Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2016-05-10T11:19:25Z No. of bitstreams: 1 TES_MARINA_PETERSEN_GEHRING_COMPLETO.pdf: 5818535 bytes, checksum: 5b089589ca820b17117fd0c8e9d1cc90 (MD5) / Made available in DSpace on 2016-05-10T11:19:25Z (GMT). No. of bitstreams: 1 TES_MARINA_PETERSEN_GEHRING_COMPLETO.pdf: 5818535 bytes, checksum: 5b089589ca820b17117fd0c8e9d1cc90 (MD5) Previous issue date: 2016-01-11 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Gliomas represent the most common class of malignant tumors of the central nervous system being the most aggressive, and lethal brain tumors in primary brain tumors. Among the treatments, radiation is one of the most used therapies, but the intrinsic radioresistance of these tumors remains a critical problem in the management of these patients. Currently it is known that the effect of radiation extends beyond the directly cytotoxicity caused in tumor cells. Radiation therapy appears to induce an immunogenic cell death that among the features is ATP release. The ATP can cause cytotoxicity via P2X7 receptor and also acts as a sign of damage activating the immune system. The ATP can be hydrolyzed by enzymes of the purinergic system, among them the ectonucleotidase CD39, to adenosine, which has an opposite effect to ATP, causing immunosuppression. The radiation-induced ATP release and the ability of this nucleotide in modulate immune responses raised the hypothesis about the purinergic signaling participation in the tumor and immune cells response to radiation. Therefore, this study investigated: i) the role of ectonucleotidase CD39/NTPDase1 in the radiation-induced immune response in gliomas, ii) the importance of ATP-P2X7 receptor in the gliomas response to radiotherapy. Using knockout mice for CD39/NTPDase1, we observed that the deletion of this enzyme combined with radiotherapy significantly reduced the immunosuppressive cells Tregs in the tumor and spleen, attenuated the infiltration of myeloid derived suppressor cells caused by radiation and increased CCR7 expression in splenic dendritic cells and macrophages, indicating the presence of freshly mobilized antigen presenting cells available to differentiate in immune-effector cells that sustain a more prolonged antigen-specific T-cell?mediated immune response. Thereby, showing that blocking the activity of CD39/NTPDase1 can control immunosuppressive mechanisms generated by the tumor and promises to improve the radiotherapy response. Furthermore, in this study we observed that radiation actives the P2X7 receptor and by silencing this receptor on the GL261 glioma cell line, we have shown that radiotherapy is less efficient in vivo when compared with mice injected with GL261 WT cells, which constitutively express the P2X7 receptor. We also showed that patients with glioma that overexpress the P2X7 receptor, showed a better response to radiotherapy, revealing the importance of the expression of this receptor on glioma cells as a useful marker to analyze the tumor sensitivity to radiation and a successful radiotherapy response. In summary, our data shed light on the purinergic signaling for modulating the radiotherapy response in gliomas. / Os gliomas representam a classe mais comum de tumores malignos do sistema nervoso central, sendo o tumor cerebral mais agressivo e letal entre os tumores cerebrais prim?rios. Dentre os tratamentos, a radia??o ? uma das terapias mais utilizadas, por?m a radiorresist?ncia intr?nseca destes tumores continua a ser um problema cr?tico na gest?o de destes pacientes. Atualmente, sabe-se que o efeito da radia??o se estende al?m da citotoxicidade direta causada nas c?lulas tumorais. A radioterapia parece induzir uma morte celular imunog?nica, que entre as caracter?sticas est? a libera??o de ATP. O ATP pode causar citotoxicidade atrav?s do receptor P2X7 e tamb?m atua como um sinal de dano celular ativando o sistema imune. O ATP pode ser hidrolisado por enzimas do sistema purin?rgico, dentre elas a ectonucleotidase CD39/NTPDase1, ? adenosina, que tem um efeito aposto ao ATP, causando imunossupress?o. A secre??o de ATP induzida pela radioterapia e a capacidade deste nucleot?deo em modular a resposta imune, levantou a hip?tese da participa??o da sinaliza??o purin?rgica na resposta de c?lulas tumorais ? radia??o e na resposta imune induzida pela radioterapia. Portanto, neste estudo visou-se investigar: i) o papel da ectonucleotidase CD39/NTPDase1 na resposta imune induzida pela radioterapia em gliomas, ii) a import?ncia da via ATP-receptor P2X7 na resposta de gliomas ? radioterapia. Atrav?s de camundongos knockout para a enzima CD39/NTPDase1, observamos que a dele??o desta enzima combinada com a radioterapia reduziu significativamente as c?lulas imunossupressoras Tregs no tumor e no ba?o, atenuou a infiltra??o de c?lulas mieloides supressoras causada pela radia??o, e aumentou a express?o de CCR7 em c?lulas dentr?ticas e macr?fagos localizados no ba?o, indicando a presen?a c?lulas apresentadoras de ant?geno rec?mmobilizadas e dispon?veis para se diferenciarem em c?lulas imunes efetoras que sustentam uma resposta imune mais prolongada mediada por c?lulas T ant?geno espec?ficas. Deste modo, mostrou-se que o bloqueio da atividade da CD39/NTPDase1 pode controlar mecanismos imunossupressores gerados pelo tumor e promete melhorar a resposta ? radioterapia. Al?m disso, neste estudo observou-se que a radioterapia ativa o receptor P2X7 e atrav?s do silenciamento deste receptor na linhagem de glioma GL261, demonstramos que a radioterapia foi pouco eficiente in vivo, quando comparado com camundongos injetados com a GL261 WT, que expressa constitutivamente o receptor P2X7. Tamb?m demonstramos que pacientes com glioma que expressaram mais o receptor P2X7, apresentaram uma melhor resposta a radioterapia, revelando a import?ncia da express?o deste receptor em c?lulas de glioma como um marcador ?til para analisar a sensibilidade tumoral ? radioterapia e para uma resposta bem-sucedida ? radioterapia. Em suma, nossos dados lan?am luz sobre a sinaliza??o purin?rgica para a modula??o da resposta ? radioterapia em gliomas.

Submitted by Verena Bastos (verena@uefs.br) on 2015-08-05T22:08:06Z No. of bitstreams: 1 Disserta??o Mona Oliveira vers?o final Maio2003 PPGbiotec.pdf: 4793209 bytes, checksum: 5e62150796f4527c8d492f92de5041e2 (MD5) / Made available in DSpace on 2015-08-05T22:08:06Z (GMT). No. of bitstreams: 1 Disserta??o Mona Oliveira vers?o final Maio2003 PPGbiotec.pdf: 4793209 bytes, checksum: 5e62150796f4527c8d492f92de5041e2 (MD5) Previous issue date: 2013-05-08 / Funda??o de Amparo ? Pesquisa do Estado da Bahia - FAPEB / Glioblastomas (GBMs) are the most common and aggressive primary tumors of the CNS. The survival of patients with this diagnosis remains very low, with poor prognosis even after surgical therapy associated with radiotherapy and chemotherapy. The present work carried out a screening for 24 synthetic ?alkaloid-like? to determine their effects on cell viability of quimioresistentes human (Gl-15 and U251) glioblastoma cells and murine (C6) glioma cells. Among the alkaloids tested (100?M) RLB87 was the most cytotoxic for transformed cells, inhibiting the viability in 75.0% 97.2% 76.6% of GL-15, U251 and C6 cells, respectively, after 72 h exposure, and it did not show toxicity to normal glial cells. It was also observed that RLB87 promoted apoptosis, 24 and 72 h after treatment, in a time-dependent manner. Moreover RLB87 also inhibited cell migration and proliferation with cells arrest at G0/G1 phase, since 24 h after treatment. Additionally, the cytotoxicity of four RLB87 analogues was tested in view to elucidate important aspects in chemical structure, required for its activity. We observed positive correlation between cytotoxic effect and isomeric of phenyl functions, with ester function and also lipophilicity. These finding suggest the ?alkaloid-like? RLB87 as a promising anticancer agent as well as a prototype for new agents for treatment of malignant and recurrent gliomas. / Glioblastomas (GBMs) s?o os tumores prim?rios mais comuns e agressivos do SNC. A sobrevida dos pacientes com esse diagn?stico continua muito baixa, tendo progn?stico ruim mesmo ap?s terapia cir?rgica seguida de radio e quimioterapia. No presente trabalho, foi realizada a prospec??o de 24 mol?culas de s?ntese, alkaloids-like, para determina??o de seus efeitos sobre a viabilidade de c?lulas quimioresistentes de glioblastoma humano (GL-15 e U251) e murina (C6). Entre os compostos testados ? (100JM), RLB87 foi o mais citot?xico para c?lulas transformadas, inibindo a viabilidade em 75,0%, 97,2%, 76,6% da GL-15, U251 e C6, respectivamente e o mesmo n?o apresentou toxicidade para c?lulas gliais normais. Observou-se, que o RLB87 promoveu apoptose 24 e 72 h ap?s o tratamento de forma tempo-dependente. O RLB87 igualmente inibiu a prolifera??o celular com acumulo na fase G0/G1 do ciclo celular ap?s 24 h. A migra??o das c?lulas de glioma foi tamb?m inibida ap?s tratamento com RLB87. Adicionalmente 4 an?logos do RLB87 foram tamb?m avaliados, elucidando aspectos importantes na estrutura qu?mica, requeridos para sua atividade, que possuem correla??o positiva com regioisomeria das fenilas, presen?a da fun??o ?ster e lipofilicidade. O RLB87 ? apresentado como promissor agente antineopl?sico assim como um prot?tipo para novos agentes terap?uticos para o tratamento de gliomas malignos e recidivados.

Glioblastoma multiforme (GBM) is the most common malignant central nervous system tumor. The ability of glioma cells to rapidly disperse and invade healthy brain tissue, coupled with their high resistance to chemotherapy and radiation have resulted in extremely poor prognoses among patients. In recent years, nitric oxide (NO) has been discovered to play a ubiquitous of role in human physiology and studies have shown that, at sufficient concentrations, NO is able to induce apoptosis as well as chemosensitization in tumor cells. This thesis discusses the synthesis and characterization of targeted NO donors for the treatment of GBM. Two glioma targeting biomolecules, Chlorotoxin (CTX) and VTWTPQAWFQWVGGGSKKKKK (VTW) were reacted with NO gas to synthesize NO donors. These NO donors, CTX-NO and VTW-NO, released NO for over 3 days and were able to induce cytotoxicity in a dose dependent manner in glioma cells. The biggest advantage, a result of the targeted delivery of NO, was that the NO donors did not have toxic effects on astrocytes and endothelial cells. To characterize the chemosensitizing effects of CTX-NO, cells were incubated with CTX-NO prior to exposure to temozolomide (TMZ) or carmustine (BCNU). These drugs are the most popular chemotherapeutics used in the treatment of GBM, but have only shown modest improvements in patient survival. Viability studies showed that CTX-NO selectively elicited chemosensitivity in glioma cells, whereas the chemosensitivty of astrocytes and endothelial cells remained unaffected. Further investigation showed that CTX-NO pretreatment decreased O6-methylguanine DNA methyltransferase (MGMT) and p53 levels, suggesting that a decrease in DNA repair ability may be the mechanism by which chemosensitivity is induced. Lastly, the effects of CTX-NO on glioma cell invasion and migration were studied using Boyden chamber and modified scratch assays. Non-toxic doses of CTX-NO decreased glioma cell invasion in a dose dependent manner. Studies quantifying matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) surface expression demonstrated that while MMP-2 expression was decreased by both CTX and CTX-NO, MMP-9 expression was decreased only by CTX-NO. Furthermore quantifying MMP-2 and MMP-9 activity levels showed that NO and CTX work synergistically to decrease the activity of the enzymes. These studies demonstrate that the decrease in glioma invasion resulting from CTX-NO treatment was partially a consequence of decreased levels of surface and activated MMP-2 and MMP-9. The work presented in this thesis describes a novel approach to treating GBM that can be modified to develop treatments for various other tumors. Furthermore this is the first study to develop glioma-targeting NO donors.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

This thesis reports that checkpoint adaptation occurs in human brain cancer cells. M059K cells, after treatment with camptothecin (CPT), recruited γ-histone H2AX, phosphorylated Chk1 and arrested in the G2 phase. Strikingly, cells escaped the checkpoint, became rounded and entered mitosis as measured by phospho-histone H3 signals. Lamin A/C immunofluorescence microscopy revealed that 48% of the cells that survived checkpoint adaptation contained micronuclei. These data suggest that brain cancer cells undergo checkpoint adaptation and may have an altered genome. This thesis also explored if phosphatases participate in checkpoint adaptation. Human colon cancer cells were treated with CPT and the PP2A inhibitor cantharidin. Following treatment the cells became rounded and 65% were positive for phospho-histone H3 signals indicating that cantharidin caused cells to be in mitosis following CPT treatment. These data suggest that PP2A might have a role in checkpoint adaptation, or participate in a pathway that bypasses checkpoint adaptation. / xi, 114 leaves : ill. (some col.) ; 29 cm

Τα γλοιώματα είναι η πιο συχνή κακοήθης μορφή καρκίνου του κεντρικού νευρικού συστήματος. Δυστυχώς, είναι επίσης μεταξύ των πιο δύσκολων μορφών όσο αναφορά τη θεραπεία, με αποτέλεσμα την κακή πρόγνωση των ασθενών. Γλοιώματα παρουσιάζουν πολύπλοκη κυτταρική και γενετική ετερογένεια, περιορίζοντας σημαντικά τις στοχευμένες θεραπευτικές προσεγγίσεις. Η ανακάλυψη γονιδίων και σηματοδοτικών μονοπατιών που ρυθμίζουν την επιβίωση και την ανάπτυξη των καρκινικών κυττάρων θα βοηθήσουν στην ανάπτυξη νέων και πιο αποτελεσματικών θεραπειών. Η πρωτεΐνη απόκρισης στο κυκλικό-AMP (CREB) είναι ένα πυρηνικό μόριο που ενεργοποιείται μέσω φωσφορυλίωσης από κινάσες σερίνης/θρεονίνης και ελέγχει τη μεταγραφή πολλών γονιδίων στα νευρικά κύτταρα. Ο ρόλος του CREB στη νευρωνική λειτουργία κυμαίνεται από την επιβίωση και τον πολλαπλασιασμό σε πιο πολύπλοκες εγκεφαλικές λειτουργίες, όπως η μνήμη και οι συμπεριφορές εθισμού. Δεδομένα από πρόσφατες έρευνες δείχνουν πως ανώμαλη έκφραση ή ενεργοποίηση του CREB μπορεί να προσδώσει καρκινικές ιδιότητες σε διάφορους τύπους ιστών και κυττάρων. Επίσης το CREB συμμετέχει σε διαδικασίες επιβίωσης και μετάστασης σε μεγάλο αριθμό καρκινικών κυτταρικών σειρών. Με βάση αυτά μελετήθηκε ο ρόλος του CREB σε κύτταρα καρκίνου του εγκεφάλου, με έμφαση στις περιπτώσεις γλοιοβλαστώματος. Δείξαμε ότι τόσο τα επίπεδα ενεργοποίησης όσο και ο αριθμός των κυττάρων που στα οποία το CREB είναι φωσφορυλιωμένο/ ενεργοποιημένο είναι σημαντικά αυξημένος σε σχέση με δείγματα παρακείμενου μη καρκινικού ιστού. Επιπλέων βρέθηκε πως η αποσιώπηση του CREB σε καρκινικές σειρές γλοιοβλαστώματος, με χρήση μορίων siRNA, επέφερε σημαντική μείωση στην βιωσιμότητα τους. Τα παραπάνω ευρήματα συσχετίζουν για πρώτη φορά το μεταγραφικό παράγοντα CREB με το γλοιοβλάστωμα και του προσδίδουν ένα αρκετά σημαντικό ρόλο όσο αναφορά την επιβίωση των συγκεκριμένων καρκινικών κυττάρων. / Gliomas are the most common malignant cancers of the nervous system. Unfortunately, they are also amongst the most difficult cancers to treat, resulting in poor patient prognosis. Gliomas exhibit complex cellular and genetic heterogeneity, limiting effective targeted therapy approaches. Discovering the genes and pathways that regulate cancer cells survival and growth will aid in the development of novel and more effective treatments. The Cyclic-AMP Response Element Binding protein (CREB) is a serine/threonine kinase-regulated nuclear factor modulating the transcription of numerous genes in nerve cells and has various roles in neuronal function, ranging from survival and proliferation to more complex brain functions, such as memory and drug addiction behaviours. Given recent findings that aberrant CREB expression can impart oncogenic properties on myeloid cells, liver cells and ovarian epithelial cells, we explored the potential role of CREB in brain cancer biology by examining its expression in a panel of human patient brain tumour specimens. We show that both the level of expression and the number of cells expressing activated/phosphorylated CREB is markedly elevated in tumours compared with adjacent non-tumour control brain tissue. Moreover using siRNA molecules we knocked-down the expression of CREB in glioblastoma cell lines and then we studied the viability of these cells throughout 96 hours with the use of MTT assay. A significant reduction in viability was observed at CREB siRNA transfected cells against the control. These observations are the first to highlight a link between CREB and brain tumours. Our hypothesis is that CREB has a role in brain tumour development/growth and that at least some of CREB's neuro-oncogenic properties are due to its role in neural cells survival and proliferation.

Des mutations dans ATRX, une protéine de remodelage de la chromatine, ont été associées, dans plusieurs études cliniques, avec la voie télomérase-indépendante de maintenance des télomères (voie ALT) dans plusieurs types de cancer. Grâce à des expériences d’immunoprécipitation de chromatine (ChIP), nous avons montré qu’ATRX était localisée au niveau subtélomérique de cellules tumorales humaines en culture. Nous avons également montré, par ChIP, que l’inactivation génétique d’ATRX provoquait une diminution des quantités de cohésine/SMC1 présentes dans les régions subtélomériques. L’inactivation d’ATRX a conduit en outre à une diminution des quantités de TERRA, transcrits non codants de l’ADN télomérique. Nos données suggèrent qu’ATRX pourrait établir des interactions fonctionnelles avec la cohésine au niveau de la chromatine subtelomérique afin de contrôler les niveaux de TERRA et que l’un ou l’autre de ces évènements pourrait avoir un rapport avec la voie ALT. / Mutations in ATRX, a chromatin remodeling protein, have been found, in several clinical studies, associated with the telomerase-independent ALT pathway of telomere maintenance in several types of cancer. Using chromatin immunoprecipitation (ChIP), we have shown that ATRX localized to subtelomeric regions of human tumor cells in culture. Cohesin has recently been shown to be part of telomeric chromatin. Here, using ChIP, we showed that genetic inactivation of ATRX provoked a diminution in the amount of cohesin in subtelomeric regions of telomerase-positive glioma cells. Moreover, inactivation of ATRX also led to a diminution in the amount of TERRAs, non-coding RNAs resulting from transcription of telomeric DNA. Our data suggest that ATRX might establish functional interactions with cohesin on subtelomeric chromatin in order to control TERRA levels and that one or the other or both of these events might be important for ALT mechanisms.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2014-08-11T13:38:15Z No. of bitstreams: 1 Rute Maria Ferreira Lima. Caracterização... 2013.pdf: 5129350 bytes, checksum: c7d761267de05b2aa805d0323bc5f557 (MD5) / Made available in DSpace on 2014-08-11T13:38:16Z (GMT). No. of bitstreams: 1 Rute Maria Ferreira Lima. Caracterização... 2013.pdf: 5129350 bytes, checksum: c7d761267de05b2aa805d0323bc5f557 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / O astrocitoma grau IV ou glioblastoma multiforme (GBM) é o mais maligno e com prognóstico ruim entre os gliomas. Esse prognóstico sombrio está associado, em parte, à quimiorresistência (QR). Ao lado disso, a classificação atual dos gliomas não consegue responder a heterogeneidade da resposta ao tratamento. Assim, parece existir subtipos de GBM com características distintas. Dessa forma, o objetivo desse trabalho foi caracterizar fenotipicamente uma nova linhagem, ESP12, e, desenvolver um modelo in vitro para a avaliação da QR. Amostras obtidas de glioma humano foram estudadas quanto aos achados característicos de malignidade e subtipadas quanto aos fenótipos proliferativo e pró-neural, imunohistoquimicamente. As culturas obtidas das amostras foram mantidas a 37 ºC em atmosfera com 5% de CO2. A caracterização de ESP12 incluiu: a) subtipagem por imunocitoquímica e por citometria de fluxo; b) investigação de um fenótipo de resistência, através da identificação de células CD133+ e de proteínas de resistência às múltiplas drogas, glicoproteína-P (Pgp) e MRP1; c) avaliação da cinética de crescimento, através da determinação do tempo de duplicação celular (TDPC); d) verificação da produção do fator de crescimento endotelial vascular (VEGF); e) avaliação da viabilidade celular, através do teste com MTT, quando exposta a carmustina (BCNU), a vimblastina (VIM) e a temozolomida (TMZ). Por fim, investigamos a atividade quimiossensibilizante do 8-metoxipsoraleno (8-MOP) utilizando o modelo estabelecido. Foram obtidos 6 casos de GBM e 3 casos de gliomas de graduação III, pela Organização Mundial de Saúde, com idade média de 52,6  14,1 anos, a maioria homens. As manifestações clínicas mais frequentes foram crises convulsivas e cefaleia, e, a localização tumoral foi variada. Os achados de imagem se correlacionaram com os achados histoquímicos confirmando o diagnóstico. A sobrevida dos pacientes variou entre 11 dias e 24 meses, com mediana de 11,5 meses. As células formaram monocamadas e revelaram intenso pleomorfismo. A maior parte das amostras apresentou fenótipo proliferativo na imunohistoquímica. As proteínas que caracterizam os fenótipos pró-neural, proliferativo e mesenquimal foram detectadas tanto por imunohistoquímica quanto por imunocitoquímica, em ESP12. Quantitativamente, o fenótipo proliferativo foi mais evidente detectado por citometria de fluxo. Células CD133+ representaram menos que 1%. Além disso, 38,6% das células foram positivas para a Pgp. Não houve diferença entre a produção do VEGF por ESP12 quando comparada a outras linhagens de GBM já estabelecidas. O TDPC de ESP12 foi de 31 h. ESP12 se mostrou mais sensível do que outras linhagens de GBM já estabelecidas a BCNU, a VIM e a TMZ. Por fim, o 8-MOP mostrou atividade quimiossensibilizante significativa. / The astrocytoma grade IV also known as glioblastoma multiforme (GBM) is the most malignant and has a poor prognosis among gliomas. This poor prognosis is associated, in part, to chemoresistance (QR). Furthermore, the current classification of gliomas cannot answer the heterogeneity of treatment response. Therefore, it seems to exist GBM subtypes with distinct characteristics. The aim of this study was to characterize phenotypically a new cell line, ESP12, and to develop an in vitro model for the assessment of QR. Human glioma samples were studied by immunohistochemistry for the characteristic findings of malignancy and subtyped as to proliferative and proneural phenotypes. Primary cultures were obtained from samples and maintained at 37 °C in an atmosphere with 5% CO2. The characterization of ESP12 included: a) subtyping by immunocytochemistry and flow cytometry; b) investigation of a resistance phenotype by identifying CD133+ cells and the multidrug resistance proteins, P-glycoprotein (Pgp) and MRP1; c) evaluation of the growth kinetics, by determining the cell doubling time (TDPC); d) assaying of vascular endothelial growth factor (VEGF) production; e) the assessment of cell viability by the MTT test after exposure to carmustine (BCNU), vinblastine (VIM), and temozolomide (TMZ). Finally, we investigated the chemosensibilizing activity of 8-methoxypsoralen (8-MOP) using the established model. Six cases of GBM and 3 cases of grade III gliomas were obtained, with a mean age of 52.6  14.1 years, mostly men. The most common clinical manifestations were seizures and headache, and the tumor location was varied. Imaging findings were correlated with the histochemical findings confirming the diagnosis. The median survival was 11.5 months (range: 11 days to 24 months). The cells formed monolayers and showed intense pleomorphism. Most samples showed proliferative phenotype in immunohistochemistry. Proteins that characterize the proneural, proliferative and mesenchymal phenotypes were detected both by immunohistochemistry and by immunocytochemistry on ESP12. The proliferative phenotype was more quantitatively evident by flow cytometry. CD133+ cells represented less than 1%. Moreover, 38.6 % of cells were positive for Pgp. There was no difference between the production of VEGF between ESP12 and other GBM cell lines already established. The TDPC of ESP12 was 31 h. ESP12 was more sensitive than other cell lines already established of GBM to BCNU, TMZ and VIM. Finally, the 8-MOP showed significant chemosensibilizing activity.

En 2006 Dilmanian et al. ont proposé une méthode d'irradiation par rayonnement synchrotron innovante appelée minifaisceaux. L'irradiation de tumeur par minifaisceaux monochromatiques consiste en un motif fractionné spatialement de faisceaux de rayons X submillimétriques produits par un synchrotron. Afin d'obtenir une dose homogène dans le volume cible, deux incidences orthogonales sont entrecroisées. Le tissu sain environnant ne subit que l'irradiation fractionnée, entre les faisceaux la dose n'est dûe qu'au diffusé et l'énergie déposée y est donc 10 à 15 fois inférieure à celle déposée sur les axes des faisceaux. Cela permet un effet protecteur des tissus sains tout en distribuant de fortes doses à la tumeur. Cette thèse porte sur le développement de la méthode expérimentale des minifaisceaux monochromatiques, ce qui comprend le contrôle de la géométrie d'irradiation, la dosimétrie expérimentale et l'étude Monte Carlo correspondante. Afin d'évaluer son efficacité, des études précliniques ont été réalisées sur un modèle de tumeur cérébrale implantée chez le rat (F98). Un suivi de traitement est réalisé par de l'imagerie anatomique et fonctionnelle afin d'évaluer son efficacité. L'imagerie de perfusion cérébrale (menant aux volumes et débits sanguin cérébrales, au temps de transit moyen) est d'après la littérature un moyen efficace de pronostique du résultat du traitement. Les paramètres clés de la vascularisation cérébrale sont principalement étudiés par imagerie IRM, du fait de l'innocuité de ce type d'imagerie. La Synchrotron Radiation Computed Tomography (SRCT) est une modalité d'imagerie dont les performances sont proches des limites théoriques dans l'obtention de mesures absolues des concentrations d'agent de contraste et peut être utilisé en tant que gold-standard. Les modèles pharmacocinétiques utilisés nécessitent comme paramètre d'entrée des concentrations d'agent de contraste en fonction du temps. La relation entre le signal obtenu par IRM et la concentration d'agent de contraste est très complexe et difficilement quantitative. Une comparaison des mesures de perfusion effectuées par IRM et par SRCT a été effectuée afin de calibrer les mesures IRM. / An innovative method of synchrotron radiation therapy, called minibeams, was proposed by A. Dilmanian et al. in 2006. Minibeams consists in tumor irradiation with monochromatic submillimetric x-ray beams spatially fractionated produced by a synchrotron source. To obtain a homogeneous dose in the target volume, an interleaving is realized using two orthogonal incidences. Adjacent healthy tissue is only partially irradiated by minibeams, the areas between the beams only receive scattered radiation and therefore the energy deposited is 10 to 15 times lower than on one minibeam axis, leading to a sparing effect of healthy tissue even when a high dose is deposited in the target volume. The thesis project is the development of this experimental method of monochromatic minibeams, which involves the control of the irradiation geometry, the control of dosimetry and its modeling by Monte Carlo simulations. To evaluate the method, preclinical experiments on models of brain tumors implanted in rats (F98) are performed. Follow-up by anatomical and functional imaging is carried out to evaluate the effectiveness of the treatment. Functional imaging of cerebral perfusion (volume and cerebral blood flow, mean transit time of heavy elements) appears to be associated in the literature as a relevant method for monitoring prognostic. The key parameters of the cerebral vasculature are mainly studied in magnetic resonance imaging (MRI), because of the harmlessness of this imaging modality. The relation between MRI signal and contrast agent concentration is very complex and no quantitative relationship is well known. Synchrotron Radiation Computed Tomography (SRCT) is an imaging modality with performances to measure absolute contrast agent concentration very close to the theoretical limits and can be used as gold-standard. The used pharmacokinetic models need as input parameters a contrast agent concentration versus time. A comparison of perfusion measurements between MRI and SRCT has been done in order to calibrate MRI measurements.

Les gliomes des bas-grade sont des tumeurs cérébrales lentement évolutives, affectant principalement les jeunes adultes, qui peuvent rester des années sans symptôme. Les patients peuvent être opérés, ou traités par radiothérapie ou chimiothérapie, avec deux thérapies possibles : le PCV et le témozolomide (TMZ).Nous souhaitons montrer dans ces travaux de thèse que la modélisation mathématique, à travers l'approche de population, peut permettre l'amélioration des traitements en termes de durée et d'amplitude de décroissance pour les gliomes de bas-grade traités par chimiothérapie (PCV et TMZ).Dans un premier temps, nous nous concentrons sur la possibilité de modifier le protocole d'administration du PCV, au niveau de la population, afin de prolonger la durée de décroissance tumorale. Nous concluons qu'espacer les cycles de traitement permet de repousser de manière significative le moment de recroissance de la tumeur.Dans un second temps, nous étudions l'évolution des gliomes de bas-grade traités par TMZ. Sur la base des données de tailles tumorales de 77 patients, ainsi que d'informations génétiques, nous développons un modèle K-PD à effets mixtes permettant de décrire la dynamique tumorale avant, et suite au traitement. Nous évaluons ensuite les capacités du modèle à prédire la durée et l'amplitude de la réponse tumorale, à partir de mesures précoces de tailles de la tumeur ainsi que des informations génétiques. Ces prédictions pourraient être utilisées pour aider les cliniciens à déterminer si le traitement doit être prolongé ou non, pour un patient donné.Enfin, nous nous intéressons plus particulièrement au phénomène de résistance au traitement par TMZ. A partir des mêmes données de tailles tumorales que précédemment, nous construisons un modèle PK-PD à effets mixtes décrivant l'apparition des cellules résistantes au sein de la tumeur. Ce modèle reproduit plus précisément l'évolution du TMZ dans l'organisme et son impact sur la tumeur. Il est utilisé pour optimiser le protocole thérapeutique au niveau individuel. A l'aide d'un algorithme d'optimisation, nous déterminons l'intervalle entre chaque cycle et la dose à administrer afin de prolonger la durée de décroissance tumorale tout en limitant l'émergence de résistance. Les protocoles ainsi optimisés sont évalués à l'aide d'une approche stochastique, permettant de tester la robustesse du modèle et de l'optimisation.A travers les différents travaux de cette thèse, nous montrons l'utilité de la modélisation mathématique pour aider à l'amélioration des traitements chimiothérapiques pour les patients souffrant de gliomes de bas-grade. Nous croyons que ces résultats peuvent être transposés à d'autres types de cancers. / Low-grade gliomas are slow-growing brain tumors, mainly affecting young adults who may remain without any symptoms for years. Patients can undergo surgery, or receive radiotherapy or chemotherapy with two different treatments: PCV of temozolomide (TMZ).In our different projects, we aim to show that mathematical modeling, and population approach, can allow to improve treatments, in terms of response duration and amplitude, for low-grade gliomas treated with chemotherapy (PCV and TMZ).In a first part, we focus on the possibility to modify PCV administration protocol, on a population level, to prolong tumor decrease duration. We claim that prolonging time interval between cycles enables us to significantly postpone the time to tumor regrowth.In a second part, we study the evolution of low-grade gliomas treated with TMZ. We analyze tumor size observations of 77 low-grade glioma patients, as well as genetic information, to develop a K-PD mixed-effects model describing tumor evolution before and after treatment onset. We then evaluate model capacity to predict tumor response duration and amplitude, on the base of early tumor sizes and genetic information. These predictions could be used to help clinicians to determine if they should prolong the treatment or not, for a given patient.In a last part, we more particularly focus on the phenomenon of resistance to TMZ. We build a PK-PD mixed-effects model describing the emergence of resistant tumor cells, using the same tumor size observations as previously. This model more accurately reproduces the evolution of TMZ in the body and its effect on the tumor. It is then used to optimize TMZ therapeutic protocol, on an individual level. Using an optimization algorithm, we determine the time interval between TMZ cycles, and the dose to administer, to prolong tumor decrease duration while limiting the emergence of resistance. The optimized protocols are evaluated with a stochastic approach, allowing to test the robustness of the model and the optimization.Through these different projects, we show the utility of mathematical modeling to help to improve chemotherapy treatments of low-grade glioma patients. We believe that these results could be transposed to other types of cancers.