsin-K, VEGF-C and TGF-β1. In mouse models of OTSCC, MMP-8 protected against tumor development but was not able to prevent metastasis formation. The main findings of this study were that 1) MMP-8 action in tissue repair depends on the site of the injury and 2) in OTSCC, MMP-8 has tumor suppressive effects, but in mouse, MMP-8 does not inhibit metastasis formation. In addition, 3) four novel MMP-8 substrates (TGF-β1, Fas-L, ER-α and -β) were identified that may explain the spatial and diverse roles of MMP-8. / Tiivistelmä Kudosvaurioiden paranemiseen ja syövän etenemiseen liittyy useita samankaltaisia mekanismeja. Molemmissa toimivat soluvälitilan muokkaamiseen osallistuvat proteaasit, joista matriksin metalloproteinaaseilla (MMP) on tärkeä merkitys; ne osallistuvat lukuisiin elimistön keskeisiin fysiopatologisiin prosesseihin. Kollagenaasi MMP-8 muokkaa monentyyppisiä molekyylejä. Se on mukana ihohaavan paranemisessa ja useissa syövissä. MMP-8:n toiminta syöpätiloissa on hyvin moninainen riippuen syöpätyypistä ja sijainnista. Väitöstutkimuksessa selvitettiin MMP-8:n merkitystä suun kova- ja pehmytkudosvaurioprosesseissa sekä kielisyövässä, joissa se on ollut tuntematon. MMP-8 poistogeenisillä hiirillä tehdyissä pehmyt- ja kovakudoshaavoissa MMP-8:n vaikutusmekanismit riippuivat kohdekudoksesta. Alveoliluun paranemisen yhteydessä MMP-8 lisäsi tulehdusta ja osallistui kollageenin muokkaamiseen. Akuutin kielihaavan paranemisessa MMP-8 hidasti haavan umpeutumista ja vähensi transformoivan kasvutekijä-β1:n (TGF-β1) määrää. Kuten useissa muissakin syövissä, myös kielisyövässä todettiin MMP-8:lla olevan suojaava vaikutus. Potilaan ennuste huononi, jos kasvainsolukon matala MMP-8-taso yhdistyi korkeaan verisuonten kasvutekijä-C:n (VEGF-C) määrään. Hiiren kielen normaaleissa fibroblastiviljelmissä MMP-8 vähensi TGF-β1:n solunsisäistä signalointia välittävän fosforyloidun Smad2:n määrää sekä solujen kykyä supistaa kollageenikiekkoja. Koeputkessa MMP-8 pilkkoi TGF-β1:tä, estrogeenireseptoreja (ER) ja apoptoositekijä Fas-ligandia (Fas-L). Ihmisen kielikarsinoomasoluviljelmissä korkea MMP-8:n määrä vähensi solujen migraatiota ja invaasiota sekä muutti MMP-1:n, MMP-9:n, katepsiini-K:n, TGF-β1:n ja VEGF-C:n ilmentymistä. Migraation heikentyminen MMP-8:aa tuottavissa soluissa johtui osin vähentyneestä TGF-β1:n määrästä. Hiiren kokeellisissa kielisyövissä MMP-8 hidasti syövän muodostumista mutta ei estänyt etäpesäkkeiden muodostusta. Tässä väitöskirjatutkimuksessa on kolme päälöydöstä: 1) MMP-8:n vaikutus kudoksen paranemisprosessiin riippuu vauriokohdasta, 2) MMP-8 on kielisyövän kehittymisessä puolustuksellinen molekyyli, mutta sen lisääntynyt tuotto ei hiirikokeissa estänyt etäpesäkkeiden muodostusta, 3) MMP-8:lle löydettiin neljä uutta kohdemolekyyliä (TGF-β1, Fas-L, ER-α ja -β), joiden muokkaus saattaa osin selittää MMP-8:n monipuoliset kudos- ja prosessispesifit vaikutukset.

Abstract Chronic, non-healing wounds constitute a massive financial burden on health care system. The healing processes of these wounds and their underlying pathology are only partly understood. In this study, important biological functions performed by Retinoic acid with its regulatory protein cellular retinoic acid binding protein 2 (CRABP2) were discussed. Possibly, these biological func-tions might be linked with chronic wound therapeutic by inducing antiproliferative activity of cells which leads to reduction in migration and growth rate of fibroblast during skin regeneration pro-cess in chronic wound healing. The aim of this study was to comparatively analyze the expression pattern of CRABP2 and P16 cyclic dependent kinase inhibitor in dermal fibroblasts at mRNA levels along with their morphological pattern, migration and growth rate. Fibroblasts were cultured and their morphology were observed by phase-contrast imaging. Difference in viability, migratory capacity was examined by Cell titer blue and scratch assay respectively and expression were meas-ured by polymerase chain reaction. Interestingly, the date revealed that morphology was altered and growth rate and migration velocity was significantly lower in chronic wound fibroblasts and senescent fibroblasts when compared with their control. Expression pattern revealed that CRABP2 was highly up-regulated only by senescent cells but not in chronic wound fibroblasts which point novel function for this protein in term of replicative senescence. However, P16 was not signifi-cantly altered among all fibroblasts which demands supplementary studies to conform the role of CRABP2 in fibroblast dysfunction and cellular senescence in chronic wounds.

Le syndrome de détresse respiratoire aiguë (ARDS) est la forme la plus sévère d'agression alvéolaire aiguë (ALI) Il est caractérisé par un dommage alvéolaire diffus suivi d'une phase de réparation nécessaire à la guérison. Bien que les fibroblastes pulmonaires soient des acteurs essentiels de cette réparation, leurs caractéristiques et leurs fonctions restent mal connues chez l'homme au cours des ALI/ARDS. L’hypothèse défendue par cette thèse est qu’une réparation alvéolaire harmonieuse nécessite un recrutement adapté des fibroblastes et de leur précurseur, les fibrocytes, vers le poumon, ainsi qu' une fine régulation de leurs fonctions par l'environnement inflammatoire de l'alvéole. Les principaux résultats de nos travaux ont permis : 1) d’établir que le liquide de lavage alvéolaire (LBA) recueilli lors des ALI/ARDS stimule la production de facteurs de réparation épithéliaux comme l'Hepatocyte Growth Factor (HGF) et le Keratinocyte Growth Factor (KGF) par les fibroblastes. Cette production est principalement induite par l'IL-1ß via une boucle autocrine COX2/PGE2. 2) de montrer que les fibroblastes alvéolaires cultivés à partir des LBA d'ALI/ARDS présentent un phénotype activé, caractérisé par une production augmentée de collagène 1 et une capacité de migration majorée. La détection de fibroblastes dans les cultures de cellules du LBA est associée à une diminution des marqueurs de l'inflammation et à une réduction de la durée de ventilation des patients concernés. 3) D’analyser l’effet des LBA d'ALI/ARDS sur la migration des fibroblastes et de monter l’existence d’un équilibre entre activation et inhibition de la migration via une modulation de le voie du PDGF. La forme soluble du récepteur a au PDGF (PDGF-sRa) est un des inhibiteurs présent dans le LBA. Une augmentation de la capacité chimiotactique des LBA de patients en ALI/ARDS est associée à une augmentation de la mortalité.4) De démontrer la présence de fibrocytes, précurseurs mésenchymateux circulants d'origine hématopoïétique, dans les LBA de patients en ALI/ARDS et de mettre en évidence que leur recrutement excessif, est associé à un mauvais pronostic. Ainsi, après ajustement sur les covariables cliniques et biologiques (modèle de COX), un pourcentage de fibrocyte supérieur à 6% de la population cellulaire totale du LBA est associé à une durée de ventilation prolongée et à une mortalité augmentée. L’ensemble de ces travaux démontrent pour la première fois que les fibroblastes et les fibrocytes sont directement associés au pronostic lors des ALI/ARDS, que leur rôle est indispensable à la guérison mais que leur recrutement excessif pourrait impacter défavorablement le pronostic des patients. Les données physiopathologiques obtenues pourraient contribuer à l’identification de nouvelles cibles thérapeutiques et à proposer de nouveaux marqueurs biologiques dans la prise en charge des ALI/ARDS / Acute Respiratory Distress Syndrome (ARDS) is the most severe form of Acute Lung Injury (ALI) and is characterized by diffuse alveolar damage followed by a phase of alveolar repair necessary to recovery. Although lung fibroblasts are key players in this repair process, their characteristics and functions remain poorly understood in humans during ALI/ARDS. The hypothesis of our studies was that effective alveolar repair requires a regulated recruitment of fibroblasts and fibrocytes (circulating mesenchymal precursors of hematopoietic origin) to the lung, as well as a tight regulation of their functions by the alveolar inflammatory environment. The main results of our works have shown that: 1) The alveolar lavage fluid (BAL) from ALI/ARDS patients stimulated the production of Hepatocyte Growth Factor (HGF) and Keratinocyte Growth Factor (KGF) by fibroblasts, both of which were involved in epithelial repair. This production was mainly induced by IL-1ß via an autocrine COX2/PGE2 dependant loop. 2) The alveolar fibroblasts cultured from BAL fluids of ALI/ARDS patients demonstrated an activated phenotype, characterized by an increase of both collagen 1 production and ability to migrate. A positive BAL fibroblast cell culture was associated with reduced markers of inflammation and reduced ventilation duration of ALI/ARDS patients. 3) BAL fluids from ALI/ARDS patients modulated fibroblast migration. This effect was related to a balance between activation and inhibition of migration involving the PDGF signaling pathway. The soluble form of the PDGF a receptor (PDGF-sRa) participated in the BAL inhibitory chemotactic activity. An increased chemotactic ability of BAL from ALI/ARDS patients was associated with higher mortality. 4) Excessive recruitment of fibrocytes is associated with poor prognosis. After adjustment of clinical and biological covariates (Cox model), a percentage of fibrocytes higher than 6% of total BAL cells is associated with a prolonged ventilation duration and an increased mortality. Taken together, our works demonstrate for the first time that fibroblasts and fibrocytes are directly associated with prognosis in ALI/ARDS. Fibroblasts are essential to lung healing, but their excessive recruitment may have a negative impact on prognosis. The data presented in this thesis may help identify new therapeutic targets and suggest new biomarkers to consider in the management of ALI/ARDS