Le développement placentaire est un processus finement contrôlé dans le temps et dans l'espace. Il est caractérisé par une invasion précoce et profonde, de l'endomètre et du premier tiers du myomètre, par les cytotrophoblastes extravilleux, cellules responsables du remodelage des artères spiralées utérines et de l'établissement de la circulation fœto-maternelle. Tout déficit dans ces processus physiologiques conduit à des complications de la grossesse, telles que la Pré-Eclampsie (PE), ou le Retard de Croissance Intra-Utérin (RCIU). Les travaux récents de l'équipe suggèrent l'implication d'un nouveau facteur angiogène, nommé EG-VEGF (Endocrine Gland Derived Vascular Endothelial Growth Factor), dans le développement de la PE. L'objectif de ma thèse fut de mieux caractériser le rôle de l'EG-VEGF dans le développement placentaire normal au cours du premier trimestre de la grossesse. Pour cela trois axes ont été explorés : i) l'étude de la régulation de l'EG-VEGF par le récepteur nucléaire PPARγ (Peroxisome proliferator-activated receptor gamma), ii) la détermination de son rôle dans les différenciations hématopoïétique et angiogénique placentaires et iii) la contribution au développement d'un modèle murin de la PE. A l'issue de cette thèse, mes travaux ont montré que non seulement l'expression de l'EG-VEGF et de son récepteur PROKR2 étaient régulées positivement par PPARγ, mais aussi, que ce récepteur nucléaire est directement impliqué dans la mise en place de la vascularisation intra-placentaire, avec la participation des deux récepteurs PROKR1 et PROKR2, et que l'inhibition de l'invasion trophoblastique par PPARγ, seraient en partie contrôlée par l'EG-VEGF, via PROKR2. Mon travail a également mis en évidence que l'EG-VEGF serait impliqué dans le contrôle de la différenciation hématopoïétique et endothéliale placentaire. Il aurait un effet inhibiteur sur la différenciation des cellules hématopoïétiques et endothéliales, mais plus particulièrement sur les cellules endothéliales hémogéniques. Enfin, ma contribution au développement d'un modèle in vivo de la PE a permis de montrer qu'un maintien de la libération de l'EG-VEGF, au-delà de sa période normale de production, serait responsable du développement de la PE, suite à un défaut de l'invasion trophoblastique, entraînant la libération par le placenta, de sFlt-1 et de sEndogline. Ces derniers vont induire un dysfonctionnement rénal et une hypertension artérielle. L'ensemble de ces trois projets a contribué à l'avancée de nos connaissances actuelles sur les mécanismes physiologiques du développement placentaire, ainsi que sur un facteur clé de la placentation, l'EG-VEGF, et a également permis de mieux appréhender les causes de l'établissement des pathologies de la grossesse, comme la PE et le RCIU. / Placental development is a process that is finely controlled. It is characterized by early and deep invasion of the endometrium and the first third of the myometrium by extravillous cytotrophoblasts that participate to the remodeling of the spiral arteries and to the establishment of the feto-maternal circulation. Poor remodeling of spiral arteries by trophoblastic cells, leads to the development pregnancy pathologies such as, Preeclampsia (PE) and Intra-Uterine Growth Restriction (IUGR). During the last decade, our team has gathered interesting data that propose the new factor, EG-VEGF (Endocrine Gland Derived Vascular Endothelial Growth Factor) as a potential marker for PE. My thesis project aimed at further characterizing the role of EG-VEGF during pregnancy. Three main axis were addressed, i) The study of the regulation of EG-VEGF by PPARγ (Peroxisome proliferator-activated receptor gamma), ii) The study of the role in hematopoietic and angiogenic placental cells differentiations and iii) The development of an in vivo model of PE. My thesis showed that 1) EG-VEGF and PROKR2 expression are upregulated by PPARγ, 2) that the regulation of intra-placental vascularization and trophoblastic invasion by PPARγ is mediated by EG-VEGF through PROKR1 and PROKR2 and through PROKR2 receptors, respectively, 3) that EG-VEGF controls hematopoietic and endothelial cell differentiation and 4) that maintenance of EG-VEGF production beyond its normal period of secretion during pregnancy leads to the development of PE in a gravid mouse model. Altogether, these projects contributed to have a better knowledge about physiological mechanisms of placental development and about a key factor of placentation EG-VEGF. Moreover they improved our understanding of the origins of pregnancy diseases establishment such as PE and RCIU.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-01-20T21:18:47Z No. of bitstreams: 1 KeilaMarthaAmorimBarroso_TESE.pdf: 21818921 bytes, checksum: 6e052052d865c5daec54b63d43d919f5 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-01-22T20:15:18Z (GMT) No. of bitstreams: 1 KeilaMarthaAmorimBarroso_TESE.pdf: 21818921 bytes, checksum: 6e052052d865c5daec54b63d43d919f5 (MD5) / Made available in DSpace on 2016-01-22T20:15:18Z (GMT). No. of bitstreams: 1 KeilaMarthaAmorimBarroso_TESE.pdf: 21818921 bytes, checksum: 6e052052d865c5daec54b63d43d919f5 (MD5) Previous issue date: 2014-02-28 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / As neoplasias de gl?ndulas salivares exibem uma grande diversidade morfol?gica e comportamentos biol?gicos variados o que suscita o interesse na pesquisa destas les?es. A dissemina??o das c?lulas tumorais ? um passo inicial para a progress?o de neoplasias malignas e, dentro deste contexto, os vasos linf?ticos neoformados s?o considerados essenciais para que ocorra essa dissemina??o. O papel do VEGF (fator de crescimento endotelial vascular) na forma??o dos vasos ? fato conhecido mas, pouco se sabe a respeito de sua participa??o em tumores de gl?ndula salivar. Desta forma, o objetivo deste estudo foi avaliar a express?o do VEGF-C e VEGF-D, a densidade linf?tica tumoral (D2-40) e a prolifera??o endotelial linf?tica (dupla marca??o D2-40/Ki-67) em uma s?rie de neoplasias de gl?ndulas salivares. A amostra foi composta por 20 adenomas pleom?rficos, 20 carcinomas aden?ides c?sticos, 20 carcinomas mucoepiderm?ides e 10 casos de tecido glandular salivar com caracter?sticas de normalidade para efeito comparativo. Todos os casos estudados exibiram express?o positiva para VEGF-C em regi?o peritumoral e intratumoral, n?o sendo encontrada diferen?as de imunoexpress?o entre os grupos. No entanto, o grupo dos carcinomas aden?ides c?sticos demonstrou diferen?a significativa da imunoexpress?o do VEGF-C segundo o padr?o cribriforme e s?lido (p = 0,004). A maioria dos casos constantes do presente estudo, apresentou fraca marca??o para VEGF-D em regi?o peritumoral e intratumoral. Na avalia??o da densidade endotelial linf?tica peritumoral, intratumoral e total, os grupos estudados revelaram um gradiente crescente, com valores menores para o grupo dos adenomas pleom?rficos, seguido dos carcinomas mucoepiderm?ides e carcinomas aden?ides c?sticos. A densidade endotelial linf?tica foi maior entre os tumores malignos do que nos tumores benignos. N?o foi observada correla??o entre a imunoexpress?o de VEGF-C e VEGF-D em rela??o a densidade linf?tica tumoral e a prolifera??o endotelial linf?tica / Salivary gland neoplasms exhibit a great morphological diversity and varied biological behavior which raises the interest in the study of these lesions. The spread of tumor cells is an early step in the progression of malignancies and the neoformed lymphatic vessels are considered essential in tumor dissemination. Vascular endotelial growth fator (VEGF) is a family of proteins involved in angiogenesis e lymphangiogenesis. However, in salivar tumors we have limited information on the expression. The aim of this study was to assess the expression of VEGF-C and VEGF-D, lymphatic vessel density (single-staining D2-40) and lymphatic endothelial proliferation (double labeling D2-40/Ki-67) in a series of salivary glands neoplasms. We selected 20 cases of pleomorphic adenoma, 20 of mucoepidermoide carcinoma, 20 of adenoid cystic carcinoma and 10 tissue sample of normal salivary gland. All cases studied showed positive expression of VEGF-C in intratumoral and peritumoral region, no differences in immunoreactivity was found between the groups. However, the group of adenoid cystic carcinoma showed a significant difference in immunoreactivity of VEGF-C by the cribriform and solid pattern (p = 0.004). Most of the cases included in this study showed weak immunoreactivity for VEGF-D in intratumoral and peritumoral region. In the assessment of lymphatic endotelial density peritumoral, intratumoral and total, the groups showed an increasing gradient, with lower values for the group of pleomorphic adenomas followed by mucoepidermoid carcinoma and adenoid cystic carcinoma. Lymphatic endothelial cell density was higher in malignant than benign tumors. No correlation was observed between the immunoreactivity of VEGF-C and VEGF-D in relation to tumor lymphatic density and lymphatic endothelial proliferation

A carcinogênese epitelial ovariana tem sido foco de estudos científicos em todo o mundo desenvolvido. A angiogênese tumoral ovariana é um processo multifatorial que resulta em vários produtos pró-angiogênicos. Entre eles, o fator de crescimento vascular endotelial (VEGF) é predominante. Os objetivos deste estudo foram relacionar as dosagens do VEGF dos fluidos peritoneais, do plasma periférico e do infundíbulo pélvico aos níveis de citorredução em pacientes operadas de adenocarcinoma epitelial de ovário (CEO); formular um modelo probabilístico de citorredução e utilizar estas dosagens para estimar a probabilidade do desfecho de citorredução. Além disto, foi criada uma nova variável chamada carga de VEGF. Pelo procedimento step-wise a citorredução foi melhor descrita pela carga de VEGF, mas faltou Normalidade aos resíduos, não sendo possível a adequação de um modelo matemático. Porém, a curva ROC, forneceu uma área sob a curva de 0,84, com sensibilidade de 71,4 % e especificidade variando de 69,5 a 73,9%. O ponto de corte ótimo foi 15,52 log de picograma de carga de VEGF. A odds-ratio (OR) calculada para citorredução ótima descrita pela carga de VEGF foi de 11 (IC= 2,59 ; 46,78). No grupo com estágio avançado (III e IV), a OR foi de 6 (IC= 1,15; 31,22). Apesar do pequeno número de casos, esta nova variável pode vir a ser um auxiliar na determinação de situações onde cirurgia citorredutora deixa de ser a pedra fundamental do tratamento primário do CEO e a indução quimioterápica passe a ter o principal papel na citorredução química antes da cirugia nestes casos. / Epithelial ovarian carcinogenesis has been the focus of scientific studies in developed world. The ovarian tumor angiogenesis is a multifactorial process that results in pro-angiogenic products. Among them, vascular endothelial growth factor (VEGF) is predominant. This study aimed to relate VEGF levels in peritoneal fluids, peripheric plasma and pelvic infundibular plasma to debulking levels in patients operated on for epithelial ovarian carcinoma (EOC), formulate a probabilistic model for debulking and use these measurements to estimate the probability of the outcome of debulking. Moreover, it was created a new variable called burden of VEGF. For step-wise procedure, cytoreduction was better described by of burden VEGF, but missed Normality of residuals, so the adequacy of a mathematical model was not possible. Nevertheless, the ROC curve provided an area under the curve of 0.84, with sensitivity of 71.4 % and especificity varying from 69.5 to 73,9%. The optimal cutoff point was 15.52 log of picograms of VEGF burden. An odds-ratio for optimal cytoreduction described by the VEGF burden was 11 (CI= 2.59; 46.78). In the group with advanced stages (III & IV), the OR was 6 (CI= 1.15; 31.22). Apart from the small number of cases, this new variable might help to determine situations where cytoreductive surgery leaves behind the cornerstone of primary treatment of CEO and the chemotherapic induction comes to have main role in chemical cytoreduction prior to surgery in these cases.

Objetivos: Os objetivos deste estudo foram: 1) determinar a eficácia da transferência gênica do vetor de adenovírus sorotipo 5, carreando o gene do receptor do fator de crescimento endotelial vascular (VEGF) solúvel humano (sVEGFR1) para a glândula lacrimal (GL); 2) investigar se a expressão de sVEGFR1 interfere na neovascularização da córnea (NVC), induzida por queimadura alcalina; 3) avaliar a segurança do procedimento. Métodos: Trinta e dois ratos Wistar foram submetidos à queimadura central da córnea direita com solução de hidróxido de sódio (NaOH) 1 M. Os animais foram divididos em três grupos e injetados diretamente em sua GL direita 25 ?l de vetores virais AdVEGFR1 (1x1010 pfu) (12 animais), 25 ?l do vetor AdNull (1x1010pfu) (10 animais), ou 25 ?l de solução salina (Controle). Após sete dias, a NVC foi observada e fotografada na lâmpada de fenda. A secreção lacrimal foi medida com fenol. A presença do sVEGFR1 na GL foi testada por qPCR (quantitative polymerase chain reaction) e a coloração, por imunofluorescência. O qPCR foi também utilizado para comparar o RNA mensageiro (RNAm) de ilterleucina-1beta (IL-1?), ilterleucina-6 (IL-6) e fator de necrose tumoral alfa (TNF-?) na GL e no gânglio do trigêmeo (GT). Resultados: O vetor AdVEGFR1 transfectou 83% dos ratos. O sVEGFR1 estava presente nas células acinares da GL. A NVC foi prevenida em nove de doze animais do grupo AdVEGFR1, em comparação com o grupo Ad-Null (3:10) e o grupo Controle (1:10) (p=0,0317). A secreção lacrimal e o RNAm das citocinas na GL e no GT foram semelhantes nos três grupos (p>0,05). Conclusões: A transferência gênica do vetor adenoviral para a GL demonstrou expressão local do sVEGFR1 humano, e evitou a NVC na maioria dos olhos expostos a queimaduras alcalinas, se mostrando seguro para a estrutura e função da GL. / Purpose: The aims of this study were: 1) to determine the efficacy of adenovirus vector serotype 5 (Ad) encoding human soluble VEGF receptor 1 (sVEGFR1) gene transfer to the lacrimal gland (LG); 2) to investigate whether expression of sVEGFR1 acts in corneal neovascularization (CNV), induced by alkali burn and; 3) to evaluate the safety of the procedure. Methods: AdVEGFR1viral vectors (25 ?l, 1x1010pfu) were injected in the right LG of rats and compared with AdNull vector (25 ?l, 1x1010pfu) or 25?l saline (Control) before cornea alkali burn with 1 M NaOH. After seven days, CNV was observed and photographed in the slit lamp. Tear secretion was measured with phenol red thread. The animals were tested for human VEGFR1 mRNA and protein in the LG by qPCR and immunofluorescence staining, respectively. qPCR was also used to compare the mRNA of IL-1?, IL-6, and TNF-? in LG and ipsilateral trigeminal ganglion (TG). Results: Ad-VEGFR1 transfected 83% of the rats. VEGFR1 was present in LG acinar cells. CNV was prevented in 9 of 12 animals of Ad-VEGFR1 group, compared to Ad-Null (3:10) and Control (1:10) (p=0.0317). The tear secretion and the cytokines mRNA in LG and TG were similar all three groups (p>0.05). Conclusion: Adenoviral vector gene transfer to LG as the has shown local expression of human sVEGFR1, as It prevented CNV in most of the eyes exposed to alkali burn and was safe for LG structure and function.

Determinar o valor prognóstico e preditivo do VEGF (vascular endothelial growth factor) e do HIF-1? (Hypoxia-inducible factor-1) em relação à sobrevida livre de doença (SLD) e sobrevida global (SG) em pacientes com carcinoma de mama localmente avançado (CMLA) tratadas primariamente pela quimioterapia neoadjuvante. MATERIAIS E METODOS: VEGF e HIF foram quantificados consecutivamente em plasma de 36 pacientes com CMLA pelo método de ELISA (enzyme labeling immunoassay absorbant) para o VEGF165 e o HIF-1?. O tratamento neoadjuvante foi realizado em todas as pacientes com docetaxel e epirrubicina. O tempo médio de seguimento foi de 56 meses. RESULTADOS: Uma análise univariada demonstrou que o HIF-1? está significantemente relacionado à SLD (P =.0238) e à SG (P = .0121) com as pacientes HER-2 positivas. Não houve diferença significante para a SLD ou SG no que diz respeito aos receptores de hormônio, comprometimento axilar ou grau tumoral. Os valores de VEGF foram maiores no grupo de pacientes RE+ do que no grupo RE negativo (P =.01). Inversamente os valores de HIF-1? foram menores no grupo RE+ comparados ao grupo RE - (P =.02). Pacientes com recorrência óssea apresentaram uma tendência a apresentarem valores de VEGF menores (media, 175.7 pg/ml) do que aquelas com recorrência visceral (441 pg/ml). Uma análise multivariada demonstrou o comprometimento axilar (P =.0004), receptores de estrógeno (ER) (P < .0001), e tamanho do tumor (P = .0085) como fatores independentes de SLD. O HIF-1? foi tido como um fator independente preditivo de SG (P =.0180). Não houve diferença estatisticamente significante entre os valores plasmáticos de HIF-1? ou VEGF nos períodos pré e pós quimioterapia. CONCLUSÕES: Os resultados sugerem que o nível plasmático do HIF-1? é preditivo de SLD e SG nas pacientes com CMLA apresentando uma sobreposição as pacientes HER-2 positivas. As dosagens de VEGF podem ser preditivas de resposta e prognóstico no tratamento neoadjuvante, mas são necessários novos estudos prospectivos comparados ao HIF-1? para conclusões mais consistentes. / To determine the predictive and prognostic value of vascular endothelial growth factor (VEGF) and Hypoxia-inducible factor-1 (HIF-1?) for relapse-free survival (RFS) and overall survival (OS) in locally advanced breast cancer (LABC) primarily submitted to neoadjuvant chemotherapy. MATERIALS AND METHODS: VEGF and HIF were quantitatively measured in plasma sample from 36 consecutive patients with LABC using an enzyme immunoassay for human VEGF165 and HIF-1?. Neoadjuvant treatment was given to all patients as docetaxel and epirrubicin. The follow-up median time was 56 months. RESULTS: Univariate analysis showed that HIF-1? is a significant predictor of RFS ( P = .0238) and OS (P = 0121) in HER-2 positive patients. No significant difference was seen in RFS or OS related to hormonal receptor, axillary status or tumoral grade. The VEGF level was higher in the group of patients who ER was positive than ER negative (P = .01). On the other hand, the HIF-1? level is higher in ER negative patients than ER positive ( P=.02). Patients with bone recurrences tended to have lower VEGF plasma level (median, 175.7 pg/ml) than patients with visceral metastasis (441 pg/ml). Multivariate analysis showed nodal status (P = .0004), estrogen receptor (ER) status (P < .0001), and tumor size (P = .0085) to be independent predictors of RFS. HIF-1? was found to be an independent predictor of OS (P = .0180). No statistically differences were observed related to pre and post chemotherapy period in HIF-1? or VEGF measurements. CONCLUSION: The results suggest that high level of plasma HIF-1? is associated to HER-2 over expression and they are major predictive factors of RFS and OS in LABC. VEGF content might also predict outcome after neoadjuvant treatment, however further studies in a prospective setting with HIF-1? homologous treatments are required.

Le cancer est l'une des principales causes de morbidité et de mortalité dans le monde avec environ 14 millions de nouveaux cas et 8,2 millions de décès liés aux cancers selon l'OMS (Organisation Mondiale de la Santé). Le cancer du poumon est le premier cancer diagnostiqué chez l'homme et compte pour 20% du nombre de mort par cancer par an dans le monde. Parmi les cancers pulmonaires, on distingue les cancers à petites cellules et les cancers non à petites cellules. Ce travail a porté sur l'étude des adénocarcinomes pulmonaires lépidiques, un type de cancer non à petites cellules, chez l'homme et l'animal. Chez l'homme, il fait partie des tumeurs pulmonaires rares, pas ou peu lié au tabagisme. Il est similaire cliniquement, radiologiquement et histologiquement à l'adénocarcinome pulmonaire ovin, un cancer du poumon induit par le β-rétrovirus JSRV (Jaagsiekte Sheep RetroVirus) affectant les petits ruminants domestiques. JSRV transforme les cellules épithéliales du parenchyme via son enveloppe oncogénique. Ces tumeurs humaines et animales ne sont pas associées au développement de métastases extra-thoraciques ou pleurales. Le but de notre travail a été d'étudier les évènements moléculaires spécifiques des adénocarcinomes lépidiques humains et animaux par i. l'analyse transcriptomique de l'expression des gènes dans ces deux cancers, ii. la caractérisation de l'adénocarcinome pulmonaire ovin par l'expression des mucines et iii. l'étude d'un rôle putatif des séquences rétrovirales endogènes dans l'induction de l'adénocarcinome lépidique humain. Premièrement, notre étude a porté sur l'analyse en parallèle des mécanismes moléculaires spécifiques des adénocarcinomes lépidiques prédominants par l'analyse des profils d'expression des gènes impliqués dans les voies de signalisation BMI1, NOTCH, Hedgehog et WNT importantes dans le développement du poumon normal et au cours des cancers pulmonaires et dans la voie de l'angiogenèse, permettant la dissémination des cellules tumorales. L'augmentation de l'expression de CDH1 (E-cadherin), acteur crucial de la voie BMI1, et la diminution de l'expression des ligands et récepteurs activateurs de l'angiogenèse VEGF/VEGFR (Vascular Growth Factor), PDGF/PDGFR (Platelet-derived Growth Factor) et ANGPT/TIE (Angiopoietin/Tyrosine kinase with immunoglobulin like and EGF like domains) ont été observées dans ces deux cancers. Lors de l'analyse de l'expression des ARNm et des protéines, nous avons mis en évidence le blocage de la voie principale de l'angiogenèse dans les cancers lépidiques. En effet, l'absence d'expression du ligand VEGFA (Vascular Endothelial Growth Factor A) et de son récepteur VEGFR2 (Vascular Endothelial Growth Factor Receptor 2) a été observée. Ce blocage n'était pas compensé par la surexpression de gènes impliqués dans les voies alternatives d'angiogenèse. Le blocage de la voie de l'angiogenèse explique l'absence de métastases extrathoraciques dans les adénocarcinomes lépidiques chez l'homme et l'animal. Deuxièmement, les adénocarcinomes pulmonaires lépidiques chez l'homme sont subdivisés en deux entités majoritaires : les adénocarcinomes lépidiques prédominants non mucineux et les adénocarcinomes mucineux invasifs. Dans le contexte des analogies entre ces cancers humains et l'adénocarcinome induit par le virus JSRV, nous avons analysé l'expression des mucines dans les cancers animaux. Nous avons montré que sur 37 cancers pulmonaires induits par JSRV, 62% étaient non mucineux, qu'ils sur exprimaient MUC1 (mucin 1, cell surface associated) et que 50% exprimaient MUC5B (mucin 5B, oligomeric mucus/gel-forming). Par ces résultats, nous avons montré que les adénocarcinomes pulmonaires ovins se rapprochaient principalement des adénocarcinomes lépidiques prédominants / Cancer is the main cause of morbidity and mortality worldwide with 14 million new cases and approximately 8.2 million deaths according to the WHO (World Health Organization). Lung cancer is the most diagnosed cancer in men and account for 20% of cancer death in the world annually. Among lung cancers, there are small cell lung cancers and non-small cell lung cancers. My work focused on the lepidic adenocarcinoma, a subtype of nonsmall cell lung cancer, in humans and animals. In humans, it is a rare lung cancer and it is not or rarely linked to tobacco smoking. Lepidic adenocarcinomas share striking clinical, radiological and histopathological similarities with ovine pulmonary adenocarcinomas induced by the β-retrovirus JSRV (Jaagsiekte Sheep RetroVirus) affecting sheep and goats. JSRV transforms epithelial cells of the distal lung through its oncogenic envelope. These human and animal tumors are not associated with pleural or extra thoracic metastases. My work was to study specific molecular events in human and animal lepidic adenocarcinomas by i. a transcriptomic analysis of the gene expression in these two lung cancers, ii. ovine pulmonary adenocarcinoma characterization by mucin expression and iii. the study of a potential role for endogenous retroviral sequences in the induction of human lepidic adenocarcinomas. First, my work focused on the parallel analysis of expression patterns in genes implicated in BMI1, NOTCH, Hedgehog and WNT pathways important for lung development and for lung cancer development and the angiogenesis pathway whose activation leads to cancer cell dissemination. CDH1 (E-cadherin) up regulation and the down regulation of main angiogenic ligand and receptors VEGF/VEGFR (Vascular Endothelial Growth Factor), PDGF/PDGFR (Platelet-derived Growth Factor) and ANGPT/TIE (Angiopoietin/Tyrosine kinase with immunoglobulin like and EGF like domains) were observed. Then, when analyzing lepidic adenocarcinomas mRNA and protein expression, we highlighted the blockade of the main angiogenesis pathway. Indeed, the VEGFA ligand and its receptor VEGFR2 were absent in lepidic adenocarcinomas. Moreover, this blockade was not compensated with angiogenic alternative pathways. These results correlate with the absence of extra thoracic metastases in human and animal lepidic adenocarcinomas. Secondly, human lepidic predominant adenocarcinomas are divided in two groups, non-mucinous lepidic adenocarcinomas and invasive mucinous adenocarcinomas. To characterize ovine pulmonary adenocarcinomas and determine to which one of these two human lepidic adenocarcinomas they are close to, we analyzed mucine expression in animal lung cancer. On 37 JSRV-induced lung cancer, 62% were nonmucinous, 100% expressed MUC1 (mucin 1, cell surface associated) and 50% expressed MUC5B (mucin 5B, oligomeric mucus/gel-forming). These results highlighted that ovine pulmonary adenocarcinomas are closer to non-mucinous lepidic adenocarcinomas

Les capillaires sanguins sont des éléments essentiels pour le maintien des fonctions du tissu adipeux comme du muscle squelettique. L’obésité induit une raréfaction capillaire intense dans ces tissus, altérant leur microenvironnement et aboutissant en définitif à des dysfonctions métaboliques systémiques. Malgré l’importance de la microcirculation adipeuse, les mécanismes moléculaires régulant la plasticité de ce réseau capillaire, processus appelé angio-adaptation, restent méconnus. L’activité physique exerce de multiples effets bénéfices chez l’obèse, stimulant notamment la croissance vasculaire dans le muscle squelettique. Dans ce tissu, la réponse angio-adaptative au cours de l’obésité et à l’exercice est sous le contrôle de l’axe Mdm2/FoxOs. Ces facteurs régulent la balance entre le signal pro-angiogénique du VEGF-A et anti-angiogénique de la TSP-1, équilibrant ainsi les processus de croissance et régression capillaire. Nous proposons l’existence d’un mécanisme de signalisation similaire, impliqué dans la régulation du processus angio-adaptatif des tissus adipeux et musculaires. Nous avons alors étudié les effets de 7 semaines d’exercice physique volontaire, chez des souris C57Bl/6 rendues obèses par un régime riche en graisses et en sucre, sur la microcirculation et le microenvironnement du tissu adipeux blanc viscéral et sous-cutané, ainsi que du brun interscapulaire. Il a été retrouvé chez les souris obèses un puissant frein angiostatique, exercé par FoxOs et conduisant à une raréfaction capillaire dans les tissus adipeux. Les 7 semaines d’exercice physique ont conduit à l’augmentation de Mdm2, la levée de ce frein et une croissance vasculaire au sein des différents tissus adipeux, associés à l’amélioration du microenvironnement adipocytaire, avec une baisse de l’hypoxie, de la fibrose et une redistribution des cellules inflammatoires. De plus, la stimulation de l’angiogenèse adipeuse chez l’obèse, via l’exercice et l’action pro-angiogénique de Mdm2, a permis d’améliorer l’insulino-sensibilité du tissu adipeux viscéral, d’activer le processus de browning au sein du tissu adipeux sous-cutané et de réduire le whitening du tissu adipeux brun. Il en résulte une amélioration de l’ensemble des paramètres cardiométaboliques systémiques. Ces données démontrent l’efficacité thérapeutique de l’exercice physique dans la lutte contre l’obésité et ses pathologies associées, mais offrent également de nouvelles perspectives de thérapies moléculaires ciblant l’angio-adaptation du tissu adipeux chez l’Homme obèse. / Blood capillaries are essential elements for maintaining adipose tissue and skeletal muscle functions. Obesity induces intense capillary rarefaction in these tissues, altering their microenvironment and ultimately resulting in systemic metabolic dysfunction. Despite the importance of adipose microcirculation, molecular mechanisms regulating adipose capillary network plasticity, a process called angio-adaptation, remains unknown. Physical exercise exerts multiple beneficial effects in obese patient, including skeletal muscle vascular growth stimulation. In this tissue, angio-adaptive response during obesity and exercise is under the control of the Mdm2/FoxOs axis. These factors regulate harmonization between VEGF-A pro-angiogenic signal and TSP-1 anti-angiogenic, thus balancing growth and capillary regression processes. We made the hypothesis of the existence of a similar signaling mechanism, involved in the angio-adaptive process regulation of adipose and muscular tissues. Therefore, we studied the effects of 7 weeks-voluntary physical exercise in C57Bl/6 obese mice induced by a diet rich in fats and sugar, on microcirculation and microenvironment of visceral and subcutaneous white adipose tissue, as well as interscapular brown adipose tissue. Obese mice presented a powerful angiostatic control in all adipose tissues, under FoxOs protein regulation, leading to capillary rarefaction. Physical exercise led to the increase of Mdm2 expression, repressing the angiostatic control in favor of adipose vascular regrowth. This phenomenon was also associated with adipocytes microenvironment improvement, a decrease in hypoxia, fibrosis and redistribution of inflammatory cells. In addition, adipose angiogenesis stimulation in obese mice, through exercise and the Mdm2 pro-angiogenic action, improved visceral adipose tissue insulin sensitivity, activated browning process within subcutaneous adipose tissue and reducing whitening of brown adipose tissue. The overall result is an improvement of all systemic cardiometabolic parameters. These data demonstrate the therapeutic efficacy of physical exercise against obesity and its associated pathologies, but also offer new prospects for molecular therapies targeting the adipose angio-adaptation in obese humans.

Le potentiel invasif et métastatique d'une tumeur est contrôlé par l'angiogenèse via le VEGF (Vascular Endothelial Growth Factor) et les altérations des systèmes impliqués dans l'adhérence cellulaire via les cadhérines. Dans ce travail, nous montrons que le L-165041, agoniste du récepteur nucléaire PPARβ (Peroxisome Proliferator-Activated Receptor) régule l'expression du VEGF dans des cellules dérivées de cancer du col de l'utérus dont le facteur étiologique est un papillomavirus (HPV). Cependant, cet effet est observé uniquement dans des cellules HPV18 indépendamment de l'oncoprotéïne virale E6 et de sa cible p53. De façon intéressante, à la diflérence de ce que nous observons dans des cellules de cancer vésical, le L- 165041 ne contrôle pas la transcription mais stabilise les messagers des isoformes du VEGF (VEGF121, VEGF165, VEGF189) indépendamment du récepteur PPARβ. La stabilisation des ARNm VEGF nécessite la phosphorylation de la p38 MAPK qui conduit, seule ou suite à une cascade de phosphorylations, au déplacement de la protéine HuR du noyau vers le cytoplasme et in fine à la stabilisation des ARNm VEGF. D'autre part, l'agoniste du PPARβ interviendrait dans la« transition épithélio-mésenchymateuse » de cellules urothéliales malignes, puisqu'une augmentation de l'expression de la cadhérine E (marqueur épithélial) et une diminution de l'expression de la cadhérine N (marqueur mésenchymateux) sont observées selon le phénotype cellulaire. Elucider le mécanisme moléculaire d'action des agonistes de PPARβ est d'un intérêt crucial puisque certains ligands sont en cours d'évaluation clinique pour le traitement de maladies métaboliques (dyslipidémies diabète de tvpe II). / Tumor invasive and metastatic potential is controlled by angiogenesis through VEGF (Vascular Endothelial Growth Factor) up-regulation and altered cadherin-mediated cellular adhesion as well. In this work, we show that the L-165041 nuclear receptor PPARβ (Peroxisome Proliferator-Activated Receptor) agonist regulates VEGF expression in cervical cancer derived cells harboring a Human Papilloma Virus (HPV). However, this effect is only observed in HPVl8 positive cells through E6 viral oncoprotein and p53 independent mechanisms. Interestingly, unlike our data obtained in bladder cancer cells, L-165041 does not control VEGF transcription but stabilizes VEGF isoforms mRNA (VEGF121, VEGFl65, VEGFl89) and acts through PPARβ-independent mechanisms. This mRNA stabilization requires p38 MAPK phosphorylation leading to the nuclear/cytoplasmic shuttling ofHuR protein and consequently to the VEGF mRNA stabilization. Furthennore, the PPARβ agonist would be involved in epithelial to mesenchymal transition since it increases E-cadherin expression and decreases N-cadherin one according to the cellular phenotype. To elucidate the molecular mechanism of PPARβ agonists action is crucial because some of ligands are currently in clinical trials for the treatment of metabolic diseases (dyslipidemia, type II diabetes).

Em roedores, o aumento da permeabilidade vascular, a transformação decidual, e angiogênese são eventos cruciais para o sucesso da gestação. O fator endotelial vascular (VEGF) é um mitogênico para células endoteliais e um indutor de angiogênese. O VEGF age via dois receptores da família das tirosina quinases: VEGFR1 e VEGFR2. O objetivo deste estudo foi investigar usando o método imunohistoquímico, a expressão espacial e temporal do VEGF e os receptores VEGFR1 e VEGFR2 em células endometriais de camundongo entre o 4º e 8º dias de gestação. No 4º dia de gestação, VEGF, VEGFR1 e VEGFR2 foram expressos pelo epitélio luminal e glandular e fracamente pelo estroma endometrial. Do 5º ao 8º dias de gestação, o VEGF foi expresso nas células deciduais mesometriais. VEGFR1 e VEGFR2 foram expresso pelas células do epitélio luminal e glandular e mostraram uma marcação diferencial na decídua mesometrial e antimesometrial. Os receptores VEGFR1 e VEGFR2 foram intensamente expressos pelas células endoteliais dos capilares sinusóides mesometriais e pelas células Nk uterinas. / In rodents, increase of vascular permeability, decidual cell transformation, and uterine angiogenesis are crucial events for the success of pregnancy. Vascular endothelial growth factor (VEGF) is a mitogen for endothelial cells and an inducer of angiogenesis. VEGF acts via two tyrosine kinase family receptors: VEGFR1 and VEGFR2. The aim of this study was to investigate using the immunohistochemical method, the spatiotemporal expression of VEGF and its receptors VEGFR1 e VEGFR2 by mouse endometrial cells on days 4 to 8 of pregnancy. On day 4, VEGF, VEGFR1 and VEGFR2 were expressed mostly by the luminal and glandular epithelium. Stromal cells showed a very weak labeling. On days 5-8, VEGF and its receptors showed an increased labeling throughout the mesometrial decidua. The expression of VEGF, VEGFR1, and VEGFR2 were differentially expressed in the mesometrial cells and in the predecidual cells of the antimesometrial decidua. VEGFR1 and VEGF R2 were highly expressed by endothelial cells of the mesometrial sinusoids, and Nk uterine cells.

La lutte contre les maladies chroniques nécessite la mise en œuvre de nouvelles stratégies de prédiction du risque et de prévention. La médecine personnalisée représente une approche sophistiquée pour réussir la prise en charge des morbidités de populations vieillissantes. Dans le cadre de ces travaux de thèse inspirés par les principes de la médecine personnalisée, nous décrivons une approche qui associe plusieurs méthodologies «-omiques». Nous avons utilisé un modèle de «dénominateur commun» pour les maladies chroniques afin d'identifier des biomarqueurs associés aux facteurs de risque et aux voies biologiques de maladies courantes. Par l’étude de variants génétiques localisés dans la région comportant le gène TREM2, nous avons identifié une association entre le rs6918289 et à la fois de taux élevés de TNF-α et une augmentation de l’épaisseur intima-média de l’artère fémorale. Grâce à des études d’association panépigénomique (EWAS), nous avons identifié de nouveaux marqueurs épigénétiques liés à des facteurs de risque de maladies courantes. Un site CpG de méthylation était associé à une augmentation du tour de taille, contribuant à expliquer la régulation épigénétique de l’obésité abdominale. De plus, une étude EWAS des taux de triglycérides a permis d’identifier deux sites CpG significatifs. L’un de ces deux sites a pu être confirmé dans le tissu adipeux. Une étude EWAS a également été réalisée pour décrire la régulation épigénétique des concentrations de VEGF-A. 20 sites CpG ont pu être identifiés ainsi et leurs relations avec la régulation du VEGF-A ont été examinées par analyse bioinformatique poussée. Les liens entre le VEGF-A et 11 cytokines ont également été étudiés. Le taux de protéine VEGF-A était associé à IL-4, MCP1 et EGF. Les associations entre les cytokines et des isoformes spécifiques de l’ARNm du VEGF-A ont également été évaluées : le VEGF165 était associé à MCP1 et IL-1α, et le VEGF189 à IL-4 et IL-6. Nous avons étudié le rôle du VEGF-A et LT dans l’athérosclérose. Cela a permis d’identifier un variant génétique lié au VEGF-A associé à l’attrition des télomères qui pourrait constituer un dénominateur commun pour les maladies chroniques. L’utilisation de diverses méthodologies pour étudier les facteurs de risque et les voies impliquées dans des maladies chroniques courantes a permis d’identifier de nouveaux marqueurs diagnostiques qui pourraient améliorer la prédiction du risque de maladie basée sur le profil génétique de chaque individu. / The fight against common chronic diseases requires the implementation of new risk prediction and prevention strategies. Personalised medicine offers sophisticated approaches for management of the morbidities of the ageing population. In this thesis, inspired by the principles of personalised medicine, we describe an integrative approach combining “-omics” methodologies. We use a model of a “common denominator” for cardiovascular disease (CVD) and other chronic diseases to identify biomarkers linked with common diseases risk factors and molecular pathways. With the investigation of genetic variants, located in the region of the TREM2 gene, we identified the association of SNP rs6918289 with increased levels of TNF-α and intima-media thickness of the femoral artery. With the use of epigenome-wide association studies (EWAS), we identified novel epigenetic biomarkers related to common diseases risk factors: central obesity and lipid levels. One methylation site (CpG) was associated with increased waist circumference (cg16170243), which could explain the epigenetic regulation of central obesity. Moreover, an EWAS of the triglyceride levels identified two significant CpG sites, one of which was replicated in the adipose tissue (cg04580029), giving insights into epigenetic regulation of lipid levels. An EWAS was also used to study the epigenetics of VEGF-A levels; 20 CpG sites were identified and their relations with VEGF-A regulation were analysed through detailed bioinformatics analysis. VEGF-A was further investigated for its relation with 11 cytokines. VEGF-A protein levels were associated with IL-4, MCP1 and EGF. Specific VEGF-A mRNA isoforms were also investigated for their association with cytokines; VEGF165 showed associations with MCP1 and IL-1α and VEGF189 with IL-4 and IL-6. Together with another important biomarker, TL, we studied the role of VEGF-A in atherosclerosis and identified one VEGF-A related genetic variant associated with telomere attrition, which could present a common denominator of chronic diseases. The employment of diverse methodologies for the investigation of common chronic diseases risk factors and pathways provided new diagnostic markers and generated results, which could help to improve the diseases risk prediction based on the individual genetic “make-up”. New insights into associations between different biomarkers might help in understanding the pathophysiological pathways common between CVDs and other chronic diseases.