Brain gliomas represent a heterogeneous group of tumors of various histological subtypes which differ according to their response to treatment and prognosis. Tumors created from astrocytes and oligodendrocytes occur most often. Histological classification of gliomas is often subjective, as well as their treatment today is still problematic. The aim of this diploma thesis was to carry out a detailed molecular cytogenetic analysis of the genome of tumor cells in patients with histologically confirmed brain gliomas of different subtypes and stages of malignancy, look for recurrent aberration-specific subtypes and assess their potential role in the development and progression of cancer. To observation specific frequency known aberrations in different subtypes of brain tumors, we used the method of interphase FISH (I-FISH) with a panel of specific locus and / or centromeric DNA probes. The whole genome analysis and detection of cryptic unbalanced changes in the genome of tumor cells, we used the method of SNP array. Combining methods I- FISH and SNP array was detected not only the known chromosomal changes that are typical of the different subtypes of tumors, but also new or uncommon recurrent aberrations. In patients with low-grade gliomas are the most commonly observed acquired UPD (aUPD) on the short...

The aim of the study was to identify molecular markers that can determine progression of low grade glioma. This was done using various approaches such as IDH1 and IDH2 mutation analysis, MGMT methylation analysis, copy number analysis using array comparative genomic hybridisation and identification of differentially expressed miRNAs using miRNA microarray analysis. IDH1 mutation was present at a frequency of 71% in low grade glioma and was identified as an independent marker for improved OS in a multivariate analysis, which confirms the previous findings in low grade glioma studies. IDH1 mutation was associated with MGMT promoter methylation when partially methylated tumours were grouped with methylated tumours. Grade II and grade III tumour comparison analysis revealed 14 novel significant miRNAs with differential expression. A miRNA signature was shown for histological subtypes, oligoastrocytoma and anaplastic oligoastrocytoma, following the miRNA expression analysis in grade II and grade III tumors based on histology. Oligoastrocytoma presented a more similar profile to oligodendroglioma, but anaplastic oligoastrocytoma was more similar to anaplastic astrocytoma. Five novel miRNAs were identified in grade III tumours, when comparing IDH1 mutant and IDH1 wild type tumours. Analysis of paired samples of primary/recurrent tumours revealed that additional genomic changes may promote tumour progression. For each of the pair, the two samples were genomically different and in each case, the reccurent tumours had more copy number aberrations than the corresponding primary tumours. Cell cultures derived from the tumour biopsies were not representative of the low grade glioma in vivo, which was evident from the differences identified in the miRNA expression and copy number changes in the paired samples. IDH1 mutation present in tumour biopsies was not maintained in their respective cell cultures. These findings give an insight into the molecular mechanisms involved in the tumourigenesis of low grade glioma and also tumour progression.

Le gliome infiltrant du tronc cérébral (en anglais “diffuse intrinsic pontine glioma”, DIPG) est une tumeur pédiatrique rare et très agressive. La durée moyenne de survie après diagnostic est inférieure à un an. Une caractéristique génétique majeure des DIPG est la mutation de l’histone H3 (H3K27M). L’évolution des connaissances en épigénétique a permis de concevoir des inhibiteurs de régulateurs épigénétiques capables de modifier, voire de contrebalancer, l’effet de cette mutation. Ainsi, le panobinostat (PS), un inhibiteur des histone-désacétylases, diminue la croissance cellulaire et conduit à la mort des cellules de DIPG in vitro et in vivo. Son efficacité est en cours d'évaluation dans des essais cliniques. Mon projet de thèse avait pour objectif de déterminer le rôle d’EBP50 et d’IRSp53, deux protéines spécifiquement dérégulées dans les lignées de DIPG après traitement des cellules par le PS. EBP50 est connue pour intervenir dans la progression tumorale mais sa dualité de fonction, à la fois oncogène et suppresseur de tumeur, nous a conduits à étudier plus précisément son rôle dans les cellules de DIPG. IRSp53 a été peu étudiée dans les cancers solides, bien qu'elle semble jouer un rôle important dans la motilité cellulaire et l’invasion. La diminution de l’expression d’IRSp53 et d’EBP50 par ARN interférence dans des lignées DIPG induit la mort des cellules par apoptose et bloque leur croissance ainsi que leur motilité cellulaire, ce qui suggérerait que ces deux protéines sont oncogéniques dans ce modèle. De plus, la localisation cytoplasmique et nucléaire d’EBP50 semble en accord avec son rôle pro-oncogénique dans les cellules de DIPG. En étudiant in vitro l’effet d’un traitement combinatoire du PS avec des inhibiteurs de l’expression d’EBP50 ou d’IRSp53, j’ai mis en évidence une augmentation de la sensibilité des cellules de DIPG au traitement par le PS. Enfin, j’ai validé le traitement ciblant EBP50 in vivo dans un modèle préclinique d’embryon de poulet. En conclusion, ces deux protéines constituent de nouvelles cibles thérapeutiques dans les DIPG et un moyen d’augmenter l’efficacité du PS. / Diffuse Intrinsic Pontine Glioma (DIPG), is a rare and highly aggressive pediatric tumor. The average survival time after diagnosis is less than one year. A major genetic characteristic of this disease is the mutation of histone H3 (H3K27M). The evolution of knowledge in epigenetics has made it possible to design epigenetic regulatory inhibitors able to modify, or even offset, the effect of this mutation. For example, panobinostat (PS), a histone deacetylase inhibitor, reduces cell growth and induces DIPG cell death, both in vitro and in vivo. Its effectiveness is currently being evaluated in clinical trials. My thesis project aimed at determining the role of two proteins, EBP50 and IRSp53, deregulated in different DIPG cell lines after treatment with PS. EBP50 has already been described as involved in tumor progression but its dual function, both oncogenic and tumor suppressor, has led us to further investigate its role in the DIPG cells. IRSp53 has been poorly studied in solid cancers, though it plays an important role in cell motility and invasion. Down-regulation by RNA silencing of these two proteins in DIPG cell lines induces apoptosis, decreases cell growth and motility, leading us to the hypothesis that these two proteins are oncogenic proteins. In addition, the cytoplasmic and nuclear localization of the EBP50 protein is consistent with its oncogenic role in DIPG cells. Then, I investigated the effect of combinatorial therapy that associates PS with EBP50 or IRSp53 expression inhibitors. My results show an increase in the antitumor effect in vitro for both proteins but also in vivo for EBP50, in a preclinical model, the chicken embryo. In conclusion, these two proteins could be the targets of new treatments for DIPG tumors in combination with PS to enhance its efficacy.

Glioblastoma (GBM) é caracterizado por sua agressividade e invasão infiltrativa do tecido cerebral. Mecanismos inflamatórios demonstram associação direta com processos carcinogênicos, especialmente aqueles relacionados à produção de eicosanoides através da ativação da fosfolipase A2 (PLA2). Compostos metálicos como o rutênio associado a drogas anti-inflamatórias não esteroides (AINEs) surgem como tratamentos antitumorais promissores. O objetivo foi avaliar in vitro a ação desses complexos de rutênio em linhagens celulares de GBM. Pudemos observar que todos os complexos de Rutênio utilizados apresentaram diminuição na contagem celular, aumento de apoptose e diminuição da mitose, destaque para a linhagem U87MG para o tratamento RuIBpOTf com diminuição de 65,4% e na linhagem A172 para aumento de apoptose em todos os tratamentos, especialmente RuIBpCl com 106,8% . Em ambas as linhagens celulares verificamos que houve a captação dos complexos, inclusive em suas frações celulares como o núcleo. Alterações nas enzimas PLA2 e COX 1 e 2 também foram detectadas nos ensaios realizados ELISA e RT-PCR. Através destes resultados, concluímos que os complexos de rutênio foram eficazes na diminuição do número de células de GBM em concentrações e tempos pré-determinados, aumento da apoptose e diminuição das mitoses, características que são fortemente recomendadas para novos fármacos. / Glioblastoma (GBM) is characterized by its aggressiveness and infiltrative invasion of brain tissue. Inflammatory mechanisms demonstrate direct association with carcinogenic processes, especially those related to the production of eicosanoids through the activation of phospholipase A2 (PLA2). Metal compounds such as ruthenium associated with non-steroidal anti-inflammatory drugs (NSAIDs) appear as promising antitumor treatments. The objective was to evaluate in vitro the action of these ruthenium complexes in GBM cell lines. It was observed that all the Ruthenium complexes showed decrease in the cell count, increase of apoptosis and decrease of mitosis, highlight to the U87MG cell line for the treatment RuIBpOTf with reduction of 65.4% and in cell line A172 to increase apoptosis in all the treatments, especially RuIBpCl with 106.8%. In both cell lines we verified that there was the uptake of the complexes, including in their cellular fractions as the nucleus. Changes in PLA2 and COX 1 and 2 enzymes were also detected in ELISA and RT-PCR assays. Through these results, we conclude that ruthenium complexes were effective in decreasing the number of GBM cells at predetermined concentrations and times, increased apoptosis and decreased mitoses, characteristics that are strongly recommended for new drugs.

O glioblastoma multiforme (GBM) é uma neoplasia do sistema nervoso central (SNC), caracterizada por uma elevada capacidade proliferativa e migratória. O desenvolvimento do tumor provoca uma remodelação da matriz extracelular (MEC) que facilita a migração tumoral. Eicosanóides são moléculas lipídicas importantes na carcinogênese e a sua síntese está correlacionada com o grau de desenvolvimento do tumor. As prostaglandinas são eicosanóides envolvidas na estimulação da angiogênese, na adesão celular e proliferação celular. Este estudo tem por objetivo avaliar in vitro o efeito da PGE2 na expressão moléculas da MEC e das moléculas de adesão envolvidas na migração, em células U87MG e U251MG. As células U251MG e U87MG foram tratadas com PGE2 (10µM) e Ibuprofeno (25µM), por um período 48hs. As proteínas da MEC foram analisadas por RT-qPCR após o tratamento. Foram realizadas reações de imunohistoquímica para as moléculas da MEC. As alterações foram encontradas na expressão de laminina, fibronectina, colágeno tipo IV e as integrinas αv , α3 e α5 para células U87MG . Observamos imunomarcação nas linhas celulares para colágeno tipo IV, laminina e fibronectina. Concluímos que o tratamento com IBU e PGE2, afeta a expressão gênica de moléculas de MEC. / Glioblastoma Multiforme (GBM) is a neoplasm of the central nervous system (CNS), characterized by a high proliferative and migratory capacity. Tumor development leads to extracellular matrix (ECM) remodeling and facilitating the migration of these cells. Eicosanoids are important lipid molecules in carcinogenesis, and their synthesis often correlates with the degree of tumor development. Prostaglandins are eicosanoids involved in the stimulation of angiogenesis, cell adhesion and cell proliferation. This study is aimed to evaluate the expression of several ECM molecules involved in migration after altering the concentration of prostaglandins, using human glioma cell lines as an in vitro model. The cell lines U87MG and U251MG were treated with PGE2 (10µM) and Ibuprofen (25µM), for a predetermined period of 48hs. Proteins involved in extracellular matrix were analyzed by RT-qPCR after treatment in vitro. Immunohistochemical reactions were also performed for the ECM molecules. Changes were found in the expression of laminin, fibronectin, type IV collagen and αv, α3 and α5 integrins in cells U87MG. We observed immunostaining in cell lines to type IV collagen, laminin and fibronectin. In conclusion, Ibuprofen and PGE2, affects gene expression of ECM molecules.

Os hormônios glicocorticóides (GCs) têm sido amplamente empregados como agentes antiinflamatórios e anti-tumorais. Sua ação ocorre via receptores nucleares (GR) sendo dependente da remodelação da estrutura da cromatina. As proteínas Brm e BRG1, componentes essenciais de um complexo regulador global da transcrição (SWI/SNF), por remodelamento da cromatina, exercem um papel-chave na ação de GR. Para estudar o mecanismo de ação de GCs, foram utilizadas as linhagens celulares ST1 e P7, derivadas da linhagem celular C6, de glioma de rato. P7 é insensível ao tratamento com GC, enquanto ST1 apresenta reversão fenotípica tumoral→normal, gerando um bloqueio específico na fase G1. Um anti-soro policlonal específico para Brm e BRG1, foi gerado através da inoculaçâo de coelha com a proteína hBRG1 recombinante. Este antisoro foi utilizado para análisar os níveis destas proteínas nas duas linhagens celulares, sob ação de GC. Enquanto em ST1, Brm é induzida por GC, em células P7, o nível basal de Brm é relativamente alto, mantendo-se inalterado na presença de GC. A possíbilidade de existirem mutações no gene brm de células P7, foi investigada através de amplificação do DNA, por PCR, e seqüenciamento. A superexpressão de brm e BRG1 em células P7 mostrou que clones isolados apresentavam, de um modo geral, achatamento celular, diminuição da taxa de crescimento e da eficiência de plaqueamento em substrato sólido e semi-sólido. Alguns destes clones passaram a responder ao tratamento com GC, porém não tão drasticamente como as células ST1. Co-imunonoprecipitação mostrou algumas diferenças entre os complexos SWI/SNF de células ST1 e P7. / Glucocorticoid hormones (GCs) have been used as anti-inflammatory and anti-tumor agents, acting via nuclear receptors (GR) and being dependent on remodeling of the chromatin structure. As components of the global chromatin remodeling transcription complex (SWI/SNF), Brm and BRG-1 proteins play a key role in the action of GR. In order to study the mechanisms of action of GCs, we have been using the ST1 and P7 cell lines, derived from the C6, a rat glioma cell line. P7 is insensitive to the GC treatment, while ST1 displays a complete phenotypic reversion from tumoral to normal, including a G1-specific block in the cell cycle. A Brm and BRG1-specific polyclonal antiserum was generated, in rabbit, using recombinant hBRG1 protein as antigen. This antiserum was used to analyze the levels of Brm and BRG1 in these two cell lines, under GC treatment. While Brm is induced by GC, in ST1 cells, the basal level of Brm, in P7 cells, is relatively high, remaining unchanged under GC treatment. The possibility of brm mutations occurring in the P7 cells, was analyzed by DNA sequencing. Overexpression of brm and BRG1 in P7 cells led to morphological alterations (cell flattening) and decreased colony formation in agarose suspension and in solid substrate. Some of these clones became partially responsive to GC, when compared to the ST1 cell line. Co-immunoprecipitation assays revealed some differences in the SWI/SNF complex between ST1 and P7 cells.

Atualmente os gliomas representam cerca de 81% dos tumores cerebrais malignos, com aumento na incidência tanto em crianças, como em adultos acima dos 45 anos. Um número elevado de receptores neuroquinina tipo 1 (NK-1) estão expressos em células de glioma, estando a ligação da Substância P (SP) a esses receptores, envolvida no desenvolvimento e progressão desse tipo de tumor. A SP quelada ao DOTA (SP-DOTA), radiomarcada, vem sendo testada para utilização na terapia de gliomas, sendo o lutécio-177 (177Lu), devido a seu menor alcance tecidual, o radioisótopo mais indicado para tumores localizados em áreas críticas do cérebro. No entanto, estudos indicam a necessidade da adição de um excesso de metionina para prevenção da oxidação peptídica da SP-DOTA-177Lu, visando aumentar a estabilidade e a capacidade de ligação às células tumorais. Para superar esse desafio, surge a perspectiva da utilização de um novo análogo da SP, com estrutura modificada, para prevenir a oxidação peptídica. Neste contexto, o objetivo desse trabalho foi estudar a marcação de um novo análogo da SP com 177Lu e caracterizar suas propriedades in vitro e in vivo, visando a obtenção de um radiofármaco inédito e com potencial aplicação na terapia de tumores cerebrais e realizar estudos preliminares de marcação deste novo análogo com Ítrio-90 (90Y). O novo análogo foi obtido pela troca do aminoácido metionina (Met) pelo aminoácido norleucina (Nle) na posição 11 da cadeia peptídica da SP, sendo esses peptídeos denominados respectivamente SP(Met11)-DOTA e SP(Nle11)-DOTA. Após análise da oxidação peptídica dos dois peptídeos, os parâmetros da radiomarcação da SP(Nle11)-DOTA, com 177LuCl3, foram estudados para determinar a melhor condição de marcação. As estabilidades in vitro da SP(Nle11)-DOTA-177Lu sob refrigeração (2-8°C), no freezer (-20°C) e em soro humano (37°C) foram determinadas após radiomarcação com alta atividade, quanto ao uso de agentes estabilizantes e após diluição. A SP(Nle11)-DOTA também foi radiomarcada com 90Y, utilizando-se a condição padrão determinada, sendo a estabilidade in vitro da SP(Nle11)-DOTA-90Y sob refrigeração (2-8°C) e em freezer (-20°C), avaliada após radiomarcação com alta atividade e quanto a utilização de agente estabilizante. A capacidade de ligação in vitro às células tumorais (U-87 MG e M059J) e a biodistribuição in vivo em camundongos BALB/c sadios foram determinadas para a SP(Nle11)-DOTA-177Lu e comparadas à SP(Met11)-DOTA-177Lu. A ligação às proteínas plasmáticas e a biodistribuição em camundongos Nude com modelo tumoral também foram avaliadas. Os resultados obtidos na análise da oxidação peptídica comprovaram a importância da adição de excesso de metionina para prevenção da oxidação peptídica e indicaram uma alta estabilidade da SP(Nle11)-DOTA, durante e após o processo de radiomarcação. A adição de 148 MBq (4 mCi) da solução de 177LuCl3 em HCl 0,05N à 10 μg de SP(Nle11)-DOTA diluída em tampão acetato de sódio 0,4 M pH 4,5 seguida pela incubação a uma temperatura de 90ºC por 30 minutos, sob agitação de 350 rpm foi definida com condição padrão de marcação. O congelamento (-20°C), o uso de agentes estabilizantes e a diluição apresentaram-se como métodos efetivos para garantir uma alta estabilidade in vitro da SP(Nle11)-DOTA-177Lu, após a marcação com alta atividade. Bons resultados também foram observados para a marcação da SP(Nle11)-DOTA com 90YCl3 e para a estabilidade in vitro da SP(Nle11)-DOTA-90Y, após congelamento (-20°C) e quando utilizado ácido gentísico como estabilizante. A SP(Nle11)-DOTA-177Lu apresentou uma boa especificidade pelas células tumorais, principalmente pelas células de glioma humano M059J, sugerindo que a substituição do aminoácido metionina por norleucina na posição 11 não compromete a capacidade de ligação da SP(Nle11) às células tumorais. Uma baixa porcentagem de ligação às proteínas plasmáticas e um rápido clareamento sanguíneo foram observados para a SP(Nle11)-DOTA-177Lu, sendo esse radiofármaco eliminado preferencialmente por via renal. A SP(Nle11)-DOTA-177Lu apresentou uma boa estabilidade in vivo e se mostrou incapaz de atravessar a barreira hematoencefálica, sendo seu uso indicado por injeção intratumoral ou intracavitária. O estudo de biodistribuição em animais com modelo tumoral, mostrou que esse radiofármaco se liga às células tumorais por ligações receptor específicas. Com base nesse dados conclui-se que a SP(Nle11)-DOTA-177Lu, apresenta-se como um radiofármaco inédito que devido às suas propriedades in vitro e in vivo favoráveis, apresenta potencial aplicação na terapia de tumores cerebrais, representando uma nova possibilidade dentro do limitado arsenal terapêutico para esse tipo de tumor. / Currently gliomas represent about 81% of malignant brain tumors with increased incidence in children and in adults over 45 years. A large number of type 1 neurokinin receptor (NK-1) are expressed in glioma cells, being the binding of substance P (SP) to these receptors, involved in the development and progression of this tumor type. The SP conjugated at DOTA chelator (SP-DOTA), radiolabeled, have been tested for use in the treatment of gliomas, and the lutetium-177 (177 Lu), due to its lower tissue range, has been the most suitable radioisotope for tumors located in critical areas brain. However, studies indicate the necessity of adding an excess of methionine to prevent the peptide SP-DOTA-177Lu oxidation in order to increase the stability and capacity to bind to tumor cells. To overcome this challenge, there is the prospect of using a new analog of SP with a modified structure, to prevent peptide oxidation. In this context, the aim of this work was study the labeling of a new analog of SP with 177Lu and characterize their properties in vitro and in vivo, in order to obtain a novel radiopharmaceutical with potential application in brain tumor therapy, and perform preliminary studies labeling of this new analog with yttrium-90 (90Y). The new analog was obtained by replacement of the amino acid methionine (Met) by the amino acid norleucine (Nle) at position 11 of the peptide chain of SP, and these peptides were called SP(Met11)-DOTA and SP(Nle11)-DOTA respectively. After analysis of the oxidation for the two peptides, the radiolabeling parameters of the SP(Nle11)-DOTA with 177LuCl3 were studied to determine the best labeling condition. The SP(Nle11)-DOTA was also radiolabeled with 90Y, using standard condition, and the stability in vitro of the SP(Nle11)-DOTA-90Y assessed under refrigeration (2-8 °C) and under freezing (-20° C), after radiolabeling with high activity and use of stabilizing agent. The stabilities in vitro of the SP (Nle11)-DOTA-177Lu under refrigeration (2-8 °C), under freezing (-20 °C) and in human serum (37 °C) were determined after radiolabeling with high activity, with use of stabilizing agents and after dilution. The ability of in vitro binding to tumor cells (U-87 MG and M059J) and the biodistribution in vivo in healthy BALB/c mice were determined for the 177Lu-DOTA-SP(Nle11) and compared to 177Lu-DOTA-SP(Met11). The plasma protein binding and biodistribution in Nude mice with tumor model were also evaluated. The results obtained from analysis of oxidation for the two peptides confirmed the importance of adding excess methionine to prevent peptide oxidation and indicated a high stability of the DOTA- SP(Nle11), during and after the radiolabeling process. The addition of 148 MBq (4 mCi) of 177LuCl3 solution in 0.05N HCl at 10 μg DOTA-SP(Nle11) diluted in 0.4 M sodium acetate buffer pH 4.5 followed by incubation at a temperature of 90 °C for 30 minutes under constant agitation to 350 rpm was defined as standard labeling condition. The freezing (-20 °C), the use of stabilizing agents and the dilution were presented as effective methods to ensure high stability in vitro 177Lu-DOTA-SP(Nle11), after labeling with high activity. Good results were also observed for labeling DOTA-SP(Nle11) with 90YCl3 and for stability in vitro of the 90Y-DOTA-SP(Nle11) after freezing (-20 °C) and when gentisic acid was used as a stabilizer. The 177Lu-DOTA-SP(Nle11) showed good specificity to tumor cells, particularly human glioma cells (M059J), suggesting that substitution of the amino acid norleucine for methionine at position 11 does not compromise the capacity of SP(Nle11) binding to tumor cells. A low percentage of plasma protein binding and rapid blood clearance were observed for the 177Lu-DOTA-SP(Nle11), being this radiopharmaceutical preferably eliminated by the kidney. The 177Lu-DOTA-SP(Nle11) showed good stability in vivo and inability to cross the blood brain barrier, being its use indicated through intratumoral or intracavitary injection. The biodistribution studies in animals with tumor model showed that the radiopharmaceutical binds to the tumor cells by specific receptor binding. Based on this data was concluded that the 177Lu-DOTA-SP(Nle11), can be presented as a novel radiopharmaceutical that due to its favorable properties in vitro and in vivo, presents a potential application in the therapy of brain tumors, representing a new possibility within the limited therapeutic options for this type of tumor.

Les gliomes diffus de bas grade sont des tumeurs cérébrales des jeunes adultes. Dans cette thèse, nous nous intéressons à la segmentation et à la modélisation de ces tumeurs. Dans la première partie du manuscrit, nous étudions la segmentation des gliomes diffus de bas grade à base de différentes méthodes manuelles et semi-automatiques. La délimitation de ces tumeurs peut être problématique en raison de leur caractère très infiltrant et inhomogène. En pratique clinique, le suivi des gliomes diffus de bas grade repose sur l'estimation du volume tumoral, soit par une segmentation suivie d'une reconstruction logicielle, soit par la méthode des trois diamètres. Pour la segmentation, elle est manuelle et est exécutée par des praticiens sur des IRM en pondération FLAIR ou T2. La méthode des trois diamètres est rapide mais s'avère difficile à implémenter dans le cas de gliomes diffus de bas grade très infiltrants ou en post-traitement. La solution par segmentation manuelle et reconstruction logicielle du volume est chronophage mais demeure plus précise en comparaison de la méthode des trois diamètres. Nous étudions ici la reproductibilité de la segmentation manuelle avec le logiciel OsiriX en réalisant un test subjectif dans le Living Lab PROMETEE de TELECOM Nancy. Les résultats de cette étude montrent que ni la spécialité du praticien ni le nombre d’années d’expérience ne semblent impacter significativement la qualité de la segmentation. Nous comparons par ailleurs les résultats obtenus à ceux d'un deuxième test où nous appliquons la méthode des trois diamètres. Enfin, nous explorons deux algorithmes de segmentation semi-automatique basés, respectivement, sur les contours actifs et sur la méthode des level set. Même si la segmentation automatique semble être une voie prometteuse, nous recommandons aujourd’hui l’utilisation de la segmentation manuelle du fait notamment du caractère diffus des gliomes de bas grade qui rend le contour complexe à délimiter. La seconde partie du manuscrit est consacrée à la modélisation des gliomes diffus de bas grade eux-mêmes ou, plus exactement, à la modélisation de l'évolution du diamètre tumoral en phase de chimiothérapie. La prise en charge thérapeutique des patients atteints de ces tumeurs inclut en effet souvent une chimiothérapie. Pour ce travail, nous nous intéressons à la chimiothérapie par Témozolomide en première ligne de traitement. Une fois le traitement entamé, les praticiens aimeraient déterminer l'instant optimal d'arrêt de traitement. Nous proposons une modélisation statistique du diamètre tumoral sous chimiothérapie. Cette modélisation s'appuie sur des modèles de régression linéaire et exponentielle. Elle permet de prédire le diamètre tumoral à partir d'un jeu de données d'apprentissage et d'alerter le clinicien sur l'état d'évolution du diamètre sous traitement. Nous espérons que ces modèles pourront un jour être utilisés comme un outil dans la planification de la chimiothérapie en milieu clinique. / Diffuse low-grade gliomas are brain tumors of young adults. In this thesis, we focus on the segmentation and on the modeling of these tumors. In the first part of the manuscript, we study the segmentation of diffuse low-grade gliomas based on different manual and semi-automatic methods. The delineation of these tumors can be problematic because of their very infiltrating and inhomogeneous nature. In clinical practice, the monitoring of diffuse low-grade gliomas is based on the estimation of tumor volume, obtained either through a segmentation followed by a software reconstruction or through the three diameters method. As for the segmentation, it is manual and it is performed by practitioners on FLAIR-weighted or T2-weighted MRI.The three diameters approach is fast but it is difficult to implement in the case of highly infiltrating diffuse low grade gliomas or after a treatment. The manual segmentation and software-based volume reconstruction solution is time-consuming but it remains more accurate in comparison with the three diameters method. We investigate in this work the reproducibility of the manual segmentation with the OsiriX software by performing a subjective test in the Living Lab PROMETEE in TELECOM Nancy. The results of this study show that neither the practitioners' specialty nor their number of years of experience seem to have a significant impact on the quality of the segmentation. We also compare the results to those of a second test where we apply the three diameters method. Finally, we explore two semi-automatic segmentation algorithms which are, respectively, based on active contours and on the level set method. Even if automatic segmentation seems to be a promising avenue, we recommend for now the use of manual segmentation because of the diffuse nature of low-grade gliomas, which makes the tumor's contours complex to delineate. The second part of the manuscript is dedicated to the modeling of diffuse low-grade gliomas themselves or, to be more precise, to the modeling of the evolution of the tumor's diameter during chemotherapy. The therapeutic management of patients with these tumors often includes indeed chemotherapy. For this work, we focus on Temozolomide chemotherapy in first-line treatment. After the beginning of the treatment, the practitioners would like to determine the optimum time of discontinuation. We propose a statistical modeling of tumor diameter under chemotherapy. This modeling is based on linear and exponential regression models. It can predict the tumor diameter from a set of training dataset and can alert the clinician on the state of change in diameter under treatment. We hope that these models will, eventually, be used as a tool in the planning of chemotherapy in a clinical environment.

Submitted by Jadson Francisco de Jesus SILVA (jadson@uefs.br) on 2018-09-20T00:46:00Z No. of bitstreams: 1 DISSERTA??O FINAL ERIC.pdf: 2788345 bytes, checksum: 01d5e7c11ba1598a630e87ef0d88330b (MD5) / Made available in DSpace on 2018-09-20T00:46:00Z (GMT). No. of bitstreams: 1 DISSERTA??O FINAL ERIC.pdf: 2788345 bytes, checksum: 01d5e7c11ba1598a630e87ef0d88330b (MD5) Previous issue date: 2017-12-12 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Lippia (Verbenaceae) is a plant genus distributed throughout all Southern and Central America, and Tropical Africa. Approximately 60 species are endemic in Brazil and 22 are endemic in the state of Bahia. Lippia has a long history of use in traditional medicine, the most common use being in the treatment of respiratory diseases. Despite Lippia?s extensive history in treating diseases, little is known about its anticancer properties against brain and central nervous system tumors. This paper explores the anticancer potential of 5 species of Lippia from the state of Bahia (Brazil),Lippia alnifolia, Lippia insignis, Lippia lasiocalycina, Lippia origanoides and Lippia thymoides. To assess the cell viability of essential oils and methanol extracts derived from these species MTT assay was utilized. To assess the effects of the essential oils and methanol extracts on morphology of cancer cells a Nikon Ts-100 phase contrast microscope was used, and photos were taken with a Nikon E4300. All essential oils were evaluated at 0.1 mg.mL-1, 0.3 mg.mL-1, 0.5 mg.mL-1, and 1 mg.mL-1. All methanol extracts were evaluated at 1 ?g.mL-1, 10 ?g.mL-1, 100 ?g.mL-1, 500 ?g.mL-1, and 1000 ?g.mL-1. The ability to inhibit cell migration was assessed by wound heal assay. All methanol extracts were evaluated at 500 ?g.mL-1. All essential oils were evaluated at 0.1 mg.mL-1. To access the effects of essential oils and methanol extracts on the cell cycle flow cytometry was used. All methanol extracts were evaluated at 1000 ?g.mL-1. All essential oils were evaluated at 0.03 mg.mL-1. The essential oils derived from Lippia alnifolia, Lippia insignis, Lippia lasiocalycina, and Lippia origanoides all drastically decreased the cell viability of the C6 cell line in a concentration dependent manner. The essential oil derived from Lippia thymoides increased cell viability in a concentration dependent manner. All of the methanol extracts appear to have the ability to abruptly decrease cell viability at either 500 ?g.mL-1 or 1000 ?g.mL-1 it. Lippia alnifolia and Lippia insignis both have the ability to significantly alter the morphology of the C6 cell line. Methanol extracts derived from Lippia alnifolia, Lippia insignis, Lippia lasiocalycina, Lippia origanoides and Lippia thymoides all significantly inhibit the migration of C6 cells at a concentration of 500 ?g.mL-1. Essential oils obtained from Lippia alnifolia, Lippia insignis, Lippia lasiocalycina, Lippia origanoides and Lippia thymoides significantly inhibited the migration of C6 cells at a concentration of 0.1 mg.mL-1. All methanol extracts and essential oils tested demonstrated the ability to induce some degree of nuclear fragmentation, vacuolization, and cytoplasmic swelling. These results indicate that the constituents of both the essential oils and methanol extracts should be isolated and their anticancer potential should be further explored. / Lippia (Verbenaceae) ? um g?nero de planta distribu?do pela Am?rica do Sul, Central e a ?frica Tropical. Aproximadamente 60 esp?cies s?o end?micas do Brasil e 22 s?o end?micas do estado da Bahia. Lippia tem uma longa hist?ria de uso na medicina tradicional, sendo o uso mais comum no tratamento de doen?as respirat?rias. Apesar da extensa hist?ria de Lippia no tratamento de doen?as, pouco se sabe sobre suas propriedades antitumorais contra tumores cerebrais. Este estudo buscou explorar o potencial antiglioma de ?leos essenciais e extratos metan?licos de Lippia alnifolia, Lippia insignis, Lippia lasiocalycina, Lippia origanoides e Lippia thymoides em c?lulas de glioma da linhagem C6. Para avaliar a viabilidade celular foram utilizados o ensaio do MTT. Os efeitos das amostras na morfologia de c?lulas cancerosas foram avaliados utilizando-se um microsc?pio de contraste de fase Nikon Ts-100 e fotos com uma Nikon E4300. Os ?leos essenciais foram avaliados nas concentra??es de 0,1 mg.mL-1, 0,3 mg.mL-1, 0,5 mg.mL-1 e 1 mg.mL-1 e os extratos de metanol a 1 ?g.mL-1, 10 ?g.mL-1, 100 ?g.mL-1, 500 ?g.mL-1 e 1000 ?g.mL-1. A capacidade de inibir a migra??o celular foi avaliada pelo ensaio de cicatriza??o da ferida, na qual os extratos foram avaliados a 500 ?g.mL-1 e os ?leos essenciais a 0,1 mg.mL-1. O ciclo celular foi avaliado na citometria de fluxo nas concentra??es de 1000 ug.mL-1 para os extratos e 0,03 mg.mL-1 para os ?leos essenciais. Os ?leos essenciais obtidos de Lippia alnifolia, Lippia insignis, Lippia lasiocalycina e Lippia origanoides reduziram drasticamente a viabilidade celular C6 de uma maneira concentrac?o-dependente. J? o ?leo essencial de Lippia thymoides aumentou a viabilidade celular de uma maneira concentrac?o-dependente. Todos os extratos de metanol parecem ter a capacidade de diminuir abruptamente a viabilidade celular em 500 ?g.mL-1 ou 1000 ?g.mL-1. Lippia alnifolia e Lippia insignis parecem ter a capacidade de alterar significativamente a morfologia da linha celular C6. Os extratos metan?licos de Lippia alnifolia, Lippia insignis, Lippia lasiocalycina, Lippia origanoides e Lippia thymoides inibem significativamente a migra??o de c?lulas C6 a uma concentra??o de 500 ?g.mL-1. Os ?leos essenciais de Lippia alnifolia, Lippia insignis, Lippia lasiocalycina, Lippia origanoidese Lippia thymoides inibem significativamente a migra??o de c?lulas C6 a uma concentra??o de 0,1 mg.mL-1. Todos os extratos metan?licos e ?leos essenciais testados demonstraram a capacidade de induzir algum grau de fragmenta??o nuclear, vacuoliza??o e incha?o citoplasm?tico.Estes resultados indicam que os constituintes dos ?leos essenciais e extratos metan?licos devem ser isolados e seu potencial anticancer?geno deve ser explorado.

by Shan Sze Wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 145-166). / Abstracts in English and Chinese. / Title --- p.i / Abstract --- p.ii / 摘要 --- p.v / Acknowledgements --- p.vii / Table of Contents --- p.viii / List of Abbreviations --- p.xiv / List of Figures --- p.xvii / List of Tables --- p.xx / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- What are the general functions of cytokines? --- p.2 / Chapter 1.2 --- What is TNP-α? --- p.4 / Chapter 1.3 --- Actions of TNF-α --- p.5 / Chapter 1.4 --- General functions of TNF-α in astrocytes --- p.6 / Chapter 1.5 --- TNF-α receptors (TNF-Rs) --- p.8 / Chapter 1.6 --- Second messengers induced by TNP-α --- p.10 / Chapter 1.7 --- Glial Cells --- p.11 / Chapter 1.7.1 --- Oligodendroglia --- p.12 / Chapter 1.7.2 --- Brain Macrophages (Microglia) --- p.12 / Chapter 1.7.3 --- Astrocytes --- p.14 / Chapter 1.7.3.1 --- Functions of astrocytes --- p.15 / Chapter 1.8 --- "Brain injury, astrogliosis and scar formation" --- p.20 / Chapter 1.9 --- β-Adrenergic receptors (β-ARs) --- p.21 / Chapter 1.9.1 --- The active functional unit: the receptor complex --- p.22 / Chapter 1.9.2 --- General functions and distribution of β-ARs --- p.22 / Chapter 1.10 --- Functions of β-ARs in astrocytes --- p.24 / Chapter 1.10.1 --- Regulations of astrogliosis by β-ARs --- p.24 / Chapter 1.10.1.1 --- β-ARs are expressed in normal optic nerves and up-regulated after nerve crush --- p.24 / Chapter 1.10.1.2 --- Injury-induced alterations in endogenous catecholamine leads to enhanced β-AR activation --- p.25 / Chapter 1.10.1.3 --- β-AR blockade suppresses glial scar formation --- p.25 / Chapter 1.10.1.4 --- β-AR agonists affect the proliferation of astrocytes in normal brain --- p.26 / Chapter 1.11 --- Manganese Superoxide Dismutase (MnSOD) --- p.27 / Chapter 1.11.1 --- MnSOD is the target gene of NF-kB --- p.29 / Chapter 1.11.2 --- Induction of MnSOD by proinflammatory cytokines in rat primary astrocytes --- p.29 / Chapter 1.11.3 --- SMase and ceramides induce MnSOD in various cell types --- p.30 / Chapter 1.12 --- Why do we use C6 glioma cells? --- p.31 / Chapter 1.13 --- Aims and Scopes of this project --- p.32 / Chapter Chapter 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.36 / Chapter 2.1.1 --- Cell Line --- p.36 / Chapter 2.1.2 --- Cell Culture Reagents --- p.36 / Chapter 2.1.2.1 --- Complete Dulbecco´ةs modified Eagle medium (CDMEM) --- p.36 / Chapter 2.1.2.2 --- Rosewell Park Memorial Institute (RPMI) medium --- p.37 / Chapter 2.1.2.3 --- Phosphate buffered saline (PBS) --- p.37 / Chapter 2.1.3 --- Recombinant cytokines --- p.38 / Chapter 2.1.4 --- Chemicals for signal transduction study --- p.38 / Chapter 2.1.4.1 --- Modulators of protein kinase C (PKC) --- p.38 / Chapter 2.1.4.2 --- Modulator of protein kinase A (PKA) --- p.39 / Chapter 2.1.4.3 --- β-Adrenergic agonist and antagonist --- p.39 / Chapter 2.1.5 --- Antibodies --- p.40 / Chapter 2.1.5.1 --- Anti-TNF-receptor type 1 (TNF-R1) antibody --- p.40 / Chapter 2.1.5.2 --- Anti-TNF-receptor type 2 (TNF-R2) antibody --- p.41 / Chapter 2.1.5.3 --- Anti-βi-adrenergic receptor (βl-AR) antibody --- p.42 / Chapter 2.1.5.4 --- Anti-β2-adrenergic receptor (β2-AR) antibody --- p.42 / Chapter 2.1.5.5 --- Antibody conjugates --- p.43 / Chapter 2.1.6 --- Reagents for RNA isolation --- p.43 / Chapter 2.1.7 --- Reagents for reverse transcription-polymerase chain reaction (RT-PCR) --- p.43 / Chapter 2.1.8 --- Reagents for electrophoresis --- p.45 / Chapter 2.1.9 --- Reagents and buffers for Western blot --- p.45 / Chapter 2.1.10 --- Other chemicals and reagents --- p.47 / Chapter 2.2 --- Maintenance of rat C6 glioma cell line --- p.47 / Chapter 2.3 --- RNA isolation --- p.48 / Chapter 2.3.1 --- Measurement of RNA yield --- p.49 / Chapter 2.4 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.50 / Chapter 2.5 --- Western blot analysis --- p.52 / Chapter Chapter 3 --- RESULTS / Chapter 3.1 --- Effect of TNF-α on the expression of TNF-receptors (TNFRs) in C6 glioma cells --- p.55 / Chapter 3.1.1 --- Effect of TNF-α on TNF-R1 and -R2 mRNA expression in C6 cells --- p.56 / Chapter 3.1.2 --- The signaling systems mediating TNP-α-induced TNF-R2 expression in C6 cells --- p.57 / Chapter 3.1.2.1 --- The involvement of PKC in TNF-α-induced TNF-R2 expression in C6 cells --- p.57 / Chapter 3.1.2.2 --- Effect of PMA on the TNF-R protein levels in C6 cells --- p.63 / Chapter 3.1.2.3 --- Effect of Ro31 on the TNF-α-induced TNF-R protein level in C6 cells --- p.65 / Chapter 3.1.2.4 --- Effect of PKA activator on the level of TNF-R2 mRNA in C6 cells --- p.67 / Chapter 3.2 --- Effect of TNP-α on the expression of β1- and β2-adrenergic receptors (β1- and β2-ARs) in C6 glioma cells --- p.69 / Chapter 3.2.1 --- Effect of TNF-α on β1- and β2-ARs mRNA expression in C6 cells --- p.70 / Chapter 3.2.2 --- The signaling systems mediating TNF-α-induced β1- and β2-AR expression in C6 cells --- p.70 / Chapter 3.2.2.1 --- The involvement of PKC mechanism between TNF-α and β-ARs in C6 cells --- p.71 / Chapter 3.2.2.2 --- Effect of PMA on the β1- and β2-ARs protein level in C6 cells --- p.76 / Chapter 3.2.2.3 --- Effect of Ro31 on the TNF-α-induced β1- and β2-AR protein levels in C6 cells --- p.78 / Chapter 3.2.2.4 --- Effect of dbcAMP on the levels of βl- and β2-ARs mRNA in C6 cells --- p.80 / Chapter 3.3 --- Relationship between TN1F-R2 and β-adrenergic mechanism in C6 cells --- p.82 / Chapter 3.3.1 --- Effects of isproterenol and propranolol on endogenous TNF-α mRNA levels in C6 cells --- p.82 / Chapter 3.3.2 --- Effects of isoproterenol and propranolol on TNF-R2 mRNA levels in C6 cells --- p.83 / Chapter 3.3.3 --- Effects of β1-agonist and antagonist on endogenous TNF-α mRNA expression in C6 cells --- p.87 / Chapter 3.3.4 --- Effects of β1-agonist and antagonist on TNF-R2 mRNA expression in C6 cells --- p.91 / Chapter 3.3.5 --- Effects of β2-agonist and antagonist on endogenous TNF-α mRNA in C6 cells --- p.93 / Chapter 3.3.6 --- Effects of β2-agonist and antagonist on TNF-R2 mRNA in C6 cells --- p.100 / Chapter 3.4 --- Effect ofTNF-α on the expression of a transcriptional factor nuclear factor kappa B (NF-kB) in C6 glioma cells --- p.102 / Chapter 3.4.1 --- Effect ofTNF-α on NF-kB (p50) mRNA expression in C6 cells --- p.106 / Chapter 3.4.2 --- Effect of β-agonist and antagonist on NF-kB (p50) mRNA expression in C6 cells --- p.108 / Chapter 3.4.3 --- Effect of PMA and Ro31 on the levels of NF-kB mRNA in C6 cells --- p.109 / Chapter 3.5 --- Effects of TNF-α on the expression of manganese superoxide dismutase (MnSOD) in C6 glioma cells --- p.111 / Chapter 3.5.1 --- Effects of TNF-α on MnSOD and Cu-ZnSOD mRNAs expression in C6 cells --- p.114 / Chapter 3.5.2 --- Effects of β-agonist and β-antagonist on MnSOD mRNA expression in C6 cells --- p.115 / Chapter 3.5.3 --- Effects of PKC activator and inhibitor on the levels of MnSOD mRNA in C6 cells --- p.117 / Chapter Chapter 4 --- DISCUSSION AND CONCLUSION / Chapter 4.1 --- Effects of TNF-α on the expression of TNF-receptors (TNFRs) in C6 glioma cells --- p.122 / Chapter 4.2 --- Effects of TNF-a on the expression of β1- and β2-adrenergic receptors (β1 and β2-ARs) in C6 glioma cells --- p.126 / Chapter 4.3 --- Relationship between TNF-α and β-adrenergic mechanism in C6 cells --- p.128 / Chapter 4.4 --- Effects of TNF-α on the expression of a transcriptional factor nuclear factor kappa B (NF-kB) in C6 glioma cells --- p.131 / Chapter 4.5 --- Effects of TNF-α on the expression of manganese superoxide dismutase (MnSOD) in C6 glioma cells --- p.133 / Chapter 4.6 --- Possible sources of β-agonists --- p.136 / Chapter 4.7 --- Conclusions --- p.137 / Appendix A --- p.143 / References --- p.145