Les méthodes de simulation Monte-Carlo s’étendent avec succès à différents domaines de la physique médicale mais aussi à différentes échelles, par exemple de la planification des traitements de radiothérapie jusqu’à une prévision des effets des rayonnements au niveau des cellules cancéreuses. La plateforme de simulation Monte-Carlo GATE, basée sur l’outil Geant4, propose des fonctionnalités dédiées aux simulations en physique médicale (médecine nucléaire et radiothérapie). Pour les applications en radiobiologie, les modèles physiques Geant4-DNA implémentés jusqu’à très basse énergie (eV) permettent d’estimer des quantités micro-dosimétriques d’intérêt. Dans le but d’implémenter une plateforme de simulation Monte-Carlo multi-échelles, nous nous sommes d’abord intéressés à la validation des modèles physiques de Geant4-DNA, puis à leur intégration dans la plateforme de simulation GATE et enfin à une validation de cette implémentation dans un contexte de radiothérapie et protonthérapie. De manière à valider les modèles physiques de Geant4-DNA, des points kernels de dose en électrons mono-énergétiques (de 10 keV à 100 keV) ont été simulés en utilisant les modèles physiques de Geant4 et de Geant4-DNA et ils ont comparés au code Monte-Carlo EGSnrc. Les parcours et pouvoirs d’arrêts des électrons (de 7,4 eV à 1 MeV) et des protons (de 1 keV à 100 MeV) calculés avec Geant4-DNA (processus et modèles préalablement intégrés dans GATE) ont ensuite été validés. Nous avons alors proposé de simuler avec la plateforme GATE l’impact de faisceaux cliniques et pré-cliniques sur l’ADN cellulaire. Nous avons ainsi modélisé un faisceau de protonthérapie de 193,1 MeV, un accélérateur linéaire en mode électrons de 6 MeV et un irradiateur RX de 250 kV. Ces simulations ont d’abord été validées en milieu aqueux par une comparaison de la dose macroscopique avec des mesures expérimentales. Les faisceaux ont ensuite été utilisés pour calculer, pour chacun d’entre eux, les fréquences de dépôts d’énergie à l’ADN. La molécule d’ADN a été simulée tout d’abord grâce à des cylindres équivalents en dimension à 10 paires de base (2 nm x 2 nm), équivalents à la taille d’un nucléosome (10 nm x 5 nm) et équivalents à la taille d’une fibre de chromatine (25 nm x 25 nm). Tous ces cylindres ont été placés aléatoirement dans un volume d’eau liquide (de rayon 500 nm). Nous avons ensuite reconstruit la molécule d’ADN dans Geant4 à partir de la lecture de fichiers PDB (Protein Data Bank) représentant douze paires de base de la molécule d’ADN et un dinucléosome (347 paires de base). Enfin, nous avons développé un outil permettant de corréler les positions de dépôts d’énergie directs dans l’eau liquide avec les coordonnées des paires de base de l’ADN, afin de calculer les nombres de cassures simple et double brin de l’ADN. Tous les calculs réalisés au cours de ce travail, ont été déployés sur l’Infrastructure de Grille Européenne ; des tests de performance sont proposés pour mesurer l’intérêt de ce type d’architecture pour les calculs Monte-Carlo. / The Monte Carlo simulation methods are successfully being used in various areas of medical physics but also at different scales, for example, from the radiation therapy treatment planning systems to the prediction of the effects of radiation in cancer cells. The Monte Carlo simulation platform GATE based on the Geant4 toolkit offers features dedicated to simulations in medical physics (nuclear medicine and radiotherapy). For radiobiology applications, the Geant4-DNA physical models are implemented to track particles till very low energy (eV) and are adapted for estimation of micro-dosimetric quantities. In order to implement a multi-scale Monte Carlo platform, we first validated the physical models of Geant4-DNA, and integrated them into GATE. Finally, we validated this implementation in the context of radiation therapy and proton therapy. In order to validate the Geant4-DNA physical models, dose point kernels for monoenergetic electrons (10 keV to 100 keV) were simulated using the physical models of Geant4-DNA and were compared to those simulated with Geant4 Standard physical models and another Monte Carlo code EGSnrc. The range and the stopping powers of electrons (7.4 eV to 1 MeV) and protons (1 keV to 100 MeV) calculated with GATE/Geant4-DNA were then compared with literature. We proposed to simulate with the GATE platform the impact of clinical and preclinical beams on cellular DNA. We modeled a clinical proton beam of 193.1 MeV, 6 MeV clinical electron beam and a X-ray irradiator beam. The beams models were validated by comparing absorbed dose computed and measured in liquid water. Then, the beams were used to calculate the frequency of energy deposits in DNA represented by different geometries. First, the DNA molecule was represented by small cylinders : 2 nm x 2 nm ( 10 bp), 5 nm x 10 nm ( nucleosome) and 25 nm x 25 nm ( chromatin fiber). All these cylinders were placed randomly in a sphere of liquid water (500 nm radius). Then we reconstructed the DNA molecule in Geant4 by reading PDB (Protein Data Bank) files representing twelve base pairs of the DNA molecule and a dinucleosome (347 base pairs). Finally, we developed a tool to correlate the positions of direct energy deposit in liquid water with the coordinates of the base pairs of DNA to calculate the number of single and double strand breaks in DNA. All calculations in this work were perfomed on the European Grid Infrastructure; performance tests are available to estimate the utility of this type of architecture for Monte Carlo calculations.

L'objectif de ce travail de recherche est le développement, la mise au point et la validation d'une méthode de spectroscopie multi-modalités en diffusion élastique et autofluorescence pour caractériser des tissus cutanés cicatriciels hypertrophiques in vivo. Ces travaux sont reposés sur trois axes. La première partie des travaux présente l'instrumentation : développement d'un système spectroscopique qui permet de réaliser des mesures de multimodalités in vivo de manière automatique et efficace. Des procédures métrologiques sont mise en place pour caractériser le système développé et assurer la repétabilité les résultats de mesure. La deuxième partie présente une étude préclinique. Un modèle animal et un protocole expérimental ont été mises en place pour créer des cicatrices hypertrophiques sur lesquelles nous pouvons recueillir des spectres à analyser. La troisième partie porte sur la classification des spectres obtenus. Elle propose des méthodes algorithmiques pour débruiter et corriger les spectres mesurés, pour extraire automatiquement des caractéristiques spectrales interprétables et pour sélectionner un sous-ensemble de caractéristiques "optimales" en vue d'une classification efficace. Les résultats de classification réalisée respectivement par trois méthodes (k-ppv, ADL et RNA) montrent que la faisabilité d'utiliser la spectroscopie bimodale pour la caractérisation de ce type de lésion cutané. Par ailleurs, les caractéristiques sélectionnées par notre méthode montrent que la cicatrisation hypertrophique implique un changement de structure tissulaire et une variation de concentration de porphyrine / This research activity aims at developing and validating a multimodal system combining diffuse reflectance spectroscopy and autofluorescence spectroscopy in characterizing hypertrophic scar tissues in vivo. The work relies on three axes. The first part concerns the development of an automatic system which is suitable for multimodal spectroscopic measurement. A series of calibration procedures are carried out for ensuring the reliability of the measurement result. The second part presents a preclinical study on an animal model (rabbit ear). An experimental protocol was implemented in order to create hypertrophic scars on which we can collect spectra to analyze. The third part deals with the classification problem on the spectra obtained. It provides a series of algorithmic methods for denoising and correcting the measured spectra, for automatically extracting some interpretable spectral features and for selecting an optimal subset for classification. The classification results arched using respectively 3 different classifiers (knn, LDA and ANN) show the ability of bimodal spectroscopy in characterization of the topic skin lesion. Furthermore, the features selected my selection method indicate that the hypertrophic scarring may involve a change in tissue structure and in the concentration of porphyrins embedded in the epidermis

Das Bronchialkarzinom ist die häufigste Todesursache bei den Krebserkrankungen und weist eine schlechte Prognose auf. Die Behandlung besteht aus einer Chemotherapie mit platinbasierten Medikamenten, doch der Erfolg ist unbefriedigend. In den letzten Jahren wurden zielgerichtete Therapien gegen Proteine wie den EGFR entwickelt. Klinische Studien zeigten, dass nur Subpopulationen von den Medikamenten Erlotinib und Cetuximab profitieren. Eine bessere (Vor-)Selektion der Patienten ist wünschenswert, um unnötige Behandlungen zu vermeiden. Für diese Analysen bedarf es relevanter präklinischer Modelle. Im Rahmen dieser Arbeit wurden 25 Xenograftmodelle des Lungenkarzinoms vergleichend charakterisiert. Ein Schwerpunkt bestand im Vergleich der Xenografts mit ihren Patiententumoren. Die Analyse der Histologie, der Proliferationsmarker als auch der Genexpressionsprofile fand übereinstimmende Ergebnisse in den Patiententumoren und ihren abgeleiteten Xenografts. Mit Hilfe von mRNA-, Protein- und SNP-Profilen ressistenzassoziierter Marker der Chemotherapie konnte die Bedeutung der Modelle zur Charakterisierung von prädiktiven und prognostischen Markern aufklärt werden. Diese Arbeit untersuchte auch Marker der anti-EGFR-Therapien. mRNA- und Proteinprofile der ERBB-Rezeptoren sowie der Liganden wurden erstellt und stimmten mit publizierten klinischen Daten überein. Genexpressionsstudien in Erlotinib Respondern und Non-Respondern zur Therapieoptimierung identifizierten den Wachstumsfaktor VEGFA als Ziel für eine Kombinationsbehandlung mit dem Angiogeneseinhibitor Bevacizumab. Die Kombination von Bevacizumab mit Erlotinib führte zu einem reduzierten Tumorwachstum. Die Ergebnisse dieser Arbeit machten deutlich, dass die individuellen Tumoreigenschaften in den patientenabgleiteten Xenografts auf Gen- und Proteinebene erhalten bleiben und diese als Modelle zur Markeranalyse sowie zur Therapieoptimierung eingesetzt werden können. / Lung cancer is still one of the most frequent cancers worldwide. The treatment option is classical chemotherapy that is based upon the combination of platin-based drugs. But no further improvement seems to be possible. For some years targeted drugs against single proteins like the EGFR were developed. The clinical trials showed that only subpopulations of patients benefit from the treatment. A better selection of patients to avoid treatment would be helpful. Therefore, pre-clinical models that are suitable for analysis and that represent clinical populations of patients are required. In this work 25 patient derived xenografts from lung cancer were intensely studied. First, the xenografts were compared with their corresponding patient tumor. The analysis of the histology and the expression of proliferation and epithelial or mesenchymal markers showed concordance of the patient tumor and the derived xenograft. The gene expression profiles were also maintained. Further analysis should elucidate the relevance of the xenografts as models for the characterisation and validation of predictive and prognostic markers. SNP, mRNA and protein expression profiles of resistance markers for chemotherapy were generated and showed similarities with clinical data. As marker for the anti-EGFR targeted therapies the ERBB receptors and the ligands of the EGFR were analysed. The mRNA and protein expression profiles resemble clinical data sets. An optimisation of the therapy should be achieved with gene expression studies. The vascular endothelial growth factor A was identified for a combination treatment with the anti-angiogenic drug bevacizumab in erlotinib resistant tumors. The combination of erlotinib and bevacizumab reduced the tumor growth in selected models. In summary, the analysis could show that the individual characteristics of the patient tumor were maintained in the xenograft. The models are a reliable tool for studies designed to improve treatment strategies.

Les micro-irradiateurs pour la radiothérapie préclinique du petit animal permettent d’effectuer des irradiations au plus proche des techniques de traitement chez l’homme, facilitant la transposition de résultats d'études radiobiologiques à la clinique. La spécificité des faisceaux millimétriques de moyenne énergie (< 300 keV) utilisés génère cependant des problématiques dosimétriques inédites. Ce travail de thèse a consisté à mettre en œuvre la dosimétrie par fibre scintillante plastique pour ce domaine d’utilisation, là où peu de détecteurs conviennent. Dans une première partie, les faisceaux d’un micro-irradiateur ont été caractérisés en dose d’une part et leur spectres en énergie obtenus par simulations Monte Carlo d’autre part, afin d’étudier les performances du dosimètre prototype. La deuxième partie a montré ses excellentes caractéristiques dosimétriques telles que la répétabilité, reproductibilité et linéarité de réponse. Un des enjeux majeurs a alors été de caractériser sa dépendance en énergie, problématique inhérente à la dosimétrie à moyenne énergie et intrinsèque au scintillateur plastique en dessous de 100 keV. Une méthode d’étalonnage a été proposée pour prendre en compte cette dépendance en conditions précliniques (mini-faisceaux et petit volume diffusant), à partir de spectres en énergie simulés. Le dosimètre a ensuite été utilisé pour la vérification de plans de traitement sur fantôme puis in vivo sur des rats, avec des résultats très concluants. Il a montré des performances prometteuses pour l’évaluation en temps réel de la dose délivrée aux tumeurs soumises aux mouvements respiratoires des animaux. / Small animal micro-irradiators designed for preclinical radiotherapy experiments mimic human clinical irradiation techniques thus facilitating the transposition of radiobiological research findings to clinical practice. These devices deliver millimetric x-ray beams of medium-energy (< 300 keV) which implies specific dosimetric issues. The objective of this thesis was the implementation of plastic scintillating fiber dosimetry in this specific field of use, for which few existing dosimeters are suitable. In a first part, beams from a micro-irradiator were characterized. Dosimetric measurements along with energy spectra Monte Carlo simulations allowed the study of the dosimeter prototype performances. In the second part of this work, excellent dosimetric properties of the detector such as repeatability, reproducibility and dose response linearity were shown. Then, a major issue was to determine the detector energy dependence, which is inherent to medium-energy dosimetry and also an intrinsic property of plastic scintillator, below 100 keV. A calibration method based on the simulated energy spectra was proposed to correct this dependence in preclinical conditions (mini-beams, small scattering volume). The dosimeter showed very conclusive results for treatment plan verification in a heterogene phantom and during rats in vivo experiments. The dosimeter also demonstrated promising performances for online control of the delivered dose to mobile tumors, subject to the animal respiratory movements.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-09T14:22:46Z No. of bitstreams: 2 Dissertação - Alarisse Arçari Fachetti Zoghaib - 2015.pdf: 1188040 bytes, checksum: 9ab52dc612351ab19ab98e90e7ce6ec9 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-09T14:24:14Z (GMT) No. of bitstreams: 2 Dissertação - Alarisse Arçari Fachetti Zoghaib - 2015.pdf: 1188040 bytes, checksum: 9ab52dc612351ab19ab98e90e7ce6ec9 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-08-09T14:24:14Z (GMT). No. of bitstreams: 2 Dissertação - Alarisse Arçari Fachetti Zoghaib - 2015.pdf: 1188040 bytes, checksum: 9ab52dc612351ab19ab98e90e7ce6ec9 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-09-28 / The ruthenium (II)/amino acid complex (rutrpII) demonstrated high anticancer activity against murine breast cancer (tumor of Ehrlich) in vitro and in vivo, and increased the median survival of animals. There was then necessary to investigate the pharmacokinetic parameters of the drug prototype as a requirement to pre-clinical knowledge of it. This study aimed to obtain the pharmacokinetic profile of ruthenium (II) / tryptophan administered to rats intraperitoneally in a single dose by quantifying this substance in plasma using validated analytical methodology in UV-VIS spectrophotometer. The methodology consisted of the administration of the prototype rutrpII to 3 rats at a dose of 6 mg/kg, intraperitoneally. Samples of blood 1.0 ml were collected by cannulation of the left jugular vein with heparinized syringe, the intervals from 0 to 9 hr. After centrifugation the plasma was frozen at -20 until the time of analysis. The bioanalytical method to quantify rutrpII was developed and validated in UV-VIS at a wavelength of 417 nm. From the construction of the concentration versus time curve, the kinetic parameters were calculated (Software WinNonlin 5.0 (Pharsight ™) Results were: Tmax = 8 h, Cmax = 169.86 mg/mL, t1/2 = 1.04 ± 0.02 h, ClT/F = 1.32 ± 0.05 mL/min/kg, Vd/F = 1,98 ± 0,05 L/kg. The developed and validated bioanalytical method was suitable for the detection and quantification of RutrpII in rat plasma. The values of pharmacokinetic profiles found, and extrapolated on allometric scaling allow us to understand that the compound studied showed a slow tissue distribution profile and / or was eliminated slowly (Vd low and low value CLT), due to a possible affinity between RutrpII and plasma proteins. This affinity may be explained by the similarity of their chemical structure with iron, enabling it to be transported by biomolecules such as transferrin, albumin or any other, and having as a target the DNA of cancer cells. In general, the compounds of ruthenium (II) / amino acids may be promising drugs for the amino acids present in the complex, may facilitate the recognition of the complex as a whole by DNA, and thus less toxic. / O complexo de rutênio(II)/aminoácido (rutrpII) demonstrou alta atividade antineoplásica contra carcinoma de mama murino (tumor de Ehrlich) in vitro e in vivo, sendo que neste último, aumentou a média de sobrevida dos animais. Fez-se necessária então a investigação dos parâmetros farmacocinéticos deste protótipo de fármaco como requisito ao conhecimento pré-clínico do mesmo. O objetivo deste trabalho foi obter o perfil farmacocinético do rutênio(II)/triptofano administrado a ratos, via intraperitoneal, em dose única por meio da quantificação desta substância em plasma utilizando metodologia analítica validada em espectrofotômetro UV-VIS. A metodologia consistiu na administração do protótipo rutrpII a 3 ratos em dose de 6 mg/kg, por via intraperitonal. Foram coletadas amostras de 1,0 mL de sangue, por canulação da veia jugular esquerda, com seringa heparinizada, nos intervalos de tempo de 0 a 9 h. Após centrifugação, o plasma foi congelado a -20ºC até o momento da análise. O método bioanalítico de quantificação do rutrpII foi desenvolvido e validado em espectrofotômetro UV-VIS no comprimento de onda de 417 nm. A partir da construção da curva de concentração versus tempo, foram calculados os parâmetros cinéticos (Software Winnonlin 5.0 (Pharsight™)). Os resultados encontrados foram: Tmax= 8h, Cmax= 169,86 µg/mL, t(1/2) =1,04 ± 0,02 h,ClT/F = 1,32 ± 0,05 mL/min/kg, Vd/F = 1,98 ± 0,05 L/kg. O método bioanalítico desenvolvido e validado foi adequado para a detecção e quantificação do RutrpII em plasma de rato. Os valores dos perfis farmacocinéticos encontrados, e extrapolados em escala alométrica, permitem entender que o composto estudado apresentou um perfil lento de distribuição tecidual e/ou foi eliminado lentamente (baixo Vd e baixo valor de CLT), devido a uma possível afinidade entre o RutrpII e as proteínas plasmáticas. Esta afinidade pode ser explicada pela semelhança de sua estrutura química com o ferro, possibilitando que ele seja transportado por biomoléculas como a transferrina, albumina ou alguma outra, e tendo como alvo, o DNA da células cancerosas. De maneira geral os compostos de rutênio(II)/ aminoácidos podem ser fármacos promissores pois os aminoácidos presentes nos complexos, podem facilitar o reconhecimento do complexo como um todo pelo DNA, sendo assim menos tóxicos.

Wang, Zexi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 63-64). / Abstract --- p.i / Acknowledgement --- p.iii / Chapter 0 --- Introduction --- p.1 / Chapter 1 --- The Design of the Experiments --- p.4 / Chapter 1.1 --- Flies and the Devices --- p.5 / Chapter 1.2 --- Parameter Settings and Interested Information --- p.8 / Chapter 2 --- Video Processing --- p.11 / Chapter 2.1 --- "Videos, Computer Vision and Image Processing" --- p.11 / Chapter 2.2 --- Details in Video Processing --- p.14 / Chapter 3 --- Data Analysis --- p.20 / Chapter 3.1 --- Background --- p.20 / Chapter 3.2 --- Outline of Data Analysis in Our Project --- p.22 / Chapter 4 --- Effect of the Medicine --- p.25 / Chapter 4.1 --- Hypothesis Testing --- p.26 / Chapter 4.2 --- Two-sample t Test --- p.28 / Chapter 5 --- Significance of the Two Factors --- p.32 / Chapter 5.1 --- Background of ANOVA --- p.33 / Chapter 5.2 --- The Model of ANOVA --- p.35 / Chapter 5.3 --- Two-way ANOVA in Our Data Analysis --- p.42 / Chapter 6 --- Regression Model --- p.45 / Chapter 6.1 --- Background of Regression Analysis --- p.47 / Chapter 6.2 --- Polynomial Regression Models --- p.52 / Chapter 6.2.1 --- Background --- p.52 / Chapter 6.2.2 --- R2 and adjusted R2 --- p.53 / Chapter 6.3 --- Model Verification --- p.58 / Chapter 6.4 --- A Simpler Model As the Other Choice --- p.59 / Chapter 6.5 --- Conclusions --- p.60 / Chapter 7 --- Further Studies --- p.61 / Bibliography --- p.62

Les tumeurs mammaires sont connues pour présenter une grande hétérogénéité intratumorale qui contribue à l’échec thérapeutique et à la progression de la maladie. L’origine de dette hétérogénéité s’explique principalement par l’organisation hiérarchique des tissus tumoraux où plusieurs sous-populations de cellules souches de cancer du sein (bCSC) sont capables de s’auto-renouveler et de maintenir l’architecture oligoclonale de la tumeur. Dans la mesure où les bCSC stimulent la croissance tumorale, résistent aux thérapies conventionnelles et initient le développement des métastases, il est indispensable de développer des thérapies spécifiques ciblant ces cellules. L’élaboration d’une telle stratégie nécessite la compréhension des propriétés moléculaires intrinsèques des bCSC. Pour mieux comprendre leur biologie, nous avons isolé les bCSC de différentes xénogreffes dérivées de tumeurs de patientes et établit leurs profil d’expression génique. Nous avons identifié un programme transcriptionnel pouvant être impliqué dans la réduction du stress réplicatif (SR) des bCSC . Nous avons montré que comparé aux non-bCSC, les bCSC présentent une sur-activation de la recombinaison homologue qui leur permet de réduire leur niveau de SR. Nous avons ensuite montré en réalisant un essai clinique que l’inhibition de cette voie permet de les sensibiliser à des agents génotoxique. Ces travaux identifient le SR comme le talon d’Achille des bCSC et mettent en évidence la recombinaison homologue comme cible potentielle pour sensibiliser les BCSC aux thérapies conventionnelles. / Breast tumors are known to present a major intratumoral heterogeneity that contributes to therapy failure and disease progression. The origin of this cellular heterogeneity is mainly explained by a hierarchical organization of tumor tissues where several subpopulations of self-renewing breast cancer stem cells (bCSCs) sustain the long-term oligoclonal maintenance of the neoplasm. bCSCs drive tumor growth, resist to conventional therapies and initiate metastasis development. Thus, developing bCSC-targeting therapies is becoming a major challenge requiring the understanding of the unique molecular circuitry of bCSC as compared to non-bCSC. To better understand the biology of these cells, we isolated bCSCs from different patient–derived xenografts (PDXs), derived fom breast tumors, and established their gene expression profiles. We identified a bCSC core transcriptional program that may be implicated in the reduction of the replicative stress in CSC: overexpression of genes implicated in dNTP metabolism and homologous recombination (HR). Our results show that HR plays a major role in SR regulation of bCSC and that bCSC are more resistant to RS than non-bCSC, We realized a preclinical assay in PDX and showed that HR inhibition prevent bCSC expansion Cisplatin-induced, suggesting a sensitization of the bCSC to the chemotherapy. Our results identify replication stress as the Achilles’ heel of bCSC and highlights HR as potential targets for anti-bCSC therapy.

Les glioblastomes (GBs) sont les tumeurs primitives du SNC les plus agressives de l’adulte. Les causes d’échec thérapeutiques sont multiples, comme une résistance des cellules tumorales à l’apoptose, l’existence de cellules souches cancéreuses ou un microenvironnement pro-tumoral. La découverte de molécules thérapeutiques qui pourraient avoir une action pléiotrope est particulièrement attractive. Dans ce contexte nous nous sommes intéressés aux mimétiques de Smac (MS), antagonistes des inhibiteurs de l’apoptose (IAP), qui inhibent le plus souvent cIAP1, cIAP2, XIAP et ML-IAP. Nous avons recherché si les IAP pouvaient être des cibles thérapeutiques dans les GBs humains en étudiant leur expression et leurs valeurs pronostiques éventuelles : les IAP sont exprimés dans les GBs et ML-IAP est associé à un plus mauvais pronostic. Nous avons choisi d’utiliser pour la suite de nos expériences, un MS qui avait une action sur les IAP et en particulier ML-IAP : le GDC-0152. Le GDC-0152 induit l’apoptose in vitro, augmente la survie des souris porteuses de GB et ralentit la croissance tumorale in vivo. Ensuite nous avons recherché si l’effet du GDC-0152 pouvait être différent en fonction du taux d'oxygène. En effet, les GBs sont des tumeurs hypoxiques. Nous avons cultivé en normoxie et en hypoxie, quatre lignées de cellules souches de GBs. En normoxie, le GDC-0152 induit la différenciation des cellules souches (voie NF-κB) et en hypoxie il induit l’apoptose et diminue la prolifération cellulaire (voie ATR).Ces travaux soulignent l’importance du modèle préclinique utilisé dans la caractérisation de l'effet de nouvelles molécules et le potentiel thérapeutique des MS dans les GBs. / Glioblastomas (GBs) are the most aggressive primary brain tumors in adults. The causes of therapeutic failure are unknown and are multiples, such as tumor cell resistance to apoptosis, the presence of cancer stem cells or a pro-tumor microenvironment. Thus, the discovery of therapeutic molecules with pleiotropic action is particularly interesting. In this context, we are interested in smac mimetics (SM), antagonists of inhibitor of apoptosis proteins (IAPs) and most often antagonize cIAP1, cIAP2, XIAP and ML-IAP.We investigated whether IAPs could be attractive therapeutic targets in human GBs by studying their expression and their possible prognostic values. All IAPs were expressed in various degrees in GBs and ML-IAP was associated with a worse prognosis. Therefore, we chose GDC-0152 for the rest of our experiments because it antagonizes the different IAPs and in particular ML-IAP. We showed that GDC-0152 induces apoptosis in vitro, increases the survival of GB-bearing mice and slows tumor growth in vivo.We investigated whether the effect of GDC-0152 could be different depending on the oxygen level. Indeed, GBs are part of the most hypoxic tumors. For this purpose, four GB stem cell lines were grown in normoxia and hypoxia. We found that GDC-0152 has an anti-tumor effect regardless of oxygen level, but the signaling pathways involved were different. In normoxia, GDC-0152 induces differentiation of GB stem cells (NF-κB pathway) and in hypoxia it induces apoptosis and decreases cell proliferation (ATR pathway).This work highlights the importance of the preclinical model used in the characterization of a new molecule effects and the therapeutic potential of SM in GBs.

Made available in DSpace on 2015-05-14T13:00:16Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1932289 bytes, checksum: a18b6ccc194715163ed381d04f52ec3d (MD5) Previous issue date: 2011-02-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The study aimed to perform toxicological tests and preclinical dermal clinical phase I, the hydrogel obtained from the hydroalcoholic extract from the bark of Anacardium occidentale Linn. The dermal preclinical studies have assessed the primary irritation of the skin - acute (single dose) and acute eye irritation (single dose) and in both experiments were used rabbits albino, New Zealand, healthy adults, numbering 12, with 6 males and 6 females (control and treated) for a dose of 0.5 g of the hydrogel obtained from the alcoholic extract of Anacardium occidentale Linn. The evaluation of primary irritation of the skin demonstrated that the hydrogel from the stem bark of Anacardium occidentale Linn is not irritating to the skin of rabbits tested in our laboratory, since only 25% of the rabbits showed barely perceptible erythema in the initial phase of the experiment (first time), and no degree of swelling was observed in rabbits throughout the experiment. In relation to the simple eye irritation, the hydrogel from the stem bark of Anacardium occidentale Linn also did not showed irritant effect, because only 33,3% and 8,3% rabbits showed, respectively, conjunctival redness and swelling in the first 24 hours after application of test substance. Did not present any change in level of the iris and cornea, in any animal throughout the experiment. To investigate the clinical phase I toxicity in humans, hydrogel from the stem bark of Anacardium occidentale Linn, we selected 28 volunteers, clinically healthy, aged between 18 and 25. Study participants were divided into two groups, male and female, with 14 participants each, and treated daily, on the night shift, via dermal, with the hydrogel from the stem bark of Anacardium occidentale Linn by a period of 4 weeks. The volunteers were tested before the start of the study and eight weeks after the study, haematological (CBC) and biochemical (glucose, urea, creatinine, total cholesterol, AST, ALT, alkaline phosphatase), in order to detect possible changes arising from the use of hydrogel in patients, as well as compare the results before and after the study. There was no evidence values changed for both hematology and for biochemical variables between times and groups. During treatment with the hydrogel from the stem bark of Anacardium occidentale Linn, Some adverse reactions were observed in participants: tingling, redness, and stinging, but the number of volunteers affected was small, and the reported symptoms occurred during the first weeks of the study and did not require specific treatment and disappeared spontaneously. Only 3,5% of the female volunteers reported feeling oily skin in three weeks of the study. In contrast, 10,7% (first week) and 3,5% (second week) of male volunteers reported skin feeling soft, "cleaner". These results suggest and the low toxicity of the product and indicate that this herbal formulation can be used by the population, the dose and route of administration tested. / O estudo objetivou realizar ensaios toxicológicos pré-clínicos dermais e clínicos fase I, com o hidrogel obtido a partir do extrato alcoólico das cascas de Anacardium occidentale Linn. Os estudos dermais pré-clínicos avaliaram a irritação primária da pele efeito agudo (dose simples) e a irritação ocular aguda (dose simples) e em ambos os experimentos foram utilizados coelhos albinos neozelandeses, sadios, adultos, em número de 12, sendo 6 machos e 6 fêmeas (controle e tratado) para uma dose de 0,5 g do hidrogel obtido a partir do extrato alcoólico das cascas de Anacardium occidentale Linn. A avaliação da irritação primária da pele demonstrou que o hidrogel das cascas do caule de Anacardium occidentale Linn não é irritante para a pele dos coelhos testados em nosso laboratório, já que apenas 25% dos coelhos estudados apresentaram eritema apenas perceptível, na fase inicial do experimento (primeira hora), e nenhum grau de edema foi observado nos coelhos durante todo o experimento. Em relação à irritação ocular simples, o hidrogel das cascas do caule de Anacardium occidentale Linn demonstrou também efeito não irritativo, porque apenas 33,3% e 8,3% dos coelhos, respectivamente, apresentaram rubor e edema em conjuntiva nas primeiras 24 horas após a aplicação da substância em estudo. Não foi identificada nenhuma alteração na íris e na córnea, em qualquer dos animais em todo o experimento. Para investigar a toxicidade clínica fase I, em seres humanos, do hidrogel das cascas do caule de Anacardium occidentale Linn, foram selecionados 28 voluntários, clinicamente saudáveis, com faixa etária compreendida entre 18 e 25 anos. Os participantes do estudo foram distribuídos em dois grupos, masculino e feminino, com 14 participantes cada um, e tratados diariamente, no turno da noite, por via dermal, com o hidrogel das cascas do caule de Anacardium occidentale Linn por um período de 4 semanas. Os voluntários foram avaliados antes do início do estudo e 8 semanas após o seu término com exames hematológicos (hemograma completo), bioquímicos (glicemia, uréia, creatinina, colesterol total, AST, ALT, fosfatase alcalina), com o objetivo de detectar possíveis alterações decorrentes da utilização do hidrogel nos pacientes, bem como, comparar os resultados antes e após o término do estudo. Não foram evidenciados valores alterados, tanto para as variáveis hematológicas como para as bioquímicas entre os tempos e os grupos. Ao longo do tratamento com o hidrogel das cascas do caule de Anacardium occidentale Linn, foram observadas algumas reações adversas nos participantes: formigamento, hiperemia, e ardência, mas o número de voluntários acometidos foi pequeno, e os sintomas relatados ocorreram nas primeiras semanas do estudo, não necessitando de tratamento específico, desaparecendo espontaneamente. Apenas 3,5% dos voluntários do sexo feminino relatou sensação de pele oleosa nas 3 últimas semanas do estudo. Em contrapartida, 10,7% (primeira semana) e 3,5% (segunda semana) dos voluntários do sexo masculino referiram sensação de pele macia, mais limpa . Estes resultados sugerem a baixa toxicidade do produto e indicam que esta formulação fitoterápica pode ser utilizada pela população, na dose e via de administração testada.

Le cancer du col de l’utérus est le troisième cancer le plus fréquent chez la femme, dont l’agent étiologique majeur est l’infection par les papillomavirus humain (notamment HPV-16 et 18), qui sont de petits virus infectant les épithéliums muqueux et cutanés, et pouvant induire la formation de tumeurs. L’inducteur majeur du processus oncogène est le processus d’intégration des régions codantes pour les oncoprotéines E6 et E7, au sein de la cellule hôte suite à l’infection. Elles interférent avec le cycle cellulaire et induisent notamment l’immortalisation, voire la transformation des cellules. Les fonctions les plus connue de ces deux oncoprotéines sont la dégradation des suppresseurs de tumeur p53 et pRb, respectivement. Mon travail de thèse à consisté en la mise au point des vecteurs adénoviraux exprimant des miARN dirigés contre l’oncoprotéine E6. Exprimés in vitro ils induisent l’induction de la mort cellulaire par apoptose des cellules tumorales traitées, via l’activation de la voie de caspases, et in vivo permettent le ralentissement de la croissance de tumeurs xénogreffées à des souris Nude. De plus, la stratégie thérapeutique adénovirale a montré ses extensions possibles sur d’autres types de cancers HPV-positifs, mais également via l’expression de différentes moléculaires thérapeutiques, visant à empêcher l’interaction des oncoprotéines telles qu’E6 avec leurs partenaires cellulaires et ainsi les empêcher d’exercer les activités biologiques impliquées dans le développement de cancers. Pour finir, les adénovirus peuvent également être vu comme des outils d’extinction fonctionnels d’E6 et permettant d’étudier les répercutions sur d’autres processus cellulaires. / Cervical cancer is the third most common female cancer. Infection by human papillomavirus (mainly HPV-16 and 18), with tropisms for mucosal or cutaneous squamous surfaces, is the major etiological agent implicated in cancer and tumor development. After infection, the oncogenic process is triggered by integration of oncoproteins E6 and E7 coding regions into the host cell genome. After integration, these oncoproteins interfere with the cell cycle and induce immortalization and cellular transformation of normal cells. The best known function of these two oncoproteins is the degradation of tumors suppressors p53 and pRb respectively. My thesis project consisted in the development of adenoviral vectors expressing miRNA directed against E6 oncoprotein. Their in vitro expression resulted in cellular death by apoptosis of the treated tumor cells, and allowed reduction of tumor growth in vivo in nude mice xenografts. In addition, the adenoviral therapeutical strategy showed its possible extensions on other types of HPV-positive cancers, but also through the possible expression of different therapeutic molecules aimed at preventing the interaction of viral oncoproteins, such as E6, with their cellular partners. These abolished interactions prevent oncoproteins from exercising their biological activities implicated in the development of cancers. In conclusion, we can say that adenoviruses can also be seen as functional tools suppressing E6 and enabling to highlight the repercussions of its suppression on other cellular processes.