Nous introduisons un modèle mathématique d'évolution d'une maladie cancéreuse à l'échelle de l'organisme, prenant en compte les métastases ainsi que leur taille et permettant de simuler l'action de plusieurs thérapies telles que la chirurgie, la chimiothérapie ou les traitements anti-angiogéniques. Le problème mathématique est une équation de renouvellement structurée en dimension deux. Son analyse mathématique ainsi que l'analyse fonctionnelle d'un espace de Sobolev sous-jacent sont effectuées. Existence, unicité, régularité et comportement asymptotique des solutions sont établis dans le cas autonome. Un schéma numérique lagrangien est introduit et analysé, permettant de prouver l'existence de solutions dans le cas non-autonome. L'effet de la concentration de la donnée au bord en une masse de Dirac est aussi envisagé.Le potentiel du modèle est ensuite illustré pour des problématiques cliniques telles que l'échec des anti-angiogéniques, les protocoles temporels d'administration pour la combinaison d'une chimiothérapie et d'un anti-angiogénique et les chimiothérapies métronomiques. Pour tenter d'apporter des réponses mathématiques à ces problèmes cliniques, un problème de contrôle optimal est formulé, analysé et simulé. / We introduce a mathematical model for the evolution of a cancer disease at the organism scale, taking into account for the metastases and their sizes as well as action of several therapies such as primary tumor surgery, chemotherapy and anti-angiogenic therapy. The mathematical problem is a renewal equation with bi-dimensional structuring variable. Mathematical analysis and functional analysis of an underlying Sobolev space are performed. Existence, uniqueness, regularity and asymptotic behavior of the solutions are proven in the autonomous case. A lagrangian numerical scheme is introduced and analyzed. Convergence of this scheme proves existence in the non-autonomous case. The effect of concentration of the boundary data into a Dirac mass is also investigated.Possible applications of the model are numerically illustrated for clinical issues such as the failure of anti-angiogenic monotherapies, scheduling of combined cytotoxic and anti-angiogenic therapies and metronomic chemotherapies. In order to give mathematical answers to these clinical problems an optimal control problem is formulated, analyzed and simulated.

La grossesse est une période de majoration du risque vasculaire, participant à une morbi-mortalité maternelle et fœtale pouvant justifier des mesures de prévention primaire et secondaire. Notre travail évalue l'impact de certains déterminants et l'apport de nouveaux acteurs moléculaires impliqués dans la dysfonction vasculo-placentaire. Le but ultime étant d'optimiser les prises en charge et de développer de nouvelles stratégies thérapeutiques. Nous avons étudié les complications vasculaires placentaires associées à des marqueurs biologiques connus : mutation du facteur V Leiden, mutation du gène de la prothrombine et marqueurs conventionnels du syndrome des anticorps anti-phospholipides (SAPL). Nos résultats montrent que les femmes à antécédents de fausses couches précoces répétitives et porteuses, soit du polymorphisme du facteur V, soit du polymorphisme du facteur II, soit d'un SAPL (traité par héparine et aspirine faible dose), ont un risque élevé de fausse couche tardive lors d'une nouvelle grossesse. Les femmes à antécédent de fausse couche tardive et porteuses des mêmes particularités biologiques, traitées pendant leur grossesse selon les recommandations (héparine pour l'anomalie du facteur V ou II, héparine plus aspirine faible dose pour le SAPL), ont un risque diminué de récidive de perte fœtale tardive mais demeurent, dans le groupe SAPL, fréquemment exposées aux complications tardives de la grossesse malgré la prophylaxie antithrombotique. Nous avons évalué l'apport de nouveaux marqueurs de la dysfonction vasculaire placentaire. Nous montrons que le polymorphisme Ile89Leu du gène de la phosphatase alcaline placentaire (PLAP), enzyme exprimée par les cellules du syncytiotrophoblaste -polymorphisme associé à une augmentation de l'activité PLAP-, exerce un effet protecteur sur l'échec d'implantation et la survenue d'une fausse couche primaire. Un facteur angiogénique (brevet en cours) a également été étudié (génétique, dosage plasmatique, fécondation in vitro) et nous montrons une association de ce marqueur avec les échecs d'implantation et les fausses couches idiopathiques. L'ensemble de ces travaux suggère que ces nouveaux marqueurs moléculaires pourraient contribuer au diagnostic des complications vasculaires de la grossesse et fournir des biomarqueurs d'implantation embryonnaire et/ou de développement placentaire. Ils pourraient suggérer de nouvelles cibles et stratégies thérapeutiques, répondant aux limites des traitements disponibles. / Vascular risk increases during pregnancy, contributing to maternal and foetal morbidity and mortality, and potentially justifying primary and secondary preventive measures. Our work evaluates the impact of some determinants and the contribution of new molecular actors implicated in placental vascular dysfunction. The ultimate aim is to optimize management and to develop new therapeutic strategies. We studied the placental vascular complications associated with known biological markers: the factor V Leiden or prothrombin polymorphisms, and conventional markers of the antiphospholipid antibody syndrome (APS). Women with previous recurrent abortions carrying polymorphisms of either factor V or factor II, or with APS (treated with heparin and low-dose aspirin), had an increased risk of foetal loss during subsequent pregnancies. Women with a previous foetal loss carrying these biological markers, treated according to recommendations during a new pregnancy (heparin for the polymorphisms, heparin plus low-dose aspirin for APS) had a lower risk of foetal loss, but an excess of late complications was observed in the APS group despite prophylaxis. We evaluated the contribution of new markers of placental vascular dysfunction. The placental alkaline phosphatase enzyme (PLAP) is synthesized and expressed by syncytiotrophoblastic cells. We found that the Ile89Leu polymorphism of the PLAP gene provides protection against implantation failure and primary miscarriage and induces increased PLAP activity. We also studied (genetics, plasma determinations, in vitro fertilisation) an angiogenic factor (patent application underway), which we showed to be associated with idiopathic implantation failure and miscarriage. These findings suggest that these molecular actors are potentially useful for the diagnosis of placenta-mediated pregnancy complications and may be relevant biomarkers of embryo implantation and/or placental development. They may indicate new targets for relevant therapeutic strategies, potentially overcoming the limitations of the currently available treatments.

Diversas pesquisas têm direcionado esforços na busca de biomateriais que fossem capazes de melhorar a osseointegração e a estabilidade de implantes dentais em regiões anatômicas desfavoráveis. Entretanto, muitas das opções de terapias regenerativas, largamente empregadas em cirurgias orais reconstrutivas, estão associadas à algumas desvantagens clínicas como um grande período de recuperação, alto risco de morbidade, risco de parestesia nos pacientes, além de apresentarem alto custo. Biomateriais obtidos a partir do látex natural extraído da Hevea brasilienses (seringueira) demonstraram apresentar propriedades angiogênicas e significante aumento no reparo ósseo de alvéolos dentais e em defeitos de calota de ratos. Considerando os benefícios da angiogênese no processo de reparação óssea, o objetivo deste trabalho foi avaliar a capacidade osteogênica proporcionado pelas proteínas angiogênicas do látex na otimização do processo de osseointegração e na estabilidade de implantes de titânio instalados em defeitos ósseos cicunferenciais. Dez cães foram submetidos às cirurgias de instalação de implantes no centro de defeitos ósseos, após a exodontia dos pré-molares. Os defeitos ósseos em cada animal foram preenchidos com coágulo, osso autógeno, proteínas angiogênicas do látex associada ao gel carreador ou gel carreador (apenas gel de colágeno associado ao ácido hialurônico, sem a presença de implante). Em um dos lados da mandíbula os implantes foram recobertos por membrana não absorvível de e-PTFE sem reforço de titânio, ao passo que no lado contralateral, os implantes foram recobertos apenas pelo retalho mucoperiosteal. Metade dos animais foram sacrificados após 4 semanas e o restante após 12 semanas pós-operatórias. A estabilidade dos implantes foi avaliada através de análise de freqüência de ressonância e a formação óssea foi analisada por meio de histologia, histomorfometria, análise qualitativa e quantitativa de fluorescência. Os resultados numéricos obtidos foram submetidos à análise de variância (ANOVA) e contrastes ortogonais foram realizados para comparações múltiplas. Histologicamente, observou-se para todos os biomateriais, uma formação de osso imaturo e lamelar no tempo de 4 e 12 semanas, respectivamente. Na análise qualitativa de fluorescência, encontrou-se maior atividade óssea no período inicial de avaliação (p≤0,05). A membrana não mostrou efeito significativo no processo de regeneração óssea e não houve diferença estatística entre os diferentes biomateriais testados (p≥0,05). O gel carreador apresentou menor formação óssea quando comparado aos demais materiais, sem considerar o tempo e a presença da membrana (p≤0,05). Houve um aumento significativo da formação óssea, osseointegração e da estabilidade ao longo do tempo para os diferentes biomateriais empregados (p≤0,05), independente da presença da membrana. O grupo das proteínas angiogências do látex mostraram formação óssea similar ao coágulo e ao osso autógeno em 4 e 12 semanas, respectivamente (p≥0,05). Os biomateriais obtiveram significante formação óssea, osseointegração e estabilidade dos implantes ao longo do período de avaliação experimental. Esta pesquisa mostrou que a regeneração óssea guiada não alterou os resultados histométricos, as proteínas angiogênicas do látex promoveram deposição óssea similar ao coágulo e osso autógeno nos defeitos alveolares, observou-se um aumento do processo de ossoeintegração e da estabilidade dos implantes em todos os sítios experimentais, independente do tratamento instituído. / The development of biomaterials that can assist osseointegration and implant stability in dissatisfactory anatomical regions has been the goal of several researches in the past years. Despite the array of regenerative therapies to be used, some clinical disadvantages still persist, such as considerable patient morbidity, extended patient recovery periods, substantial cost, risk of paresthesia and residual defects in the donor site. Natural latex biomaterials from Hevea brasiliensis (rubber tree) have shown promising advantages, such as angiogenic properties, significantly high bone repair in dental alveoli, easy acquisition and handling, biocompatibility, reduced cost, dispensability of donor sites and very little chance of morbidity. Considering the well-known benefits of angiogenesis in the bone healing process, the purpose of this study was to evaluate the osteogenic capacity of angiogenic latex proteins in accelerating osseointegration and implant stability, when placed in circumferential bone defects. Ten male dogs were underwent an implant placement in center of the defects after pre-molar extractions. The circumferential gaps around the implant were filled either with coagulum, particulate autogenous bone graft, latex angiogenic proteins mixed in a gel carrier, or with collagen and hyaluronic acid gel carrier without implants. Circumferential defects were covered with a non-absorbable e-PTFE membrane on one side and left uncovered on the other. Half the animals were sacrificed 4 and 12 weeks after implant placement. Implant stability was evaluated using resonance-frequency analysis and bone formation was analyzed by histological, histometric, qualitative and quantitative bone labeling analysis. Data were analyzed statistically using ANOVA and orthogonal contrasts for multiple comparisons. Immature and lamellar bone were observed at 4 and 12 weeks, respectively. Bone labeling analysis showed more bone activity on early period of healing (p≤0.05). The membrane did not show significant effect on bone regeneration and there was not statistical difference among the different biomaterials, considering the different time evaluation periods (p≥0.05). Gel carrier group showed less bone formation than other filling materials, not considering recovery time and membrane (p≤0.05). Latex angiogenic proteins showed bone regeneration similar to coagulum and bone graft at 4 and 12 weeks, respectively (p≥0.05). There was significant bone formation, osseointegration and implant stability of biomaterials during the entire healing period. This research showed that guided bone regeneration technique did not change the histometric results; latex angiogenic proteins promoted bone deposition similar to coagulum and bone graft on alveolar bone defects; osseointegration process and implant stability occurred in all experimental sites, regardless of the treatment performed.

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No. of bitstreams: 1 DANIELE_CRIST?V?O_ESCOUTO.pdf: 2805745 bytes, checksum: c76c0d3c115aae92a7015f9312fd9604 (MD5) Previous issue date: 2018-03-19 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Introdu??o ? A distin??o adequada dos casos de alto risco para eventos graves nas doen?as hipertensivas gestacionais, n?o apenas pr?-ecl?mpsia, ? um desafio cl?nico. O modelo fullPIERS ? uma ferramenta simples e de baixo custo que utiliza vari?veis cl?nicas para estratificar a probabilidade de eventos adversos em gestantes com pr?-ecl?mpsia. O fator de crescimento placent?rio (PlGF) ? um biomarcador com concentra??es reduzidas no plasma de mulheres com pr?-ecl?mpsia e com crescente emprego na avalia??o de gestantes com suspeita de pr?-ecl?mpsia. Objetivos ? O objetivo deste estudo ? estimar a acur?cia do modelo fullPIERS e do biomarcador PlGF como preditores de desfechos adversos maternos em gestantes com doen?a hipertensiva gestacional. M?todos ? Estudo de coorte prospectiva em um hospital terci?rio em Porto Alegre, Brasil, que incluiu gestantes admitidas com press?o arterial sist?lica ? 140 e/ou press?o arterial diast?lica ? 90 mmHg a partir da 20? semana de gesta??o. Os piores valores de vari?veis cl?nicas e laboratoriais dentro das primeiras 48 horas de admiss?o foram coletados. Desenvolvimento de eventos adversos foi acompanhado por um per?odo de 14 dias. Concentra??es plasm?ticas maternas de PlGF do momento da admiss?o foram mensuradas. Resultados ? 405 gestantes foram inclu?das no estudo. Entre as 351 mulheres inclu?das na an?lise do modelo fullPIERS, 20 (5%) desenvolveram pelo menos um evento adverso materno dentro de 14 dias de interna??o. O modelo fullPIERS teve pouca capacidade discriminativa para prever desfechos em 48 horas [AUC 0,639 (95% CI 0,458-0,819)]. A acur?cia do modelo foi ainda mais baixa dentro de sete semanas da admiss?o [AUC 0,612 (95% CI 0,440-0,783)]; a capacidade discriminativa manteve-se similar dentro de 14 dias da admiss?o [AUC 0,637 (95% CI 0,491-0,783)]. A calibra??o do modelo fullPIERS tamb?m foi ruim: inclina??o 0,35 (95% CI 0,08-0,62) e intercepto 1,13 (95%CI -2,4-0,14). A an?lise do PlGF incluiu 392 gestantes. PlGF <5? percentil esteve associados a eventos adversos maternos dentro de 48 horas em gestantes inclu?das antes de 35 semanas com sensibilidade de 0,80 (0,4-0,96), valor preditivo negativo (VPN) de 0,98 (0,9-0,99) e AUC ROC de 0,672 (IC 95% 0,5-0,9). PlGF <100 pg/mL apresentaram sensibilidade de 0,8 (0,4-0,96), especificidade de 0,6 (0,5-0,7) e VPN de 0,99 (0,94-0,99) em mulheres ap?s 37 semanas de gravidez. PlGF apresentou bom desempenho para prever parto at? 14 dias em gestantes inclu?das antes de 35 semanas. PlGF <5? percentil esteve associado a rec?m-nascido pequeno para idade gestacional (PIG) com sensibilidade de 0,75 (0,6-0,9), especificidade 0,65 (0,5-0,7), NPV de 0,87 (0,79-0,94) e AUC ROC 0,698 (0,6-0,79), em gestantes com <35 semanas, a acur?cia diminuiu com o aumento das idades gestacionais. Conclus?es ? O modelo fullPIERS e a concentra??o de PLGF mostraram baixa acur?cia na predi??o de desfechos adversos maternos em mulheres com doen?a hipertensiva gestacional, incluindo pr?-ecl?mpsia. O modelo fullPIERS teve desempenho inferior na nossa amostra quando comparado com o estudo que validou este teste. O PLGF parece ser um biomarcador para uso como ferramenta adicional na predi??o de parto dentro de 14 dias e rec?m-nascidos PIG, especialmente em gestantes antes da 35? semana gestacional. / Introduction - Singling out high-risk patients from the diverse hypertensive disorders of pregnancy, and not only preeclampsia, is a challenge for clinicians. The fullPIERS model is a simple and low-cost evaluation instrument using clinical variables to stratify the adverse outcomes probability of pregnant women with high-risk preeclampsia. Placental growth factor (PlGF) levels are reduced in preeclampsia and are increasingly being used as a biomarker in the assessment of this disease. Objectives - The aim of the study is to evaluate the performance of the fullPIERS model and PlGF to predict adverse outcomes in women with hypertensive disorders of pregnancy. Methods - A prospective cohort study carried out at a teaching hospital in Porto Alegre, Brazil enrolling pregnant women admitted with a systolic blood pressure ? 140 mmHg and/or a diastolic blood pressure ? 90 mmHg from the 20th week of gestation. First 48 hours of admission worst clinical and laboratory data were recorded and the development of adverse maternal and perinatal outcomes scrutinised up to 14 days. Admission maternal plasma PlGF concentrations were measured. Results ? A total of 405 women were enrolled. From the 351 women included in the fullPIERS model analysis, 20 (5%) developed at least one of the combined maternal adverse outcomes. The fullPIERS model had poor outcomes discrimination at 48h [AUC 0.639 (95% CI 0.458-0.819)]. At the seventh admission day, the model?s accuracy was even lower [AUC 0.612 (95% CI 0.440-0.783)]; the model?s discriminative ability remained similar [AUC 0.637 (95% CI 0.491-0.783)] at 14 days. Calibration of the fullPIERS model was poor: slope - 0.35 (95% CI 0.08-0.62), intercept -1.13 (95%CI -2.4-0.14). PlGF analysis included 392 women. PlGF < 5th percentile predicted maternal adverse outcomes within 48h in women with gestation < 35 weeks with sensitivity of 0.80, NPV of 0.98 and AUC ROC of 0.672 (CI 95%0.5-0.9). The threshold of <100 pg/mL, had best accuracy in women after 37 weeks of pregnancy, sensitivity of 0.8, specificity of 0.6, negative predictive value of 0.99 and PPV of 0.04. PlGF had good performance to predict delivery within 14 days in women presenting before 35 weeks. PlGF <5th percentile predicted delivery of a SGA infant with sensitivity of 0.75, specificity 0.65, PPV of 0.45, NPV of 0.87, and AUC ROC 0.698, in women with gestation < 35 weeks, accuracy decreased at later gestational ages. Conclusion - In conclusion, in our sample the fullPIERS model and PlGF were limited predictors of maternal adverse outcomes in pregnant women with hypertensive disorders of pregnancy, including preeclampsia. The performance of the fullPIERS model in our sample was inferior to that of the original cohort. PlGF as a biomarker appears to be an additional tool to predict delivery within 14 days and SGA newborn in women before 35 weeks gestation.

Apesar de sua importância clínica e epidemiológica, a fisiopatologia da préeclâmpsia ainda não foi completamente compreendida. Sabe-se que a doença constitui-se de uma fase pré-clínica e um estágio clínico. Durante a última década muito esforço tem se concentrado na identificação precoce da doença, ainda em sua fase pré-clínica. A literatura científica tem demonstrado claramente um desequilíbrio na regulação da angiogênese das gestantes com pré-eclâmpsia, marcado por níveis elevados do fator antiangiogênico soluble fms-like tyrosine kinase-1 (sFlt-1) e níveis diminuídos do fator pró-angiogênico placental growth fator (PlGF). Embora um número crescente de estudos em populações de alto risco tenha avaliado o papel desses biomarcadores no diagnóstico de pré-eclâmpsia, dados sobre sua utilização para a predição de pré-eclâmpsia superajuntada, cujo diagnóstico pode ser particularmente difícil, permanecem relativamente escassos e controversos. Com o presente estudo pretendemos avaliar o desempenho de medidas seriadas dos níveis maternos circulantes dos fatores sFlt-1 e PlGF, bem como da razão sFlt-1/PlGF, para predição de pré-eclâmpsia superajuntada e compará-lo ao seu desempenho na predição de pré-eclâmpsia em sua forma \"pura\", não superajuntada. Para este propósito, estudamos uma coorte prospectiva composta de dois braços, um de gestantes com hipertensão arterial crônica e outro de gestantes normotensas, e avaliamos os níveis séricos de sFlt-1 e de PlGF e a razão sFlt-1/PlGF nas idades gestacionais de 20, 26, 32 e 36 semanas, tendo como desfecho principal o diagnóstico de pré-eclâmpsia. Um total de 97 gestantes foram acompanhadas, 37 normotensas e 60 com hipertensão arterial crônica. Entre elas, 4 (10,8%) desenvolveram pré-eclâmpsia e 14 (23,3%) desenvolveram pré-eclâmpsia superajuntada. Para predição de pré-eclâmpsia, a análise ROC (Receiver Operating Characteristics) apresentou área sob a curva (AUC - area under curve) de 0,83 (IC 95% = 0,68-0,99, P = 0,035) para dosagem de PlGF com 20 semanas e AUC = 0,92 (IC 95% = 0,81 - 1,00, P = 0,007) para a razão sFlt-1/PlGF com 26 semanas de gestação. A variação percentual dos níveis de PlGF entre 26 e 32 semanas de gestação apresentou AUC = 0,96 (IC de 95% = 0,89-1,00, P = 0,003). Para a predição de pré-eclâmpsia superajuntada, a razão sFlt-1/PIGF na idade gestacional de 32 semanas apresentou AUC = 0,69 (IC de 95% = 0,53-0,85, P = 0,039). Entre 20 e 26 semanas de gestação, a variação percentual do PIGF e da razão sFlt-1/PlGF apresentaram, respectivamente, AUC = 0,74 (IC de 95% = 0,58-0,90, P = 0,018) e AUC = 0,71 (IC 95% = 0,52-0,91, P = 0,034). Por nossos resultados podemos concluir que, embora os níveis de PlGF e a razão sFlt-1/ PlGF tenham apresentado bons desempenhos na predição de pré-eclâmpsia, é preciso ter cuidado ao usá-los para a predição de pré-eclâmpsia superajuntada. Nessas gestantes, a dosagem dos fatores angiogênicos apresenta capacidade de predição menor e mais tardia. Avaliações seriadas dos fatores podem melhorar o desempenho dos testes para predição de pré-eclâmpsia superajuntada em idades gestacionais mais precoces / Despite being a major public health problem, the pathophysiology of preeclampsia is incompletely understood. Preeclampsia progression comprises a pre-clinical stage and a clinical stage. During the last decade much work has focused on identifying the pre-clinical stage of preeclampsia. Many researchers have clearly demonstrated an anti-angiogenic imbalance that is marked by higher levels of soluble fms-like tyrosine kinase-1 (sFlt-1) and lower levels of placental growth factor (PlGF) in the subjects who develop preeclampsia compared with those who do not. Although a growing number of studies in the high-risk population have shown the role of these biomarkers in diagnosing preeclampsia, superimposed preeclampsia, which can be a challenging diagnosis, remains partially understudied and the literature regarding this subject continues to be relatively scarce as well as controversial. By this study, we aimed to evaluate the performance of serial measurements of maternal circulating sFlt-1 and PlGF levels for the prediction of superimposed preeclampsia in chronic hypertensive subjects and to compare it to the prediction of preeclampsia in normotensive control subjects. For this purpose, we evaluated a two-armed prospective cohort of women with normotensive and chronic hypertensive pregnancies and assessed the serum levels of sFlt-1 and PlGF and the sFlt-1/PlGF ratio at gestational ages of 20, 26, 32 and 36 weeks, having preeclampsia as the primary outcome to be predicted. A total of 97 women were followed-up, 37 in the normotensive group and 60 in the chronic hypertensive group. Among them, 4 (10.8%) women developed preeclampsia and 14 (23.3%) developed superimposed preeclampsia. For predicting preeclampsia, PlGF at 20 gestational weeks presented an AUC=0.83 (CI 95% = 0.68 - 0.99, P=0.035) and the sFlt-1/PlGF ratio at 26 gestational weeks presented an AUC=0.92 (CI95% = 0.81 - 1.00, P=0.007). The percent change of the PlGF levels between 26 and 32 gestational weeks presented an AUC=0.96 (CI 95% = 0.89 - 1.00, P=0.003). For predicting superimposed preeclampsia, the sFlt-1/PlGF ratio at 32 gestational weeks presented an AUC=0.69 (CI 95% = 0.53 - 0.85, P=0.039). Between 20 and 26 gestational weeks, the percent change of PlGF and the sFlt-1/PlGF ratio presented, respectively, an AUC=0.74 (CI 95% = 0.58 - 0.90, P=0.018) and an AUC=0.71 (CI 95% = 0.52 - 0.91, P=0.034). By our results, we concluded that, although the PlGF level and the sFlt-1/PlGF ratio present good performances in the prediction of preeclampsia, caution is required when using them for the prediction of superimposed preeclampsia. Sequential assessments slightly improve the test performances for predicting superimposed preeclampsia at earlier gestational ages

OBJETIVOS: Comparar a expressão placentária de células natural killer deciduais (dNK), e a expressão sérica e placentária de suas citocinas reguladoras em gestações gemelares com pré-eclâmpsia (grupo pré-eclâmpsia, GPE) e sem comorbidades (grupo-controle, GC). MÉTODOS: Estudo transversal do tipo caso-controle, desenvolvido na Clínica Obstétrica do HC-FMUSP no período de julho de 2015 a junho de 2017. Foram obtidas amostras das regiões deciduais placentárias, para avaliação, por meio de técnica de imuno-histoquímica, da expressão de células dNK e suas interleucinas (IL) 10, 12 e 15, em pacientes que contemplaram os critérios de inclusão e concordaram em participar do estudo. Além disso, estas pacientes tiveram amostra sérica colhida no terceiro trimestre para dosagem de IL-10, IL-12 e IL-15 - por meio de kit comercial Milliplex®, que utiliza a tecnologia Luminex® xMAP®, da EMDMillipore (Merck Millipore Co., Alemanha) - e de fatores relacionados à angiogênese, como soluble fms-like tyrosine kinase-1 (sFlt-1) e placental growth factor (PlGF) - por meio de ensaio com imunoanalisador COBAS e411 (Roche Diagnostics, Alemanha). Os valores obtidos para as análises placentárias e séricas foram comparados entre o GPE e o GC, e a significância estatística estabelecida foi p < 0,05. RESULTADOS: Foram selecionadas 30 pacientes, sendo 20 no GC e 10 no GPE. Não se observaram diferenças significativas com relação às características maternas, gestacionais e de desfechos perinatais entre os dois grupos, exceto pela idade gestacional de início do pré-natal, menor no GPE (12,5 vs. 20,0 semanas, p = 0,015). Quanto à avaliação placentária, houve maior expressão de IL-15 no GPE (p = 0,001), e não houve diferença entre os grupos quanto à expressão placentária local de células dNK (p = 0,999), IL-10 (p = 0,063) e IL-12 (p = 0,135). Com relação às interleucinas séricas maternas, demonstrou-se aumento significativo nos níveis de IL-10 (22,7 vs. 11,9 pg/mL, p = 0,024) e IL-15 (15,9 vs. 7,4 pg/mL, p = 0,024) no GPE com relação ao GC, sem diferença entre os grupos para IL-12 (102,5 vs. 61,5 pg/mL, p = 0,373). A dosagem dos fatores relacionados à angiogênese demonstrou maiores níveis séricos maternos de sFlt-1 (15920 vs. 7978 pg/mL, p = 0,009) e da razão sFlt-1/PlGF (88,71 vs. 24,63, p = 0,002), e menores valores de PlGF (193,0 vs. 340,6 pg/mL, p = 0,036) no GPE. CONCLUSÃO: Observou-se maior concentração sérica materna tanto de fatores pró quanto anti-inflamatórios no GPE, quando comparado ao GC. Entretanto, não foram observadas diferenças entre os grupos quanto à expressão placentária de IL-10, importante fator anti-inflamatório. Estes achados podem sugerir que a tentativa sérica materna de equilibrar estas interleucinas não alcançou resposta localmente na placenta, contribuindo para o desenvolvimento da doença no GPE / OBJECTIVES: To compare the placental expression of decidual natural killer cells (dNK) and serum and placental expression of their regulatory cytokines in twin pregnancies with preeclampsia (preeclampsia group, PEG) and uncomplicated twin pregnancies (control group, CG). METHODS: This was a case-control study, developed in a tertiary referral center, from July 2015 to June 2017. Samples of the placental decidual region were obtained and analyzed by immunohistochemistry technique for the expression of dNK cells and interleukins (IL) 10, 12 and 15, in patients who met the inclusion criteria. In addition, maternal serum sample was collected in the third trimester for the dosage of IL-10, IL-12 and IL-15 - by means of a commercial Milliplex® kit using Luminex® xMAP® technology from EMDMillipore (Merck Millipore Co., Germany) - and angiogenesis factors, such as soluble fms-like tyrosine kinase-1 (sFlt-1) and placental growth factor (PlGF) - by COBAS e411 immunoassay (Roche Diagnostics, Germany). The values obtained for the placental analyzes and maternal circulating factors were compared between PEG and CG and the statistical significance was set at p < 0.05. RESULTS: Thirty patients were selected, 20 in CG and 10 in PEG. There were no significant differences in maternal, gestational and perinatal outcomes between the two groups, except for the gestational age at the onset of prenatal care, which was lower in PEG (12.5 vs. 20.0 weeks, p = 0.015). PEG showed strong immunostaining for IL-15 (p = 0.001) when compared to CG, with no difference between the groups concerning the placental expression of dNK (p = 0.999), IL-10 (p = 0.063), and IL -12 (p = 0.135). Relating to maternal circulating interleukins, a significant increase in IL-10 (22.7 vs. 11.9 pg/mL, p = 0.024) and IL-15 (15.9 vs. 7.4 pg/mL, p = 0.024) was observed in PEG, with no difference between the groups for IL-12 (102.5 vs 61.5 pg/mL, p = 0.373). We also demonstrated higher maternal levels of sFlt-1 (15920 vs. 7978 pg/mL, p = 0.009) and sFlt-1/PlGF ratio (88.71 vs. 24.63, p = 0.002) and lower levels of PlGF (193 vs. 340.6 pg/mL, p = 0.036) in PEG. CONCLUSION: A higher maternal serum concentration of both pro- and anti-inflammatory factors was observed in the PEG. However, no difference was found between the groups regarding the placental expression of IL-10, an important anti-inflammatory factor. These findings may suggest that the maternal serum attempt to balance these interleukins did not reach local placental response, which contribute to the development of the disease in the PEG

OBJETIVOS: Comparar a expressão placentária de células natural killer deciduais (dNK), e a expressão sérica e placentária de suas citocinas reguladoras em gestações gemelares com pré-eclâmpsia (grupo pré-eclâmpsia, GPE) e sem comorbidades (grupo-controle, GC). MÉTODOS: Estudo transversal do tipo caso-controle, desenvolvido na Clínica Obstétrica do HC-FMUSP no período de julho de 2015 a junho de 2017. Foram obtidas amostras das regiões deciduais placentárias, para avaliação, por meio de técnica de imuno-histoquímica, da expressão de células dNK e suas interleucinas (IL) 10, 12 e 15, em pacientes que contemplaram os critérios de inclusão e concordaram em participar do estudo. Além disso, estas pacientes tiveram amostra sérica colhida no terceiro trimestre para dosagem de IL-10, IL-12 e IL-15 - por meio de kit comercial Milliplex®, que utiliza a tecnologia Luminex® xMAP®, da EMDMillipore (Merck Millipore Co., Alemanha) - e de fatores relacionados à angiogênese, como soluble fms-like tyrosine kinase-1 (sFlt-1) e placental growth factor (PlGF) - por meio de ensaio com imunoanalisador COBAS e411 (Roche Diagnostics, Alemanha). Os valores obtidos para as análises placentárias e séricas foram comparados entre o GPE e o GC, e a significância estatística estabelecida foi p < 0,05. RESULTADOS: Foram selecionadas 30 pacientes, sendo 20 no GC e 10 no GPE. Não se observaram diferenças significativas com relação às características maternas, gestacionais e de desfechos perinatais entre os dois grupos, exceto pela idade gestacional de início do pré-natal, menor no GPE (12,5 vs. 20,0 semanas, p = 0,015). Quanto à avaliação placentária, houve maior expressão de IL-15 no GPE (p = 0,001), e não houve diferença entre os grupos quanto à expressão placentária local de células dNK (p = 0,999), IL-10 (p = 0,063) e IL-12 (p = 0,135). Com relação às interleucinas séricas maternas, demonstrou-se aumento significativo nos níveis de IL-10 (22,7 vs. 11,9 pg/mL, p = 0,024) e IL-15 (15,9 vs. 7,4 pg/mL, p = 0,024) no GPE com relação ao GC, sem diferença entre os grupos para IL-12 (102,5 vs. 61,5 pg/mL, p = 0,373). A dosagem dos fatores relacionados à angiogênese demonstrou maiores níveis séricos maternos de sFlt-1 (15920 vs. 7978 pg/mL, p = 0,009) e da razão sFlt-1/PlGF (88,71 vs. 24,63, p = 0,002), e menores valores de PlGF (193,0 vs. 340,6 pg/mL, p = 0,036) no GPE. CONCLUSÃO: Observou-se maior concentração sérica materna tanto de fatores pró quanto anti-inflamatórios no GPE, quando comparado ao GC. Entretanto, não foram observadas diferenças entre os grupos quanto à expressão placentária de IL-10, importante fator anti-inflamatório. Estes achados podem sugerir que a tentativa sérica materna de equilibrar estas interleucinas não alcançou resposta localmente na placenta, contribuindo para o desenvolvimento da doença no GPE / OBJECTIVES: To compare the placental expression of decidual natural killer cells (dNK) and serum and placental expression of their regulatory cytokines in twin pregnancies with preeclampsia (preeclampsia group, PEG) and uncomplicated twin pregnancies (control group, CG). METHODS: This was a case-control study, developed in a tertiary referral center, from July 2015 to June 2017. Samples of the placental decidual region were obtained and analyzed by immunohistochemistry technique for the expression of dNK cells and interleukins (IL) 10, 12 and 15, in patients who met the inclusion criteria. In addition, maternal serum sample was collected in the third trimester for the dosage of IL-10, IL-12 and IL-15 - by means of a commercial Milliplex® kit using Luminex® xMAP® technology from EMDMillipore (Merck Millipore Co., Germany) - and angiogenesis factors, such as soluble fms-like tyrosine kinase-1 (sFlt-1) and placental growth factor (PlGF) - by COBAS e411 immunoassay (Roche Diagnostics, Germany). The values obtained for the placental analyzes and maternal circulating factors were compared between PEG and CG and the statistical significance was set at p < 0.05. RESULTS: Thirty patients were selected, 20 in CG and 10 in PEG. There were no significant differences in maternal, gestational and perinatal outcomes between the two groups, except for the gestational age at the onset of prenatal care, which was lower in PEG (12.5 vs. 20.0 weeks, p = 0.015). PEG showed strong immunostaining for IL-15 (p = 0.001) when compared to CG, with no difference between the groups concerning the placental expression of dNK (p = 0.999), IL-10 (p = 0.063), and IL -12 (p = 0.135). Relating to maternal circulating interleukins, a significant increase in IL-10 (22.7 vs. 11.9 pg/mL, p = 0.024) and IL-15 (15.9 vs. 7.4 pg/mL, p = 0.024) was observed in PEG, with no difference between the groups for IL-12 (102.5 vs 61.5 pg/mL, p = 0.373). We also demonstrated higher maternal levels of sFlt-1 (15920 vs. 7978 pg/mL, p = 0.009) and sFlt-1/PlGF ratio (88.71 vs. 24.63, p = 0.002) and lower levels of PlGF (193 vs. 340.6 pg/mL, p = 0.036) in PEG. CONCLUSION: A higher maternal serum concentration of both pro- and anti-inflammatory factors was observed in the PEG. However, no difference was found between the groups regarding the placental expression of IL-10, an important anti-inflammatory factor. These findings may suggest that the maternal serum attempt to balance these interleukins did not reach local placental response, which contribute to the development of the disease in the PEG

A manutenção do estado progenitor durante o cultivo de células mesenquimais progenitoras derivadas do tecido adiposo (MSCs-TA), caracterizado pelo potencial de diferenciação e da capacidade de autorrenovação, é atualmente um dos maiores desafios da terapia celular. Sabendo da influência da angiogênese no desenvolvimento de tecidos de origem mesenquimal, avaliamos se um ambiente pro-angiogênico mimetizado em cultura forneceria condições para manutenção de um estado progenitor durante o processo de expansão celular. Utilizando como modelo de um ambiente pró-angiogênico o cultivo no meio EGM-2, o qual é suplementado pelos fatores de crescimento EGF, FGF-2, IGF e VEGF, nós demonstramos que a presença de tais fatores pró-angiogênicos é fundamental para a manutenção do estado progenitor de MSCs-TA em cultura. Verificamos que a presença de tais fatores de crescimento possibilitaram às MSCs-TA apresentarem um alto potencial de diferenciação adipogênico e osteogênico em comparação ao meio convencional DMEM/F12 e ao meio EBM, ausente de fatores. Além disso, o cultivo na presença de fatores pró-angiogênicos aumentou o potencial clonogênico das MSCs-TA, ao mesmo tempo em que aumentou a capacidade proliferativa destas células. Dentre os fatores de crescimento, EGF e FGF-2 foram responsáveis pelos efeitos mais robustos. Ao mesmo tempo, células cultivadas nas presença destas citocinas foram capazes de manter a morfologia fibroblastóide e apresentaram alta expressão do fator de pluripotência Klf-4. Em concordância com estes achados, o transplante subcutâneo de MSCs-TA cultivadas nestas condições mostrou que aquelas mantidas em EGM-2 geram um tecido semelhante ao tecido formado pela fração estromal vascular não cultivada. Estes resultados reforçam o papel do ambiente pró-angiogênico na manutenção do estado progenitor de MSCs-TA, e que tal estado foi proporcionado pela ação dos fatores de crescimento pró-angiogênicos EGF, FGF-2, IGF e VEGF nas células em cultivo, com destaque para as citocinas EGF e FGF-2. Em conclusão, o uso do ambiente pró-angiogênico no cultivo de MSCs-TA mostrou-se como uma abordagem promissora para a manutenção do estado progenitor destas células in vitro. / The maintenance of the progenitor state in the culture of adipose tissue derived- mesenchymal progenitor cell (TA-MSCs), characterized by the differentiation potential and self-renewal capability, is currently one of the major challenges of cell therapy. The information that the angiogenesis influences the development of mesenchymal tissues, has led us to evaluate how a pro-angiogenic environment mimicked in culture would provide conditions for maintaining a progenitor state during the cell expansion process. We designe a model for a pro-angiogenic environment in which cells grown in EGM-2 supplemented with the following growth factors: EGF, FGF-2, IGF and VEGF, and demonstrated that the presence of such pro-angiogenic growth factors was crucial for maintenance of the progenitor of AT-MSCs in culture. We observed that the presence of such growth factors allowed to AT-MSCs a high potential of adipogenic and osteogenic differentiation compared to conventional DMEM/F12 medium and the EBM medium, in the absence of the factors. Furthermore, the culture in presence of pro-angiogenic growth factors increased the clonogenic potential of AT-MSCs and increased the proliferative capability of these cells. Among the growth factors, EGF and FGF-2 were responsible for most robust effects. At the same time, cells cultured in the presence of these cytokines were able to maintaining the fibroblastoid morphology and presented high expression levels of Klf-4 pluripotency factor. In agreement with these observations, the subcutaneous transplantation of AT-MSCs cultured under these conditions showed that those cells kept in EGM-2 generated a tissue-like to tissue formed by the stromal vascular fraction uncultivated. These results reinforce the role of the pro-angiogenic environment in the maintenance of the progenitor state of AT-MSCs, and that such a state was provided by the action of the pro-angiogenic growth factors EGF, FGF-2, IGF and VEGF in cultured cells, highlighting EGF and FGF-2 cytokines. In conclusion, we showed that the use of a pro-angiogenic environment in AT-MSCs culture is a promising approach to the maintain the progenitor state of these cells in vitro.

L’Aminopeptidase-N (APN ou CD13) [EC.3.4.11.2] est une ectoenzyme homodimérique de nature glycoprotéique appartenant à la famille M1 des zinc-aminopeptidases. Elle est surexprimée à la surface des cellules endothéliales angiogéniques, ainsi que sur un certain nombre de cellules tumorales. Et il existe une corrélation étroite entre l’élévation de l’expression de l’APN, une activité enzymatique accrue et le pouvoir invasif de nombreux types de cellules tumorales. Des inhibiteurs puissants et sélectifs de l’APN, appartenant à la famille des composés de type amino-benzosubérone, ont été synthétisés au laboratoire. Ces composés ont été testés in vitro et in vivo, et il est apparu qu’ils présentaient une affinité variant du nano au picomolaire. En parallèle à ces essais, un nouveau projet a débuté il y a quelques années au laboratoire, visant à déterminer la structure tridimensionnelle de l’APN humaine. La connaissance de cette structure constitue un enjeu majeur car des co-cristallisations avec ces inhibiteurs permettraient de résoudre le mode de liaison de cette nouvelle famille de composés à l’APN. La difficulté de cette étude réside dans le fait que l’APN est une glycoprotéine membranaire particulièrement difficile à purifier à partir de tissus ; de plus, cette protéine étant ancrée dans la membrane de la cellule, sa cristallisation en est d’autant plus complexe. Plusieurs stratégies de clonage et de surexpression de l’APN humaine ont été envisagées, avec pour objectif final, l’obtention d’une protéine cristallisable, glycosylée ou non. / Aminopeptidase-N (APN or CD13) [EC.3.4.11.2] is a highly glycosylated type II membrane-bound ectoenzyme that belongs to the M1 family of zinc-dependent aminopeptidases. The members of this family have a thermolysin-like catalytic domain with the consensus HEXXH-X18-E zinc-binding sequence and an exopeptidase motif, GXMEN, in the active site. APN/CD13 is a widespread enzyme, located in many tissues, organs and cells, whose multiple functions dependent on its location. It is overexpressed on the endothelial cells of angiogenic, but not normal, vasculature, as well as on numerous tumor cells. As it was demonstrated that APN plays a critical role in tumor cell angiogenesis and metastasis, this protein was identified as a potential target for cancer therapy. In this context, highly potent and selective non-peptidic APN inhibitors, with Ki values ranging from micro to nanomolar, were previously designed and synthesized in the laboratory. In vitro and in vivo efficacy of these novel amino-benzosuberone derivatives was tested. In parallel to these works, a new project was started a few years ago which consists to solve the 3D structure of mammalian APN. Co-crystallizations with amino-benzosuberone derivatives would determine the binding mode of these novel inhibitors. Nevertheless solving the structure of a membrane protein like mammalian APN still remains a challenge. Therefore several cloning and expression strategies for human APN production were developed.

Le glioblastome (GB), tumeur primitive du cerveau, la plus agressive, et la plus fréquente chez l’adulte, présente une prolifération vasculaire importante. Des agents thérapeutiques innovants ciblant à la fois l'angiogenèse et les cellules tumorales sont recherchés, ainsi que des systèmes pour augmenter leur délivrance dans la tumeur cérébrale. Un de ces agents est le sorafénib (SFN), un inhibiteur de tyrosine kinase. Sa mauvaise solubilité aqueuse et ses effets secondaires indésirables limitent son utilisation. Le premier objectif de cette thèse était d'encapsuler cet agent dans des nanocapsules lipidiques (NCL) pour contrer ces inconvénients. Nous avons développé des NCL avec une haute efficacité d'encapsulation du SFN qui inhibaient in vitro l'angiogenèse et la viabilité de la lignée de GB humain U87MG. La délivrance intratumorale de SFN-NCL chez des souris porteuses d’une tumeur intracérébrale U87MG induit une normalisation vasculaire tumorale précoce qui pourrait améliorer l'efficacité de la chimiothérapie et de la radiothérapie. Le second objectif était de définir si la délivrance intranasale de NCL pouvait constituer une voie non-invasive alternative. Nous avons étudié via le transfert d'énergie par résonance de type Förster, le devenir des NCL chargées d’un fluorochrome à travers des monocouches de cellules Calu-3, un modèle de l'épithélium nasal. L'utilisation de NCL augmente le passage du fluorochrome à travers les cellules Calu-3, mais les particules sont rapidement dégradées après leur capture. Ces données mettent en évidence que les NCL sont appropriées pour la délivrance locale du SFN mais doivent être modifiées pour une délivrance intranasale. / Glioblastoma (GB), the most aggressive, and the most frequent primary tumor of the brain in adults, present a prominent vascular proliferation. Innovative therapeutic agents targeting both angiogenesis and tumor cells are urgently required, along with competent systems for their delivery to the brain tumor. One such agent is sorafenib (SFN), a tyrosine kinase inhibitor. However, poor aqueoussolubility and undesirable side effects limit its clinical application. The first objective of this thesis was to encapsulate this drug inside lipid nanocapsules(LNCs) to overcome these drawbacks. We developed LNCs with a high SFN encapsulation efficiency (>90%) that inhibited in vitro angiogenesis and the viability of the human U87MG GB cell line. Intratumoral delivery of SFN-LNCs in mice bearing intracerebral U87MG tumors induced early tumor vascular normalization which could be used to improve the efficacy of chemotherapy and radiotherapy in the treatment of GB. The second objective was to define whether intranasal delivery of LNCs could be an alternative non-invasive route. In this regard, we investigated through Förster resonance energy transfer, the fate of dye-loaded LNCs across Calu-3 cell monolayers, a model of the nasal mucosa. We showed that employment of LNCs dramatically increased the delivery of the dye acrossCalu-3 cell monolayer but they were rapidly degraded after their uptake. These data highlight that LNCs are suitable nanocarriers for the local delivery of SFN but must be redesigned for enhancing their nose-to-brain delivery.