The success of regenerative cardiac therapy requires reestablishing a capable blood supply via vasculature. The objective of this study was to develop an optimal scaffold formulation for de novo collateral vessel growth of aortic grafts using modified fibrin clots. This ex vivo vascular outgrowth model can be used to interrogate the complex cell or tissue interactions on the angiogenic front as vessels are formed. Based on formulation constraints, the methods used here may provide a clinically applicable option for guided collateral formation. Once understood, the methods and procedures can be tested and modified as necessary for in vivo, in situ regenerative therapy. Aortic segments from wild-type (C57BL/6J) and apolipoprotein-E deficient (ApoE) atherosclerosis-prone mice were cultured in a 3D environment created by various formulations of PEGylated fibrin. Aortic outgrowth was assessed and the optimal formulation was chosen to test the formation of de novo vascular circuits -- the first step necessary for collateral artery formation. The cultures were examined by conventional and confocal microscopy as well as by optical coherence tomography. Experiments testing the relationship between fibrin PEGylation and aortic vascular outgrowth showed that PEGylating fibrinogen prior to clot formation increased outgrowth over non-PEG control (n=6, p<.05) at lower fibrin concentrations. Lowering fibrin concentration to 10, 5, or 2.5mg/ml resulted in significantly higher outgrowth that was 1.92, 2.04, or 2.20 times that of 20mg/ml PEGylated fibrin gels. When multiple aortic segments are cultured in proximity, microvascular outgrowths visually anastamose suggesting that aorta-aorta conduits can be formed in fibrin based hydrogels. Anastomosing circuits appeared between wild-type aortic segments as well as between wild-type and atherosclerotic prone ApoE knockout segments. Fibrin gels, with or without PEGylation, form scaffolds suitable for regenerative vascular outgrowth ex vivo in normal and atherogenic environments. PEGylating fibrin prior to thrombin-initiated polymerization will allow the incorporation of growth factors or other bioactive components, making this a customizable therapy for guided collateral formation. Additionally, the incorporation of PEG itself does not limit and may actually increase the outgrowth from aortic segments in lower density gels. Finally, PEGylated fibrin gels offer an environment that will promote vascular extensions that visually anastamose, making this a viable model for ex vivo collateral formation. / text

Ocular neovascularization (NV) associated with corneal NV, ischemic retinopathies and age-related macular degeneration is a leading cause of severe vision loss. While numerous contributing factors have been identified, the potential role of the CD36 scavenger receptor has been largely overlooked notwithstanding its crucial involvement in normal retinal function. Accordingly, the central aim of this work was to elucidate the contribution and regulation of CD36 during ocular NV using the cornea as a model. / Initial work investigating the role of CD36 10 maintaining corneal avascularity, an important feature of the normal cornea, revealed that genetic ablation of CD36 elicits age-related corneal NV. Subsequent studies using a pathophysiologically relevant model of inflammatory corneal NV showed constitutive expression of CD36 in the normal cornea with marked induction in the neovascularized cornea. Importantly, activation of CD36 suppressed and induced regression of corneal NV, effects that proceeded via concerted inhibition of VEGFA, JNK-1, and cJun. / Because hypoxia is a fundamental stimulus for angiogenesis, it was pertinent to explore the role and regulation of CD36 during hypoxia. We demonstrate that CD36 expression was significantly elevated in hypoxia-exposed corneal and retinal tissue and in hypoxic retinal pigment epithelial cells. Essential contributions of hypoxia-inducible factor (HIF)-1 and reactive oxygen species were also established. Functional consequences were depicted by augmentations in CD36 phagocytic and anti-angiogenic activities. / Collectively, data disclose CD36 as an important modulator of corneal avascularity and inflammatory corneal NV; this imparts several interesting avenues for future research on the involvement of CD36 in neovascular diseases of the eye. Novel data further identify CD36 as a hypoxia and HIF-1 regulated gene thus creating a framework for future elucidation of the regulatory aspects of this receptor.

Cette thèse vise à utiliser la modélisation mathématique avec les outils du contrôle avancé, afin de guider les thérapies pour assurer la contraction de la tumeur. Les buts de cette thèse sont la contribution au développement des méthodes de la théorie des ensembles pour le contrôle robuste des systèmes non linéaires et le développement d’outils numériques pour l’analyse et le contrôle de la croissance tumorale en présence de chimiothérapie et=ou de traitement anti-angiogénique. Génériquement, dans le contexte de la théorie du contrôle, les techniques qui sont théoriquement basées sur certaines propriétés des sous-ensembles de l’espace d’état du système pourraient être désignées comme des méthodes de la théorie des ensembles. Dans la première partie, nous passons en revue les définitions, concepts et outils de la théorie des ensembles existants dans la littérature pour réponde efficacement à des problématiques de contrôle des systèmes linéaires et non linéaires avec contraintes dures et incertitudes. Dans ce cadre, nous nous intéressons à deux propriétés des ensembles qui sont l’invariance et la contraction. Les problèmes liés à la stabilité des systèmes peuvent être formulés en termes de calcul de leurs domaines d’attraction. Pour des fins de développement, nous rappelons les méthodes de la littérature pour la caractérisation de ces domaines d’attraction pour les systèmes linéaires et non linéaires. Une application importante de ces méthodes est le contrôle de la croissance tumorale en présence de différents traitements. Car dans cette application, plusieurs contraintes peuvent être posées pour éviter l’intoxication des patients pendant les traitements et les méthodes de la théorie des ensembles peuvent les prendre en compte facilement. Pour cette application, nous proposons une méthodologie pour déterminer les domaines d’attraction pour les modèles mathématiques choisis pour simuler la croissance tumorale. Dans la deuxième partie, nous proposons des méthodes de la théorie des ensemble pour la caractérisation des domaines d’attraction pour les systèmes non linéaires incertains. Au début, nous développons des conditions suffisantes pour l’invariance et la contraction d’un ellipsoïde pour des systèmes saturés. Ces conditions permettent de déterminer implicitement une fonction de Lyapunov quadratique locale. Nous montrerons que l’approche proposée est moins conservatrice que celles de la littérature, et donnerons un algorithme pour la caractérisation de l’ellipsoïde invariant et contractif. Pour les systèmes non linéaires incertains, nous développons une condition suffisante pour l’invariance contrôlable robuste pour le cas des incertitudes paramétriques. Une méthode basée sur cette condition est développée pour la caractérisation des domaines d’attraction des systèmes avec ces incertitudes. Ensuite, nous nous concentrons sur l’étude des systèmes non linéaires avec incertitudes additives, et nous donnons également une autre méthode pour la caractérisation de leurs domaines d’attraction. Ces méthodes sont des méthodes facilement traitables en utilisant les outils de l’optimisation convexe. Dans la troisième partie, nous développons des outils numériques pour la caractérisation des domaines d’attraction pour les modèles de la croissance tumorale en présence de traitements, en particulier la chimiothérapie et le traitement anti-angiogénique. Ces domaines contiennent tous les états des patients pour lesquels ils existent des protocoles de traitement efficaces. Dans ce cadre, nous considérons que les modèles sont incertains car les paramètres exactes qui les définissent sont en pratique inconnus. Ces outils sont basés sur les méthodes rappelées et développées dans cette thèse. Plusieurs informations utiles pour une thérapie tumorale efficace peuvent être extraites de ces domaines. / This thesis aims at using the mathematical modeling with advanced control tools to guide therapies for the contraction of the tumor. The aims of this thesis are the contribution to the development of the set-theoretic methods for robust control of nonlinear systems and the development of analytical tools for the analysis and control of tumor growth in presence of chemotherapy and/oranti-angiogenic therapy. Generically, in the context of control theory, techniques that are theoretically based on some properties of subsets of the system state space could be referred as set-theoretic methods.In the first part, we review the definitions, concepts and tools of the existing set-theoretic methods in the literature to respond effectively to the control issues of linear and nonlinear systems with hard constraints and uncertainties. In this context, we are interested in two properties of sets that are invariance and contractiveness. The problems associated with the stability of the systems may be formulated in terms of calculation of their domain of attraction. For development purposes, we recall methods from the literature for characterizing these domains of attraction for linear and nonlinear systems. An important application of these methods is the control of tumor growth in the presence of different treatments. For this application, several constraints can be imposed in order to avoid the patient intoxications during the treatments and the set-theoretic methods can consider easily these constraints. For this latter application, we propose a methodology to estimate the domains of attraction for the mathematical models chosen to simulate the tumor growth.In the second part, we propose set-theoretic methods for the characterization of the domains ofattraction for linear and nonlinear uncertain systems. At the beginning, we develop sufficient conditions for the invariance and contractiveness of an ellipsoid for saturated systems. These conditions allow implicitly determining a local Lyapunov function. We will show that the proposed approach is less conservative than those in the literature, and we give an algorithm for characterizing the invariant ellipsoids. For uncertain nonlinear systems, we develop a sufficient condition for the robust controlled invariance in the case of parametric uncertainties. A method based on this condition is developed for characterizing the domains of attraction for nonlinear systems with these uncertainties. Then we focus on the study of nonlinear systems with additive uncertainties, and we also give a method for the characterization of their domains of attraction. These methods are easily treatable using convex optimization tools.In the third part, we develop numerical tools for characterizing the domains of attraction for themodels of tumor growth in the presence of treatments, particularly chemotherapy and anti-angiogenictreatment. These domains contain all the states of the patients for whom effective treatment protocols exist. In this context, we consider that the models are uncertain and in particular the parameters that are unknown in practice. These tools are based on the methods developed in this thesis. Several useful informations for effective tumor therapy can be extracted from these domains.

Introdução: Sabe-se que os fatores angiogênicos e antiangiogênicos encontram-se alterados nas gestações com pré-eclâmpsia (PE), mas se desconhece seu comportamento nestes recém-nascidos (RNs). Objetivo: Dosagem do vascular endothelial growth factor (VEGF), soluble fms-like tyrosine kinase-1 (sFtl-1) e heterodímero vascular endothelial growth factor/placental growth factor (VEGF/PlGF) em pré-termos filhos de mães com PE. Métodos: Incluídos: RNs com peso de nascimento < 2.000 g e idade gestacional (IG) ≤ 34 semanas, divididos em dois grupos: filhos de mães com e sem PE. Excluídos: RNs transferidos de outra instituição com mais de 72 horas de vida, óbito antes da coleta dos exames, malformação congênita maior, erros inatos de metabolismo, gestações múltiplas, mães com infecção do grupo sífilis, toxoplasmose, rubéola, citomegalovírus, herpes (STORCH) ou vírus da imunodeficiência humana (HIV) e doença autoimune. Coletado sangue nas primeiras 72 horas de vida, e nos RNs que permaneceram internados, foi realizada uma segunda coleta com 28 dias. Foi utilizado método ELISA para a dosagem do VEGF, sFlt-1 e VEGF/PlGF. Resultados: Incluídos: 88 pacientes (37 filhos de mães com PE, 51 sem PE) com IG de 29,12 ± 2,96 semanas e peso de nascimento de 1223,80 ± 417,48 g. O VEGF foi menor no grupo com PE [32,45 (6,36-85,75) x 82,38 (35-130,03) pg/mL], p = 0,001 e o sFlt-1 foi maior no grupo com PE [1338,57 (418,8-3472,24) x 318,13 (182,03-453,66) pg/mL], p < 0,001. Na análise multivariada, o VEGF foi 80% menor e sFlt-1 13,48 vezes maior no grupo com PE. O sFlt-1 foi maior nos RNs pequenos para idade gestacional (PIG) do que nos adequados para idade gestacional (AIG) [1044,94 (290,64-3472,24) x 372,67 (236,75-860,14) pg/mL], p = 0,013. No grupo com PE, houve um aumento [≠ 151,71 (76,55-226,86); p < 0,001] entre as dosagens do VEGF entre a primeira e a segunda coleta com 28 dias, já o sFlt-1 diminuiu [≠ 1941,44 (2757,01-1125,87); p < 0,001] entre as duas dosagens. O VEGF/PlGF foi maior nos filhos de mães com PE [20,69 pg/mL (12,79-52,86) x 12,19 pg/mL (0,03 -21,58)], p = 0,003. Esses achados mantiveram-se na análise multivariada, com o VEGF/PlGF 1,05 vezes maior nos filhos de mães com PE. Os níveis de VEGF/PlGF foram inversamente proporcionais ao peso de nascimento, com p < 0,001 e r = - 0,418. Na segunda coleta com 28 dias de vida não houve diferença entre os dois grupos. Conclusão: Os maiores níveis de sFlt-1 e VEGF/PlGF e menores níveis de VEGF no grupo com PE, assim como maiores concentrações de sFlt-1 nos PIG refletem uma predominância dos mecanismos antiangiogênicos na PE e na restrição de crescimento. Os níveis de VEGF/PlGF também foram relacionados ao peso de nascimento, sendo inversamente proporcionais. O estado antiangiogênico da PE tende à normalização com 28 dias de vida. / Background: It is known that angiogenic and antiangiogenic factors are altered in pregnant women with preeclampsia (PE), but the pattern of expression of these factors in their newborn infants remains unknown. Objective: To measure vascular endothelial growth factor (VEGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and vascular endothelial growth factor/placental growth factor (VEGF/PlGF) heterodimer levels in preterm neonates born to mothers with PE. Methods: Neonates with birth weight < 2,000 g and gestational age ≤ 34 weeks were included and divided into two groups: born to mothers with and without PE. Exclusion criteria were as follows: the neonate was transferred from another institution after 72 hours of life; the neonate died before blood collection; major congenital anomalies; inborn errors of metabolism; congenital infections (STORCH screen); HIV-positive mothers; multiple pregnancies; and mothers with autoimmune disease. Blood was collected from neonates within the first 72 hours of life, and a second sample was collected at 28 days of life from those who remained hospitalized. VEGF, sFlt-1 and VEGF/PlGF levels were measured using the ELISA method. Results: A total of 88 neonates were included (37 born to mothers with and 51 without PE), with mean gestational age of 29.12 ± 2.96 weeks and birth weight of 1223.80 ± 417.48 g. VEGF was lower in the group with PE [32.45 (6.36-85.75) vs. 82.38 (35-130.03) pg/mL] (p = 0.001), and sFlt-1 was higher in the group with PE [1338.57 (418.8-3472.24) vs. 318.13 (182.03-453.66) pg/mL] (p < 0.001). In the multivariate analysis, VEGF was 80% lower and sFlt-1 was 13.48 times higher in the group with PE. sFlt-1 concentration was higher in neonates small for gestational age (SGA) than in those appropriate for gestational age (AGA) [1044.94 (290.64-3472.24) vs. 372.67 (236.75-860.14) pg/mL] (p = 0.013). In the group with PE, VEGF levels increased [≠151.71 (76.55-226.86); p < 0.001) between the first and second collection (at 28 days), while sFlt-1 levels decreased [≠1941.44 (2757.01-1125.87); p < 0.001] between the two measurements. Median VEGF/PlGF levels were significantly higher among infants born to mothers with PE (20.69 pg/mL [12.79-52.86] vs. 12.19 pg/mL [0.03-21.58], p = 0.003). These findings held on multivariate analysis, with VEGF/PlGF levels 1.05-fold higher in the PE group. VEGF/PlGF levels were inversely proportional to birth weight (p < 0.001, r = - 0.418). There were no between-group differences in blood samples collected at age 28 days. Conclusion: Higher sFlt-1 and VEGF/PlGF and lower VEGF levels in the group with PE, as well as higher sFlt-1 levels in SGA neonates, reflect a predominance of antiangiogenic mechanisms in PE and growth restriction. The VEGF/PlGF levels also affected the weight at birth, with VEGF/PlGF levels inversely proportional to birth weight. This antiangiogenic state of PE shows a trend toward normalization within 28 days of life.

Au cours des dernières années, des thérapies anti-cancéreuses ciblant l'angiogenèse tumorale ont été développées et ont démontré un bénéfice en terme de survie globale pour les patients atteints de certains cancers métastatiques. Cependant, dans de nombreux cas, les tumeurs acquièrent des résistances échappent au traitement. Le développement de nouveaux composés anti-angiogène est donc une réelle nécessité pour être proposés en seconde ligne thérapeutique. Dans ce travail, notre objectif était d'identifier de nouvelles molécules anti-angiogènes par le criblage à haut débit, de la chimiothèque académique de l'Université de Grenoble. Nous avons adapté le test de blessure sur cellules endothéliales au format des plaques de 96 puits et avons identifié une famille de molécules qui inhibent spécifiquement leur fermeture. L'activité anti-angiogène de la molécule leader (COB223) a été confirmée dans des modèles d'angiogenèse tridimensionnels in vitro, et, chez la souris, dans un modèle d'angiogenèse sous-cutanée. Nous avons testé l'activité anti-tumorale de COB223 dans un modèle de xénogreffe chez la souris et observé une diminution significative de la taille des tumeurs dans les souris traitées. A la recherche de son mécanisme d'action, nous avons observé que COB223 inhibe la prolifération cellulaire et diminue les phosphorylations de MEK et Raf, de ERK1/2 induites par le VEGF-A dans les cellules endothéliales. Nous avons également montré que COB223 n'inhibe pas les phosphorylations du VEGFR2 et de PLC. D'après ces résultats, nous proposons que la cible de COB est localisée dans la voie de signalisation VEGF/ PLC /PKC/ERK entre PKC et Ras. / Several anti-tumoral therapies targeting angiogenesis have been developed over the recent years and have demonstrated benefits for several metastatic cancers. However, in many cases, resistances to these treatments appear over time, allowing tumor escape. The development of new anti-angiogenic compounds is thus dramatically urged in order to propose second-line anti-angiogenic treatments. In this work, our aim was to identify new anti-angiogenic compounds through high throughput screening of the academic library from the University of Grenoble. We adapted the endothelial cell scratch assay to 96-well plates. We identified a family of molecules that specifically inhibited endothelial cell migration. The anti-angiogenic activity of the leader molecule (COB223) was confirmed in vitro in 3D cellular models of angiogenesis and in vivo using a mouse model of subcutaneous sponge implantation. We tested the anti-tumoral activity of COB223 on a mouse xenograft model. We observed that tumor growth was significantly reduced in treated mice correlated with decreased microvessel density. In search for its mechanism of action, we observed that COB223 inhibits cell proliferation and reduces VEGF-A-induced phosphorylation of MEK and ERK1/2 in endothelial cells. We also showed that COB223 did not affect VEGFR2 and PLC phosphorylation but reduces Raf phosphorylation responsible for its activity. These results allow us to propose that the molecular site of action of COB223 is located in the VEGF/ PLC /PKC/ERK pathway, between PKC and MEK.

Introdução: Sabe-se que os fatores angiogênicos e antiangiogênicos encontram-se alterados nas gestações com pré-eclâmpsia (PE), mas se desconhece seu comportamento nestes recém-nascidos (RNs). Objetivo: Dosagem do vascular endothelial growth factor (VEGF), soluble fms-like tyrosine kinase-1 (sFtl-1) e heterodímero vascular endothelial growth factor/placental growth factor (VEGF/PlGF) em pré-termos filhos de mães com PE. Métodos: Incluídos: RNs com peso de nascimento < 2.000 g e idade gestacional (IG) ≤ 34 semanas, divididos em dois grupos: filhos de mães com e sem PE. Excluídos: RNs transferidos de outra instituição com mais de 72 horas de vida, óbito antes da coleta dos exames, malformação congênita maior, erros inatos de metabolismo, gestações múltiplas, mães com infecção do grupo sífilis, toxoplasmose, rubéola, citomegalovírus, herpes (STORCH) ou vírus da imunodeficiência humana (HIV) e doença autoimune. Coletado sangue nas primeiras 72 horas de vida, e nos RNs que permaneceram internados, foi realizada uma segunda coleta com 28 dias. Foi utilizado método ELISA para a dosagem do VEGF, sFlt-1 e VEGF/PlGF. Resultados: Incluídos: 88 pacientes (37 filhos de mães com PE, 51 sem PE) com IG de 29,12 ± 2,96 semanas e peso de nascimento de 1223,80 ± 417,48 g. O VEGF foi menor no grupo com PE [32,45 (6,36-85,75) x 82,38 (35-130,03) pg/mL], p = 0,001 e o sFlt-1 foi maior no grupo com PE [1338,57 (418,8-3472,24) x 318,13 (182,03-453,66) pg/mL], p < 0,001. Na análise multivariada, o VEGF foi 80% menor e sFlt-1 13,48 vezes maior no grupo com PE. O sFlt-1 foi maior nos RNs pequenos para idade gestacional (PIG) do que nos adequados para idade gestacional (AIG) [1044,94 (290,64-3472,24) x 372,67 (236,75-860,14) pg/mL], p = 0,013. No grupo com PE, houve um aumento [≠ 151,71 (76,55-226,86); p < 0,001] entre as dosagens do VEGF entre a primeira e a segunda coleta com 28 dias, já o sFlt-1 diminuiu [≠ 1941,44 (2757,01-1125,87); p < 0,001] entre as duas dosagens. O VEGF/PlGF foi maior nos filhos de mães com PE [20,69 pg/mL (12,79-52,86) x 12,19 pg/mL (0,03 -21,58)], p = 0,003. Esses achados mantiveram-se na análise multivariada, com o VEGF/PlGF 1,05 vezes maior nos filhos de mães com PE. Os níveis de VEGF/PlGF foram inversamente proporcionais ao peso de nascimento, com p < 0,001 e r = - 0,418. Na segunda coleta com 28 dias de vida não houve diferença entre os dois grupos. Conclusão: Os maiores níveis de sFlt-1 e VEGF/PlGF e menores níveis de VEGF no grupo com PE, assim como maiores concentrações de sFlt-1 nos PIG refletem uma predominância dos mecanismos antiangiogênicos na PE e na restrição de crescimento. Os níveis de VEGF/PlGF também foram relacionados ao peso de nascimento, sendo inversamente proporcionais. O estado antiangiogênico da PE tende à normalização com 28 dias de vida. / Background: It is known that angiogenic and antiangiogenic factors are altered in pregnant women with preeclampsia (PE), but the pattern of expression of these factors in their newborn infants remains unknown. Objective: To measure vascular endothelial growth factor (VEGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and vascular endothelial growth factor/placental growth factor (VEGF/PlGF) heterodimer levels in preterm neonates born to mothers with PE. Methods: Neonates with birth weight < 2,000 g and gestational age ≤ 34 weeks were included and divided into two groups: born to mothers with and without PE. Exclusion criteria were as follows: the neonate was transferred from another institution after 72 hours of life; the neonate died before blood collection; major congenital anomalies; inborn errors of metabolism; congenital infections (STORCH screen); HIV-positive mothers; multiple pregnancies; and mothers with autoimmune disease. Blood was collected from neonates within the first 72 hours of life, and a second sample was collected at 28 days of life from those who remained hospitalized. VEGF, sFlt-1 and VEGF/PlGF levels were measured using the ELISA method. Results: A total of 88 neonates were included (37 born to mothers with and 51 without PE), with mean gestational age of 29.12 ± 2.96 weeks and birth weight of 1223.80 ± 417.48 g. VEGF was lower in the group with PE [32.45 (6.36-85.75) vs. 82.38 (35-130.03) pg/mL] (p = 0.001), and sFlt-1 was higher in the group with PE [1338.57 (418.8-3472.24) vs. 318.13 (182.03-453.66) pg/mL] (p < 0.001). In the multivariate analysis, VEGF was 80% lower and sFlt-1 was 13.48 times higher in the group with PE. sFlt-1 concentration was higher in neonates small for gestational age (SGA) than in those appropriate for gestational age (AGA) [1044.94 (290.64-3472.24) vs. 372.67 (236.75-860.14) pg/mL] (p = 0.013). In the group with PE, VEGF levels increased [≠151.71 (76.55-226.86); p < 0.001) between the first and second collection (at 28 days), while sFlt-1 levels decreased [≠1941.44 (2757.01-1125.87); p < 0.001] between the two measurements. Median VEGF/PlGF levels were significantly higher among infants born to mothers with PE (20.69 pg/mL [12.79-52.86] vs. 12.19 pg/mL [0.03-21.58], p = 0.003). These findings held on multivariate analysis, with VEGF/PlGF levels 1.05-fold higher in the PE group. VEGF/PlGF levels were inversely proportional to birth weight (p < 0.001, r = - 0.418). There were no between-group differences in blood samples collected at age 28 days. Conclusion: Higher sFlt-1 and VEGF/PlGF and lower VEGF levels in the group with PE, as well as higher sFlt-1 levels in SGA neonates, reflect a predominance of antiangiogenic mechanisms in PE and growth restriction. The VEGF/PlGF levels also affected the weight at birth, with VEGF/PlGF levels inversely proportional to birth weight. This antiangiogenic state of PE shows a trend toward normalization within 28 days of life.

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

Introdução: Sabe-se que os fatores angiogênicos e antiangiogênicos encontram-se alterados nas gestações com pré-eclâmpsia (PE), mas se desconhece seu comportamento nestes recém-nascidos (RNs). Objetivo: Dosagem do vascular endothelial growth factor (VEGF), soluble fms-like tyrosine kinase-1 (sFtl-1) e heterodímero vascular endothelial growth factor/placental growth factor (VEGF/PlGF) em pré-termos filhos de mães com PE. Métodos: Incluídos: RNs com peso de nascimento < 2.000 g e idade gestacional (IG) ≤ 34 semanas, divididos em dois grupos: filhos de mães com e sem PE. Excluídos: RNs transferidos de outra instituição com mais de 72 horas de vida, óbito antes da coleta dos exames, malformação congênita maior, erros inatos de metabolismo, gestações múltiplas, mães com infecção do grupo sífilis, toxoplasmose, rubéola, citomegalovírus, herpes (STORCH) ou vírus da imunodeficiência humana (HIV) e doença autoimune. Coletado sangue nas primeiras 72 horas de vida, e nos RNs que permaneceram internados, foi realizada uma segunda coleta com 28 dias. Foi utilizado método ELISA para a dosagem do VEGF, sFlt-1 e VEGF/PlGF. Resultados: Incluídos: 88 pacientes (37 filhos de mães com PE, 51 sem PE) com IG de 29,12 ± 2,96 semanas e peso de nascimento de 1223,80 ± 417,48 g. O VEGF foi menor no grupo com PE [32,45 (6,36-85,75) x 82,38 (35-130,03) pg/mL], p = 0,001 e o sFlt-1 foi maior no grupo com PE [1338,57 (418,8-3472,24) x 318,13 (182,03-453,66) pg/mL], p < 0,001. Na análise multivariada, o VEGF foi 80% menor e sFlt-1 13,48 vezes maior no grupo com PE. O sFlt-1 foi maior nos RNs pequenos para idade gestacional (PIG) do que nos adequados para idade gestacional (AIG) [1044,94 (290,64-3472,24) x 372,67 (236,75-860,14) pg/mL], p = 0,013. No grupo com PE, houve um aumento [≠ 151,71 (76,55-226,86); p < 0,001] entre as dosagens do VEGF entre a primeira e a segunda coleta com 28 dias, já o sFlt-1 diminuiu [≠ 1941,44 (2757,01-1125,87); p < 0,001] entre as duas dosagens. O VEGF/PlGF foi maior nos filhos de mães com PE [20,69 pg/mL (12,79-52,86) x 12,19 pg/mL (0,03 -21,58)], p = 0,003. Esses achados mantiveram-se na análise multivariada, com o VEGF/PlGF 1,05 vezes maior nos filhos de mães com PE. Os níveis de VEGF/PlGF foram inversamente proporcionais ao peso de nascimento, com p < 0,001 e r = - 0,418. Na segunda coleta com 28 dias de vida não houve diferença entre os dois grupos. Conclusão: Os maiores níveis de sFlt-1 e VEGF/PlGF e menores níveis de VEGF no grupo com PE, assim como maiores concentrações de sFlt-1 nos PIG refletem uma predominância dos mecanismos antiangiogênicos na PE e na restrição de crescimento. Os níveis de VEGF/PlGF também foram relacionados ao peso de nascimento, sendo inversamente proporcionais. O estado antiangiogênico da PE tende à normalização com 28 dias de vida. / Background: It is known that angiogenic and antiangiogenic factors are altered in pregnant women with preeclampsia (PE), but the pattern of expression of these factors in their newborn infants remains unknown. Objective: To measure vascular endothelial growth factor (VEGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and vascular endothelial growth factor/placental growth factor (VEGF/PlGF) heterodimer levels in preterm neonates born to mothers with PE. Methods: Neonates with birth weight < 2,000 g and gestational age ≤ 34 weeks were included and divided into two groups: born to mothers with and without PE. Exclusion criteria were as follows: the neonate was transferred from another institution after 72 hours of life; the neonate died before blood collection; major congenital anomalies; inborn errors of metabolism; congenital infections (STORCH screen); HIV-positive mothers; multiple pregnancies; and mothers with autoimmune disease. Blood was collected from neonates within the first 72 hours of life, and a second sample was collected at 28 days of life from those who remained hospitalized. VEGF, sFlt-1 and VEGF/PlGF levels were measured using the ELISA method. Results: A total of 88 neonates were included (37 born to mothers with and 51 without PE), with mean gestational age of 29.12 ± 2.96 weeks and birth weight of 1223.80 ± 417.48 g. VEGF was lower in the group with PE [32.45 (6.36-85.75) vs. 82.38 (35-130.03) pg/mL] (p = 0.001), and sFlt-1 was higher in the group with PE [1338.57 (418.8-3472.24) vs. 318.13 (182.03-453.66) pg/mL] (p < 0.001). In the multivariate analysis, VEGF was 80% lower and sFlt-1 was 13.48 times higher in the group with PE. sFlt-1 concentration was higher in neonates small for gestational age (SGA) than in those appropriate for gestational age (AGA) [1044.94 (290.64-3472.24) vs. 372.67 (236.75-860.14) pg/mL] (p = 0.013). In the group with PE, VEGF levels increased [≠151.71 (76.55-226.86); p < 0.001) between the first and second collection (at 28 days), while sFlt-1 levels decreased [≠1941.44 (2757.01-1125.87); p < 0.001] between the two measurements. Median VEGF/PlGF levels were significantly higher among infants born to mothers with PE (20.69 pg/mL [12.79-52.86] vs. 12.19 pg/mL [0.03-21.58], p = 0.003). These findings held on multivariate analysis, with VEGF/PlGF levels 1.05-fold higher in the PE group. VEGF/PlGF levels were inversely proportional to birth weight (p < 0.001, r = - 0.418). There were no between-group differences in blood samples collected at age 28 days. Conclusion: Higher sFlt-1 and VEGF/PlGF and lower VEGF levels in the group with PE, as well as higher sFlt-1 levels in SGA neonates, reflect a predominance of antiangiogenic mechanisms in PE and growth restriction. The VEGF/PlGF levels also affected the weight at birth, with VEGF/PlGF levels inversely proportional to birth weight. This antiangiogenic state of PE shows a trend toward normalization within 28 days of life.

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&le;0,05). A membrana não mostrou efeito significativo no processo de regeneração óssea e não houve diferença estatística entre os diferentes biomateriais testados (p&ge;0,05). O gel carreador apresentou menor formação óssea quando comparado aos demais materiais, sem considerar o tempo e a presença da membrana (p&le;0,05). Houve um aumento significativo da formação óssea, osseointegração e da estabilidade ao longo do tempo para os diferentes biomateriais empregados (p&le;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&ge;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&le;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&ge;0.05). Gel carrier group showed less bone formation than other filling materials, not considering recovery time and membrane (p&le;0.05). Latex angiogenic proteins showed bone regeneration similar to coagulum and bone graft at 4 and 12 weeks, respectively (p&ge;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.

Cette thèse présente des travaux de modélisation mathématique de la croissance tumorale. On détaille dans ce manuscrit la construction d’indicateurs de bio-imagerie, destinés à quantifier l’hétérogénéité tumorale. Un modèle d’équations aux dérivées partielles constitué de deux types de cellules tumorales est étudié par la suite. Le paramétrage de ce modèle est propre à chaque patient et à chaque lésion. Il est effectué grâce à des données d’imagerie médicale (IRM ou scanner), ce qui constitue une méthode non invasive pour le patient. Les indicateurs ainsi que le modèle décrit ont été utilisés dans le cadre du suivi des métastases des lésions rénales de 5 patients traités avec un médicament anti-angiogénique. Enfin, la dernière partie a pour objectif de distinguer différents types de lésions rénales (malignes ou non) grâce à l’imagerie afin de limiter les chirurgies inutiles. On s’est particulièrement attaché à distinguer les carcinomes rénaux à cellules claires des angiomyolipomes pauvres en graisse. / This document deals with mathematical modelling of tumour growth. Biological indicators based on medical images are constructed in order to quantify tumoral heterogeneity. In the first part, a partial differential equations model made of two distinct cell subtypes is being studied. The model’s parameters are unique for each patient and each lesion. They are computed thanks to medical images (MRI or scan), which is a non-invasive method for the patient. Both the indicators and the model described are used on the cases of 5 patients treated with an anti-angiogenic medicine. The last part of the document aims at distinguishing different renal tumour subtypes that can be malignant or benign. Angiomyolipomas and renal cells carcinomas were particulary studied in the last part of the document.