Thèses sur le sujet « Gliomi »

Gliomas are considered among the most puzzling problems of medicine due to the complexity of their anatomic localization, and the lack of a real therapeutic treatment. The main topic of this work concerns the possibility to use bovine herpesvirus type 4 (BoHV-4) as a vector for gene therapy for the treatment of glioma, and that the cAMP-dependent protein kinases (PKA) pathway as a possible target for such therapy. This study is based on a research project that involved both University of Padova and University of Parma. Here BoHV-4 to is shown to infect rat glioma cells in vitro and in vivo, in addition, BoHV-4 can infect human immortalized glioma cells in vitro and human brain tumour primary cell cultures. The study of the relationship between gliomas and the PKA pathway shows a peculiar distribution of the PKA regulatory subunits in glioma cells in mouse, rat and human. The present data suggest that the PKA pathway modulation may be targeted for the treatment of gliomas. The present Study pave the way to the use of a safe and efficient BoHV-4-based vector for the delivery of therapeutic genes or for the targeting of specific abnormal pathways, like the PKA-mediated one, for the treatment of the hopeless high-grade glioma.<br>L’ipotesi alla base di questo lavoro è che vi sia la possibilità di impiegare quale vettore virale per la terapia genica dei gliomi l’herpesvirus bovino di tipo 4 (BoHV-4) e di determinare se la via di trasduzione del segnale mediata dalle protein chinasi AMPc-dipendenti (PKA) possa essere utilizzata come bersaglio terapeutico. Infatti i gliomi sono ritenuti essere uno dei più importanti e stimolanti problemi irrisolti della medicina. Questo sia per la evidente complessità della sede anatomica di insorgenza, sia per il fatto che, nonostante gli enormi sforzi in cui gli scienziati di tutto il mondo si sono profusi non si è ancora riusciti a giungere alla messa a punto di un protocollo curativo realmente efficace. Il progetto di ricerca su cui si è basato il presente studio è il risultato della convergenza di due linee di ricerca preesistenti presso le Università di Padova e di Parma. I risultati conseguiti hanno dimostrato la capacità di BoHV-4 di infettare in vitro e in vivo cellule di glioma di ratto. Inoltre tale capacità è stata confermata in vitro sia su cellule immortalizzate di glioma umano che su colture primarie di tumore cerebrale umano. Per quanto riguarda lo studio della relazione tra PKA e tumori cerebrali, è stato in primo luogo rilevata una distribuzione peculiare delle diverse subunità regolatorie delle medesime che è caratteristica delle cellule di glioma. Inoltre diversi esperimenti suggeriscono che la modulazione di tale via possa essere impiegata per il trattamento dei gliomi oltre che per fini diagnostici. Tutti i risultati ottenuti suggeriscono di proseguire ed ampliare il progetto facendo convergere entrambe le sue linee costituenti in un modello da poter esportare nella pratica clinica nel minor tempo possibile.

Si sono studiati i correlati neurali dei sistemi di memoria verbale in pazienti con glioma cerebrale. Si tratta di pazienti sottoposti ad asportazione di lesioni cerebrali dell’emisfero dominante, in cui le funzioni mnestiche sono state valutate mediante prove specifiche prima e dopo l’intervento chirurgico, per verificare l’impatto funzionale dell’espanso e della rimozione della lesione. L’indagine anatomo-funzionale si è inoltre avvalsa delle tecniche di mappaggio cerebrale in awake surgery laddove l’intervento è stato eseguito in anestesia asleep-awake per la rimozione dei tumori estesi alle aree del linguaggio. Nel primo studio si esamina l’impatto della crescita tumorale sulla memoria verbale: le valutazioni neuropsicologiche effettuate pre-trattamento hanno permesso di identificare difficoltà conclamate o lievi compromissioni, che variano in funzione delle aree di infiltrazione tumorale; la prospettiva anatomo-clinica ha fornito una conferma dell’esistenza di sistemi di memoria verbale in distretti anatomo-funzionali differenti. Il secondo studio esamina i correlati neurali della memoria verbale a breve termine in un gruppo di pazienti sottoposti a mappaggio cerebrale in awake surgery: la stimolazione corticale e sottocorticale diretta ha permesso di identificare un circuito fronto-parietale coinvolto nei processi di MBT; l’analisi degli errori registrati durante i test intraoperatori ha permesso inoltre di distinguere il substrato neurale delle due componenti del circuito fonologico. Il terzo studio valuta gli effetti della lobectomia temporale sulla memoria a lungo termine; le prove mnestiche somministrate ad una settimana dall’intervento e dopo tre mesi hanno evidenziato prestazioni patologiche che non vanno incontro ad un rapido recupero nei pazienti che hanno subito la rimozione di un circuito cortico-sottocorticale esteso dalle regioni temporo-polari a quelle temporali mesiali; lo studio anatomico mediante voxel-based lesion-symptom mapping delle aree asportate ha permesso di verificare i correlati neurali di memoria semantica e apprendimento verbale. Questi risultati confermano che i pazienti con tumore cerebrali possono fornire dati interessanti nello studio dei deficit neuropsicologici.

La Risonanza magnetica di diffusione (dMRI) sta diventando lo strumento più adatto per indagare la microstruttura del cervello umano in vivo. Modellando le proprietà della diffusione dell’acqua nei tessuti cerebrali, è infatti possibile ottenere delle misure simili a quelle derivate dall’istologia, come la densità di fibre, la loro conformazione e la loro direzione di propagazione, in maniera non invasiva. In più, misure locali di integrità e di orientazione della materia bianca possono essere usate da algoritmi di trattografia per ricostruire globalmente il percorso seguito dalle fibre in tutto il cervello, permettendo di studiare come le varie regioni corticali sono connesse. Nonostante ciò, l’utilizzo della dMRI deve essere condotto con attenzione in presenza di patologie che alterano drasticamente la fisiologia del cervello, come nel caso dei tumori cerebrali. La varietà di microambienti cellulari che caratterizza questo tipo di patologie invalida alcune ipotesi sul quale si fondano i modelli di microstruttura basati sulla dMRI. In più, il processo di ricostruzione della trattografia nel cervello presenta particolari difficoltà tecniche nelle regioni affette dalla patologia. Date queste limitazioni, vi è del valore nell’utilizzare tecniche basate sulla dMRI in questo complesso ambiente patologico? Negli ultimi tre anni, ho avuto modo di esplorare diverse di queste metodologie in una popolazione di pazienti con tumore cerebrale. La presente tesi vorrebbe quindi essere una sintesi di questo lavoro, che costituisce una base verso l’integrazione di tecniche di diffusione avanzate all’interno della pratica neuro-oncologica. Nella sua interità, la tesi presenta tre lavori, organizzati come segue: La prima parte presenta uno studio analitico su due noti modelli di microstruttura, Neurite Orientation Dispersion and Density Imaging (NODDI) e la Spherical Mean Technique. Questo lavoro è volto alla quantificazione della bontà del fit e precisione parametrica delle due tecniche all’interno della lesione tumorale. Alcuni lavori, concentrati principalmente su NODDI, usano queste tecniche come modelli di segnale e non biofisici, cercando di trovare biomarker capaci di caratterizzare aspecificamente il tessuto patologico. L’analisi qui svolta supporta i risultati di letteratura da un punto di vista tecnico, senza considerazioni sul significato biologico di questi modelli. La seconda parte contiene uno studio di confronto tra due diversi metodi per la quantificazione di regioni di materia bianca sconnessa a causa del tumore. Due categorie di approcci qui sono stati studiati: approcci diretti, e approcci indiretti. I primi fanno uso della trattografia singolo-soggetto per investigare quali fasci di fibre siano affetti nel loro decorso dalla presenza del tumore. I secondi invece non hanno bisogno di acquisizioni dMRI, e utilizzano un atlante normativo di fasci di materia bianca per investigare, probabilisticamente, quali di questi potrebbero essere affetti data la locazione e l'estensione della zona tumorale. Utilizzando noti strumenti di analisi dell’immagine, i due approcci vengono qui confrontati, discutendo pregi e difetti di ciascun metodo. Nella terza e ultima parte della tesi, viene studiata la relazione tra alterazioni di matrici di connettività strutturale (SC) di pazienti tumorali e variazioni regionali di metabolismo misurate usando la Tomografia ad Emissione di Positroni (PET) con tracciante [18F]-FDG. All'interno di questo studio, viene prima proposta una procedura per la selezione dell’algoritmo di trattografia ottimale per le analisi. A seguire, viene sviluppata una metodologia statistica per rilevare le loro connessioni della matrice SC alterate dalla presenza del tumore. La presenza di queste alterazioni viene infine correlata con la PET, e si discutono i risultati ottenuti, ponendo particolare attenzione alle limitazioni di entrambe queste modalità di imaging.<br>Diffusion-based Magnetic Resonance Imaging (dMRI) is rapidly becoming the instrument of choice to probe the structure of the human brain in vivo. By modelling the properties of water diffusion inside cerebral tissues, it is indeed possible to extract surrogates of histological measures, such as fibre density, conformation and preferential direction, in a non-invasive manner. Furthermore, local orientational features can be used to reconstruct axonal pathways that link different brain regions, allowing the study of how they are structurally connected. Nevertheless, the quantification of dMRI measures must be cautious when the physiological environment of brain tissues is drastically altered. Such is the case of brain tumours. The microstructure of brain tumours is highly heterogeneous, being diverse between and inside specific types and malignancy grade. The wide spectrum of cellular environments they feature invalidates several hypotheses on which diffusion-based microstructure models are built and, contemporarily, poses difficulties in the process of tracking white matter in affected regions. Given these limitations, are these techniques worth using in this complex pathological environment? During the last three years I explored several state of the art diffusion-based methodologies in a cohort of patients suffering from a range of brain tumours. Hence, this thesis strives to be a summary of this work, laying the foundation for future studies aiming to integrate the use of advanced dMRI in the clinical neuro-oncological practice. The thesis is divided in three main parts, which are organized as follows: In the first part, an assessment is made whether two widely known diffusion advanced models, Neurite Orientation Dispersion and Density Imaging (NODDI) and the Spherical Mean Technique (SMT) are properly fitted in the tumoral lesion in terms of goodness-of-fit and parameter precision. Several works, concentrating mainly on NODDI, used such techniques not as biophysical models but as signal representations, trying to find biomarkers that differentiate more and less isotropic environments which contribute to the totality of the diffusion signal in ‘tumoral’ voxels. These studies were performed without first checking whether these diffusion metrics are mathematically reliable. This issue is here assessed from a technical point of view, without giving specific biophysical meaning to the models in exam inside the tumoral tissues The second part features a comparison study between methods for the identification of structurally disconnected white matter (WM) in brain tumour patients. Here, two branches of methodologies were identified, namely direct and indirect approaches. The formers use single-subject tractography to directly investigate which fibre bundles may be affected by the presence of the tumour. The latters, instead, embed the focal lesion on a normative atlas of white matter tracts, identifying the probability of a WM voxel being disconnected by the pathology. Employing known image analysis metrics, both approaches are discussed, highlighting points of convergence, but also of disagreement, in terms of the physio-pathological information they can convey. In the third and last part of this thesis, tumour-related anomalies of diffusion-based structural connectivity (SC) matrices are put in relationship with metabolic measures from [18F]-FDG PET. A procedure for tractography algorithm selection was firstly performed, and after the SC quantification, a statistical method of detecting altered connections in the tumour-affected SC matrix is presented. Within such a framework, the amount of affected SC entries was eventually quantified in the available cohort of patients and put in relationship with standardized uptake values from PET. Finally, a discussion of the results of this association is provided, paying particular attention to the limitations of these imaging modalities in the brain oncological field.

Gliomas constitute 36% of all primary brain tumors and 81% of all primary malignant brain tumors. The overall prognosis in patients with gliomas depends mainly on the location and histologic grade of the tumor.<p>The World Health Organization classification of gliomas is the primary basis for guiding therapy and assessing overall prognosis in gliomas. This classification system, based on histological features, often falls short of predicting therapeutic response of individual tumors within the same histological grade. Yet, it still remains the grading method for both research and clinical prospects.<p>Unlike any other organ the brain has multiple protective layers such as the skull that ensure a homeostatic environment. The resulting reduced access to the brain and the absence of plasmatic brain tumor markers bring neuroimaging in a central position in diagnosis and management of brain tumors. Moreover, neuroimaging has evolved from a purely morphologic investigation into a diagnostic tool that allows characterization of particular physical alterations within brain tissue. Understanding the relationship between the physical characteristics of tumor tissue, studied by MR imaging, and biological characteristics of the tumor is therefore important for the appropriate integration of neuroimaging in brain tumor management. The general objective of this work is to define the relationship between physiologybased MR imaging and biological features of glial tumors. Diffusion and perfusionweighted imaging, physiologybased MR techniques provide the data based on physical characteristics of the tissues. Diffusion weighted imaging (DWI) allows the measurement of water molecules diffusivity within the brain tissue by means of apparent diffusion coefficient (ADC) measurements. Perfusion weighted imaging (PWI) is based on changes of MR signal during the passage of contrast material through the intravascular space and allows hemodynamic measurements such as those of cerebral blood volume (CBV)within the brain tissue.<p>Highgrade diffuse gliomas are currently differentiated from low grade diffuse gliomas by increased cellularity with nuclear atypia, mitotic activity, endothelial proliferation and necrosis. Components of the extracellular matrix and angiogenesis constitute some other features of gliomas, which have established links with oncogenic processes that influence the proliferative and infiltrative potentials of these tumors. We have specifically targeted these features in our comparative studies with the working hypothesis that physiologybased MR measurements, obtained in vivo, might provide information that is pertinent in terms of tumor malignancy.<p>We chose to approach the biology of brain tumors in two ways: in vivo, by means of metabolic imaging techniques such as positron emission tomography (PET) and ex vivo, by means of histological and immunohistochemical analyses of tumor specimens.<p>Many studies have investigated the relation between ADC values and cellularity in gliomas. The underlining theory is based on the premise that water diffusivity within the 9 extracellular compartment is inversely related to the content and attenuation of the constituents of the intracellular space. Therefore when cellularity increases, intracellular space volume increases with a relative reduction of the extracellular space, leading to restricted diffusion of water molecules. However other factors may affect the value of ADC in gliomas such as the extracellular matrix which contains various amounts of hydrophilic macromolecules susceptible to change water molecules diffusivity. Hyaluronic acid is one highly hydrophilic component of the extracellular matrix of gliomas and its level of expression changes significantly during the progression to anaplasia in gliomas. Our hypothesis was that hyaluronan may influence ADC values in high and low grade gliomas.<p>We demonstrated a positive correlation between ADC values and the immunohistochemical level of hyaluronan in glial tumors.<p>Previous studies have confirmed the utility of positron emission tomography using C11 Methionine (PETMET) as a prognostic tool in patients with gliomas. Higher MET uptake is associated with greater proliferative potential and a higher level of malignancy in gliomas.<p>The increased aminoacid uptake in gliomas seems to reflect increased transport mediated by aminoacid carriers located in the endothelial cell membrane. Our hypothesis was that CBV measurements, index of tumor vascularity, may be related to tumor aminoacid metabolism.<p>We found a positive correlation between maximum CBV values and maximum MET uptake values in gliomas.<p>A limitation to these preliminary studies was lack of direct correlation between MRbased measurements and histologic and metabolic data. Indeed, glial tumors are known for their remarkable tissue heterogeneity across different grades, within the same grade, and even within a single given tumor. Therefore we used image coregistration and stereotactic biopsies to further assess the relationship between MRbased imaging data and both metabolic and histologic analysis.<p>The second part of our studies was based on measurements at the exact same localization on both MR and PET images where biopsy specimens were performed. We found a local relationship between CBV and MET uptake values. Both measurements correlated with mitotic activity and endothelial proliferation; two features of tumor aggressiveness.<p>In order to quantify tumor cellularity and tumor angiogenesis, we respectively measured cell density and vessel density using immunohistochemical markers to identify vessels. We found a regional relationship between CBV and cell density, as well as vessel density in gliomas whereas no correlation was found regionally between ADC and cell density.<p>We concluded that CBV measurements may be used locally as indices of angiogenesis and cellularity in gliomas; whereas local ADC measurements are more variable and may not be used as a marker of cellularity in heterogeneous tumors such as gliomas.<br>Doctorat en Sciences médicales<br>info:eu-repo/semantics/nonPublished

Introduction. Neurofibromatosis type I, the most common form of Neurofibromatosis, presents with a wide phenotypic variability, both inter- and intra-familiar; a comparative analysis between type/site of mutation and phenotype cannot identify a correlation between genotype and clinical features in affected patients. Pilocytic astrocytomas (PAs) are the tumors that occur most often in NF1 patients; they affect children and young adults and occur preferentially along the optic pathway. These tumors are defined as Grade I gliomas (low-grade) and their growth is slow, but a small number of these tumors continue to grow, behave in an aggressive fashion and cause loss of vision or hypothalamic dysfunction. It is not known which changes determine whether an NF1-associated pilocytic astrocytoma will remain stable or exhibit a clinical progression: genetic alterations associated with NF1-associated optic glioma pathogenesis have not been well characterized. Aim of the study. The aim of the study was to analyze a group of NF1 patients affected with optic glioma and a group of patients affected with optic glioma in which the diagnosis of NF1 was excluded, in order to evaluate the presence of specific mutations in the NF1 gene and in the tumor-suppressor gene CDKN2A; to analyze polymorphisms in the tumor-suppressor genes CDKN2A and TP53, that are reported in the literature to be associated with risk and tumor progression, in the study patients and in a healthy control group; to evaluate results with statistical methods; to establish if the glioma formation is associated with some specific mutations or polymorphisms in the studied genes. Materials and methods. A mutation/SNP screening in NF1, CDKN2A and TP53 genes was performed in 25 NF1 patients affected with optic glioma diagnosis and 21 non-NF1 patients affected with optic glioma. Blood samples were obtained after informed consent from all of the patients and a control group of 50 unrelated adult healthy individuals from the Padova Clinical Genetic Service database. Mutation analysis was done by DHPLC after amplification by PCR of all the exons of NF1 and CDKN2A genes, in all of the patients, and direct sequencing of the samples showing some alteration at the DHPLC analysis. The SNPs analysis was done in CDKN2A and TP53 genes in all the patients and in the control group by RFLP and HRMA, respectively, after amplification by PCR of the target sequences. Results. Mutation scanning in the NF1 gene by DHPLC analysis has identified mutations in 60% of the NF1 patients with optic glioma diagnosis. A preferential type of nucleotidic alteration was not observed but the majority (67%) of them causes the formation of a truncated protein. No mutations were found in the non-NF1 patients with optic glioma diagnosis. These results confirm the lack of a genotype–phenotype (optic glioma) correlation in patients affected with NF1. Mutation scanning in the CDKN2A gene by DHPLC analysis has identified two nucleotidic alterations in three NF1 patients: the G442A polymorphism in exon 2, known in the literature, and a new substitution in 3’ UTR of the gene (C520G) that could represent a new polymorphism. SNPs analysis (C500G and C540T in 3’ UTR of CDKN2A gene and IVS 6+62 G/A and 12256 G/C, in the promoter region of the TP53 gene) was done by two different techniques: RFLP and HRMA, respectively. We have compared the two methods and define that HRMA is a better instrument for SNPs analysis because of its sensitivity and specificity and because it is a cost-effective and simple post-PCR technique. A statistical analysis of allelic and genotypic frequences of the CDKN2A and TP53 genes polymorphisms in patients and control groups was done using the ?2 test. The results seem to suggest a non-association between the presence of these SNPs and an increased risk of optic glioma growth in the studied population (P>0,05). However the number of individuals analized is not sufficient for exhaustive conclusions. Future studies should be done with more polymorphisms and a larger patients group.

Les gliomes sont des tumeurs du système nerveux central. Leur plus haut degré de malignité est le glioblastome (GBM), le plus fréquent des cancers du cerveau. Les patients atteints de GBM sont d’abord opérés (si possible) puis traités par la radiothérapie avec témozolomide concomitant et adjuvant. Ce traitement n’est cependant pas curatif, en partie en raison d’une radiorésistance primaire élevée des cellules de GBM. La voie de signalisation JAK/STAT3 (Janus Kinase/Signal Transducer and Activator of Transcription 3) semble contribuer à la gravité des GBM. STAT3 est une protéine intracellulaire de transduction du signal. Elle est activée par phosphorylation de ses résidus tyrosine 705 (pSTAT3-Y705) et sérine 727 (pSTAT3-S727). L’activation de la tyrosine 705 se produit en aval du signal induit par la liaison de la cytokine interleukine 6 (IL-6) à son complexe récepteur transmembranaire gp130-IL-6Rα. Les mécanismes d’activation de la sérine 727 sont moins bien caractérisés. Le rôle de l’activation de STAT3 dans la radiorésistance des GBM a été ici étudié. Une évaluation du niveau basal de pSTAT3-Y705, pSTAT3-S727 et de la radiorésistance intrinsèque a été faite sur un panel de 15 lignées de GBM humain. L’activation de STAT3 dans les lignées cellulaires de gliomes a été évaluée par western blot et la radiorésistance par la survie cellulaire à l’irradiation. En plus de la description de l’état basal d’activation de STAT3 dans les lignées cellulaires de gliomes, cette étude a mis en évidence pour la première fois, une corrélation de pSTAT3-S727 avec la radiorésistance intrinsèque des GBM. Une stratégie de blocage pharmacologique de STAT3 nous a permis d’identifier le Gö6976 comme inhibant la phosphorylation Y705 de STAT3 dans les cellules de GBM. Celui-ci s’est avéré inhiber aussi la phosphorylation S727 mais seulement dans les lignées de GBM pSTAT3-Y705 négatives. Le traitement par le Gö6976 ralentit la croissance des cellules de GBM, indépendamment du statut d’activation de STAT3. De façon intéressante, le Gö6976 a montré un pouvoir radiosensibilisant très significatif sur les lignées pSTAT3-Y705 négatives, ce qui est concordant avec la baisse du niveau de pSTAT3-S727. La pertinence de ces résultats est confortée par un marquage immunohistochimique sur des échantillons cliniques de GBM, montrant la présence à des degrés variables de pSTAT3-S727 dans toutes les cellules cancéreuses de tous les patients. En parallèle, une étude in vitro des fonctions de pSTAT3-S727 utilisant des dominants positif et négatif est en cours. En somme, nous avons démontré que pSTAT3-S727 participe à la radiorésistance intrinsèque et que pSTAT3-Y705 est un marqueur prédictif négatif de la réponse des cellules de GBM au Gö6976 à la fois comme inhibiteur de pSTAT3-S727 et radiosensibilisant. L’ensemble de nos résultats conforte l’intérêt d’une inhibition spécifique de pSTAT3-S727 pour radiosensibiliser les GBM et ainsi améliorer le traitement des patients<br>Gliomas are tumors of the central nervous system. The highest degree in glioma malignancy is Glioblastoma (GBM) that is the most frequent of the brain cancers. GBM patients are treated by surgery at first (if it is possible), followed by radiotherapy and concomitant and adjuvant temozolomide. However, this treatment is not curative in part because GBM cells display an outstanding primary radioresistance. The JAK/STAT3 (Janus Kinase/Signal Transducer and Activator of Transcription 3) signaling pathway seems to be involved in the GBM aggressiveness. STAT3 is an intracellular signal transducer protein. It is activated by phosphorylation on its tyrosine 705 (pSTAT3-Y705) and serine 727 (pSTAT3-S727) residues. The tyrosine 705 activation is produced downstream the signal induced by the binding of interleukine-6 (IL-6) cytokine to its gp130-IL-6Rα transmembrane receptor complex. The mechanisms of the serine 727 phosphorylation are less characterized. The role of STAT3 activation in the radioresistance of GBM was studied here. Basal levels of pSTAT3-Y705, pSTAT3-S727 and intrinsic radioresistance were evaluateded in a panel of 15 GBM cel lines. Activation of STAT3 in the glioma cell lines was assessed by western blotting and radioresistance through cell surviving fraction to irradiation. In addition to the description of the basal activation of STAT3 in the glioma cell lines, this study evidenced, for the first time, a correlation between pSTAT3-S727 and GBM intrinsic radioresistance. Using a pharmacological inhibition strategy, we identified Gö6976 as a chemical blocking Y705 phosphorylation of STAT3 in GBM cells. Gö6976 also inhibited pSTAT3-S727 but only in the pSTAT3-Y705-negative cell lines. Treating GBM cell with Gö6976 slowed their growth regardless of STAT3 activation status. Interestingly, Gö6976 showed a highly significant radiosensitizing effect on pSTAT3-Y705-negative cell lines that was consistent with the down-modulation of pSTAT3-S727. The relevance of these results is strengthened by immunohistochemical assay performed of GBM clinical samples that showed a variable level of pSTAT3-S727 positive staining in all tumor cells of all the patients. Furthermore, we are currently running on an in vitro study of the pSTAT3-S727 biological function by the mean of STAT3 dominant positive and dominant negative proteins. In summary, we showed that pSTAT3-S727 is involved in the intrinsic radioresistance and that pSTAT3-Y705 is a negative predicting marker of GBM cell response to Gö6976 as both a pSTAT3-S727 inhibitor and a radiosensitizer. Altogether, our results strengthen the clinical relevance of a specific inhibition of pSTAT3-S727 to radiosensitize GBM and then improve the patient treatment

Les gliomes de haut-grades sont des les tumeurs primitives les plus fréquentes du système nerveux central. Le traitement de cette pathologie associe chirurgie, radiothérapie et chimiothérapie. Les principales faiblesses de ces traitements sont le caractère infiltrant de la tumeur au sein d’un parenchyme hautement fonctionnel, l’existence de la barrière hémato-encéphalique limitant le passage trans-vasculaire de la chimiothérapie et la radiorésistance naturelle des cellules gliomateuses.Parmi les stratégies proposées pour outre-passer cette barrière hémato-encéphalique, une injection directe au sein du parenchyme a été évoquée. Afin d’optimiser cette délivrance le concept d’infusion convective a été développé, il s’agit d’une injection intra-parenchymateuse à un débit lent et contrôlé.Le bevacizumab est un anticorps dirigé contre le VEGF-A, un des principaux facteurs angiogéniques. Le but de ce traitement est de lutter contre l’ angiogénèse et de freiner la croissance tumorale.Dans un premier temps, la pharmacocinétique d’une injection intracérébrale de bevacizumab a été étudiée en comparaison avec une administration systémique plus classique. Les résultats permettent de mettre en évidence une concentration locale équivalente avec des concentrations systémiques beaucoup plus faibles avec une injection intra-tumorale. Un point important de cette étude est que la concentration dans l’hémisphère controlatéral à l’injection est aussi importante que lors d’une injection systémique.Puis l’efficacité d’une injection intratumorale de bevacizumab a été comparée à un traitement systémique sur un modèle de gliome chez la souris. L’efficacité du traitement est claire sur la survie de l’animal avec un avantage pour une injection intratumorale par rapport à une injection systémique. D’un point de vue microscopique cet avantage de survie peut être corrélé à une angiogénèse et une prolifération tumorale moins importante an cas d’injection directe au sein de la tumeur.Contrairement aux études pré-cliniques chez les rongeurs, les principaux essais cliniques n’ont pas permis de mettre en évidence un avantage d’une injection intra-tumorale directe. Principalement du à une mauvaise délivrance liée à des fuites et des reflux. Une des limites du modèle petit animal est l’absence de sillon cortical, vecteur de fuite. Le développement d’un modèle de gliome anatomiquement pertinent permettrait de simuler au mieux ces fuites et simultanément la mise au point de technologies de délivrance implantable à l’échelle humaine. Nous avons donc développé le premier modèle de gliome chez le porc. L’immunotolérance a été induite par un traitement par ciclosporine, des cellules de gliomes humains U87 et G6 ont été implantés, permettant se développer des tumeurs.Afin de dépister une mauvaise délivrance et anticiper les fuites ou les reflux, nous avons étudié les profils de pression le long de la ligne d’injection corrélés à l’existence de fuites ou de reflux. Nous avons pu identifier un profil pressionnel typique d’une injection de qualité. Les injections ne répondant pas à ces critères ont systématiquement conduits à des fuites ou reflux.L’étape suivante a été l’injection au sein d’une tumeur chez des porcs grâce à un système innovant implanté. Cette injection a été possible sans complication infectieuse avec une bonne tolérance locale et neurologique.La dernière étape de ce travail est l’étude anatomique de la diffusion d’un colorant injecté par une technique d’infusion convective. Cette étude s’intéressait notamment à la diffusion depuis la corona-radiata vers les différentes voies de substance blanche. La diffusion est anisotrope le long des fibres de substance blanche cependant la diffusion suit des voies différentes en fonction de la position du cathéter par rapport à elle. L’injection semble ouvrir des voies d’impédances rhéologiques faibles préférentielles nécessitant une adaptation anatomique aux voies qui seront la cible du traitement<br>High grade gliomas are the most frequent primitive central nervous system tumor. The standard treatment is an association of surgery, radiotherapy and chemotherapy. The mains issues with these treatments are the infiltrative properties of the tumour in a highly functional parenchyma, the blood-brain barrier limiting the transvascular transport of chemotherapy and the inherent radioresistance of glioma cells.Upon different strategy to overpass the blood-brain barrier, a direct injection in the brain was advocated. In order to maximize this delivery, the concept of convection enhanced delivery was developed; it consists in a direct injection in the parenchyma with a low flow-rate.Bevacizumab is an anti-VEGF A antibody, VEGF is one of the most important angiogenic factors. The goal of this treatment is to inhibit the angiogenesis and slow down the tumor growth.We propose to study the use of this antibody in a direct intra-cerebral infusion.First, we focalize on the pharmacokinetic properties of an intratumoral injection by convection –enhanced delivery compared to a systemic administration. This shows an equivalent intratumoral concentration with systemic concentrations significantly lower with the intra-tumoral injection. An important result is the similar concentration in the controlateral hemisphere with the two routes of infusion. Convection-enhanced delivery is suitable to carry far from the infusion site high molecular weight proteins. An intra-tumoral bevacizumab may theoretically provide similar efficiency with less systemic side-effect.Then, the efficiency of an intra-tumoral infusion of bevacizumab is compared to a systemic injection on a mouse glioma model. In terms of survival the intra-tumoral treatment is significantly more efficient with an important decrease of angiogenesis and tumoral proliferation.If convection-enhanced delivery rodent study were promising, clinical trials failed to show any efficiency of intra-tumoral injection mainly due to inadequate delivery secondary to backflows and leakages. One of the limits of the rodent model is the absence of cortical sulci, main leakage provider. The development of a model anatomically relevant could simulate real conditions of injection and develop implantable device of injection in realistic conditions. We have developed the first induced model of glioma in a large animal. We choose the pig for the similarity of its brain anatomy and its size. The animals have been treated with ciclosporin to induce an immunosuppression, human glioma cells have been implanted, leading to the development of brain tumor.We have studied the pressure on the infusion line and correlate it to backflow and leakage. We have identified a pattern of pressure for successful infusion. Different pressure pattern have systematically led to backflow or leakage. These pressures criteria could permit to us an early detection of inadequate infusion to replace the catheter and avoid the failure of precedent clinical trials.Next step have been the intra-tumoral injection via an implanted device on pig glioma model. No infectious complication has been related with a good local and neurologic tolerance. The injections have led to a relevant diffusion through the tumor with a rapid flow to the periphery due to the interstitial pressure gradient between the tumor and the periphery.Last step of this work have been the anatomical study of a dye distribution by convection-enhanced delivery in a human encephalon. Indeed if pig brain is similar to human brain, human white matter structure is unique. This work is focalized on the diffusion from the corona-radiata to the main white matter tracts. The distribution is anisotropic following white matter but the diffusion is different depending on the position of the catheter. The infusion seems to open low rheological impedance paths the position of the catheter have to be adapted to the white matter tract to target

Les gliomes diffus de bas grades sont des tumeurs qui affectent des régions fonctionnelles du cerveau chez des jeunes patients. Malgré leur faible taux de prolifération ces tumeurs peuvent dégénérer en des tumeurs plus agressives après leurs exérèses. Le gène IDH1 est très fréquemment muté dans les DLGG. Cette mutation confère à l’enzyme isocitratedeshydrogénase (IDH1) la propriété de produire du 2-OH-glutarate (2-HG) au lieu de l’α-cétoglutarate (α-KG). L’oncométabolite 2HG rentre alors en compétition avec l’α-KG pour les enzymes de déméthylation conduisant à une hyperméthylation de l’ADN et de l’histone H3 concourant à un blocage de la différenciation cellulaire. Mon projet de thèse consiste à la caractérisation des cellules tumorales et la compréhension des voies de signalisation impliquées dans la progression tumorale ainsi que l’identité du microenvironnement tumoral. Les récepteurs tyrosine kinase, PDGFRα et EGFR, sont abondamment exprimés par les cellules tumorales mais ne sont pas activés. En revanche, une forte phosphorylation de la protéine Erk p42/44 a été détectée dans les tumeurs. Cette phosphorylation a une double origine : les cellules tumorales et leur environnement. L’utilisation d’une série de marqueurs m’a permis de mieux définir l’état de différenciation des cellules tumorales et de mettre en évidence une préférence pour l’expression de Sox8 dans les oligodendrogliomes tandis que Sox9 est prédominant dans les astrocytomes. Dans une seconde partie, j’ai mis au point des méthodes pour la culture des gliomes diffus de bas grade et isolé cinq lignées de gliomes portant la mutation récurente IDH1 R132H. Récemment, la société Agios a identifié des inhibiteurs très spécifiques (notamment l’AGI-5198) de l’enzyme mutée IDH1 qui, utilisés dans un modèle de gliome murin, provoquent une déméthylation des histones H3K9me3 associées à une augmentation de l’expression de gènes de différenciation ainsi qu’à une réduction de la masse tumorale. A contrario, j’ai montré que l’AGI-5198 augmente la croissance cellulaire sur les lignée de patients, modifie la migration cellulaire ainsi que différentes voies de signalisation.Ces travaux apportent un nouvel éclairage sur le phénotype des cellules tumorales, leur diversité et les mécanismes moléculaires régissant leur prolifération<br>Low grade gliomas are low proliferating tumors affecting functional regions of young patients. In most cases, they tend to transform into a more malignant state following surgery. These tumors carry a key mutation in isocitrate dehydrogenase (70-80% of DLGG). Gliomas with IDH1 mutation have improved prognosis compared togliomaswith wild type IDH1. IDH1 protein acquires the ability to convert α-Ketoglutarate (α-KG) to 2-OH-glutarate (2-HG). The new onco-metabolite can interfere with the normal function of α-KG, leading to a general hypermethylation of the genome, thus inducing a blockage of the cellular differentiation. Very good reviews on the molecular mechanisms underlying high grade glioma invasion already exist but little is known about the cellular and molecular mechanisms in diffuse low grade gliomas. To that end, I characterized the profile of IDH1 mutated cells in the different types of DLGG. I have demonstrated that the tyrosine kinase, PDGFRα and EGFR receptors are abundantly expressed by tumor cells eventhough they are not activated. In contrast, a strong phosphorylation of Erk p42 / 44 proteins was detected in these tumors. This phosphorylation has a dual origin: tumor cells and their environment. The use of a series of markers allowed me to better define the state of differentiation of cancerous cells and to demonstrate a preferential expression of Sox8 in oligodendrogliomas while Sox9 is predominant in astrocytomas. In a second time, I have developed a method for the culture of low-grade diffuse gliomas and isolated five cell lines carrying the recurrent mutation IDH1 R132H. Recently Agios has identified very specific inhibitors (particularly AGI-5198) of the mutated IDH1 enzyme which, used in a murine glioma model, contributed to the demethylation of H3K9me3 histones with an increased expression of differentiation related genes as well as a reduction of the tumor mass. On the contrary, I have shown that AGI-5198 increases cell growth of patient cell lines, modifies the cellular migration and various signaling pathways.These studies shed new light on the phenotype of tumor cells, their diversity and The molecular mechanisms governing their proliferation

Le glioblastome qui correspond au stade de malignité le plus élevé des gliomes est associé à un très mauvais pronostic car il envahit le parenchyme cérébral de manière diffuse, ce qui rend son exérèse complète généralement impossible, et il résiste aux traitements conventionnels en raison de sa résistance intrinsèque aux stimuli pro-apoptotiques. Le cancer de l’œsophage est également un cancer très agressif car invasif et résistant également aux stimuli pro-apoptotiques. <p>Les chémokines sont des cytokines chémotactiques responsables de la migration des leucocytes et exprimées en réponse à des cytokines inflammatoires, à des facteurs de croissance et à des stimuli pathogènes. De nombreux cancers possèdent un réseau complexe de ces chémokines. Les chémokines de type CXCL et plus particulièrement CXCL8 et CXCL12 sont impliquées dans la biologie des gliomes et du cancer de l’oesophage. Au cours de mon travail de thèse de doctorat, nous avons étudié l’expression des 15 chémokines CXCL et des 9 récepteurs aux chémokines CXCL dans divers modèles de gliomes et de cancers de l’œsophage. Cette étude menée par RT-PCR nous a permis de mettre en évidence la présence d’un patron d’expression complexe de ces chémokines CXCL dans les divers modèles analysés. Nous avons observé une expression plus importante des chémokines CXCL pro-angiogéniques par rapport aux chémokines anti-angiogéniques dans ces deux types de cancers. Nous avons également pu mettre en évidence une implication potentielle des chémokines CXCL2, CXCL3 et CXCL8 dans l’acquisition de la résistance au traitement par témozolomide des gliomes d’origine astrogliale. <p>Les glioblastomes et les cancers de l’œsophage étant deux types de cancers résistants aux stimuli pro-apoptotiques, et le témozolomide étant la seule molécule dotée de bénéfices thérapeutiques réels dans le cas du glioblastome, nous avons également testé le témozolomide dans nos modèles de cancer de l’œsophage in vitro et in vivo. Nous avons pu ainsi montrer un bénéfice thérapeutique réel apporté par cette molécule in vivo sur des animaux immunodéficients greffés avec des cellules humaines de carcinome épidermoïde de l’œsophage. Ce bénéfice thérapeutique peut être expliqué en partie par différents mécanismes d’action tels que l’induction de processus soutenus d’autophagie suivis par de l’apoptose mais également par des effets anti-angiogéniques. Enfin, nous avons pu montrer que la diminution d’expression même transitoire de la chémokine CXCL2 dans nos modèles in vitro de glioblastome et de carcinome épidermoïde de l’œsophage entraîne une diminution de la croissance de ces populations cellulaires cancéreuses, suggérant un rôle important de cette chémokine dans la biologie de ces deux types de cancers. Enfin, nous avons démontré un effet anti-angiogénique in vivo pour le témozolomide dans un modèle de xénogreffes de cancers oesophagiens humains chez la souris immunodéficiente.<p><p>En conclusion, l’ensemble de nos résultats suggèrent que le témozolomide, bien qu’il devienne bientôt un générique sous sa forme d’administration i.v. (la forme orale étant déjà générique), pourrait représenter une molécule d’intérêt pour combattre le cancer de l’œsophage, comme on le sait déjà depuis 2005 en ce qui concerne les glioblastomes. Nos résultats montrent ensuite l’importance du patron d’expression des chémokines CXCL dans la biologie des cellules gliales tumorales et des cellules cancéreuses de l’œsophage. Enfin, nos résultats montrent que le témozolomide détruit en partie ce réseau de chémokines CXCL au sein de ces deux types de cancers.<p><br>Doctorat en Sciences biomédicales et pharmaceutiques<br>info:eu-repo/semantics/nonPublished

Une radiothérapie efficace nécessite un dépôt de dose localisé à la tumeur, et donc un contraste entre le tissu tumoral et les tissus sains environnants. Une irradiation de basse énergie monochromatique au synchrotron d'une tumeur chargée en éléments lourds permet de maximiser l'interaction photoélectrique dans la tumeur et d'épargner les tissus sains, car les photoélectrons et les électrons Auger produits ont un TEL très élevé et déposent leur énergie autour des éléments lourds, augmentant fortement le dépôt de dose. Ils peuvent induire des dommages à l'ADN (cassures double brin) fortement létaux. Un autre phénomène permet également de promouvoir l'émission d'électrons Auger et d'augmenter ainsi la dose, l'effet Mössbauer. Cette interaction résonante et sans recul spécifique à certains isotopes dont le 57Fe présente une section efficace 450 fois plus importante que celle de l'effet photoélectrique. Ce travail de doctorat a évalué l'utilisation in vitro de nanoparticules de magnétite combinées à ces deux effets physiques. Les nanoparticules présentent une internalisation et une distribution dans les cellules F98 qui sont très propices à la radiosensibilisation : de grandes concentrations proches du noyau des cellules, et peu de toxicité ont été obtenues. Ceci a permis d'obtenir par photoactivation des NPFe un facteur d'augmentation de 3 ce qui est considérable. Ce travail multidisciplinaire rassemble des expériences de physique, de biologie et de chimie, pour évaluer les applications de nanoparticules de fer à la radiothérapie<br>An efficient radiotherapy needs a localized dose to the tumour, which means a high contrast between tumorous and healthy tissues. A synchrotron low energy monochromatic irradiation of a tumour charged in high-Z elements allows maximizing photoelectric interactions in the tumour and spare the healthy tissues. Photoelectrons and high LET Auger electrons thus produced deposit their energy locally, enhancing radiation dose to tumor cells. Another interaction allows to enhance the dose by Auger electrons: the Mössbauer effect. This resonant and recoilless interaction specific to some isotopes like 57Fe has a cross section 450 times bigger than photoelectric effect. This thesis evaluates the in vitro use of magnetite nanoparticles combined with those 2 types of interactions. The nanoparticles evaluated present a high internalisation and a perinuclear distribution inside F98 cells. A dose-enhancement factor of 3 was obtained by photo activation of the iron Nps, this represents a huge increase. This multidisciplinary work encompasses experiments in chemistry, physics and biology in order to evaluate the applications of magnetite nanoparticles to radiotherapy

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<br>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

Hedgehog-signalling in malignant gliomas The Hedgehog signalling pathway is important for the development of the central nervous system. On the other hand, aberrant induction is observed in different tumors. Immunofluorescence and real-time qRT-PCR confirmed that in some gliomas, specifically in Glioblastoma multiforme (GBM), Gli1, a transcription factor activated by signalling, is present. In general, the hedgehog pathway is initiated by binding of extracellular ligands to the transmembrane receptor Patched and leads finally to the activation of the transcription factors Gli1, Gli2, Gli3 and Gli4. Whereas Gli1 acts as an activator, Gli2 appears to be an activator but retains some repressor activities and Gli3 and Gli4 are believed to act only as inhibitors. Therefore, the determination of hedgehog activity at the level of transcription requires additional experiments measuring gene activation. For that reason, cells isolated from 13 tumors of patients with glioblastoma (WHO Grade IV) and cells from two different glioma cell lines were transfected with reporter genes. These reporter genes carried the luciferase gene from Gaussia princeps under the control of two promoters (pT109 and pT81) conjugated to Gli binding sites. The activity of the reporter genes was compared to a control plasmid with mutant Gli-binding sites. In addition reporter gene activity was analysed in the absence and presence of the hedgehog signalling inhibitor cyclopamine and the effect of cyclopamine on cellular metabolism was studied. The analysis revealed that the two cell lines and cells from 6 glioblastomas exhibited enhanced reporter gene activity compared to the activity mutant control. This points towards an enhanced expression of Gli1. In three cultures a repression was detected suggesting that Gli3 may be active in these cells. Four cultures did neither show activation nor repression. This could provide evidence that Gli1 and Gli3 effects cancel each other out or that there is no effect at all. Enhanced luciferase activity in cells from the line T98G and in cells from four primary cultures was not influenced by the hedgehog inhibitor cyclopamine, whereas one cell line significantly responded to its presence with a decreased activity. Interestingly, ATP level was suppressed by cyclopamine in cells from the line T98G and also in cells from one primary culture that responded to the inhibitor. This may point towards an effect of cyclopamine independent of smo. Since cyclopamine is a potential new substance for the treatment of tumors, the observed effect of this inhibitor even in cells without an indication of hedgehog signalling activity should be investigated in further experiments in more detail<br>Der Hedgehog (Hh) -Signalweg spielt während der Embryonalentwicklung eine wichtige Rolle, so auch bei der Entstehung des zentralen Nervensystems (Varjosalo &amp; Taipale 2008). Andererseits führt seine unregulierte Aktivität zur Ausbildung verschiedenster Tumore (Bailey et al. 2009; Fiaschi et al. 2009; Shaw et al. 2009; Velcheti &amp; Govindan 2007). Vorausgegangene Studien wiesen durch Immunfluoreszenz und real-time qRT-PCR nach, dass auch in Gliomen, speziell in Glioblastoma multiforme, dem agressivsten Hirntumor des Menschen, Effektoren des Signalweges (Gli1) überexprimiert werden (Wang et al. 2010). Die Aktivierung des Signalweges geschieht über Bindung des Hh-Liganden an den Rezeptor Ptch und endet mit der Aktiverung der Transkriptionsfaktoren der Gli Familie (Kinzler &amp; Vogelstein 1990; Stone et al. 1996). Die aktuell bekannten Vertreter dieser Familie sind der Aktivator der Transkription Gli1, Gli2, der als Aktivator und Repressor agieren kann sowie Gli3 und Gli4, die die Transkription inhibieren (Marine et al. 1997; Ruppert et al. 1988). Ziel dieser Arbeit war es, herauszufinden, inwieweit die Transkriptionsfaktoren der Gli-Familie in Zellen von Glioblastoma multiforme aktiv sind. Dafür wurden Zellen aus Tumormaterial isoliert und daraus Primärkulturen hergestellt. In diese 13 Primärkulturen, wie auch in zwei Gliom-Zelllinien, wurden mittels transienter Transfektion Reporterplasmide eingebracht. Diese enthielten ein Gen der Gaussia-Luciferase, das unter der Kontrolle zweier verschiedener Promotoren (pT109 und pT81) mit Bindungsmotiven für die Transkriptionsfaktoren der Gli-Familie stand. Weiterhin wurde der Einfluss des Inhibitors des Hh-Signalweges Cyclopamin auf die Gli-Aktivität und die Metabolische Aktivität der Zellen untersucht. Die Beobachtungen ergaben, dass die zwei Zelllinien und sechs der primären Kulturen eine erhöhte Luciferaseaktivität und damit gesteigerte Aktivität von Gli1 zeigten. Weiterhin wiesen vier Kulturen eine verminderte Luciferaseaktivität auf. Dies ließ darauf schließen, dass in diesen Zellen Gli3 aktiv war. In den restlichen vier Kulturen zeigte sich keine Veränderung der Luciferaseaktiviät, was für einen Aufhebungseffekt von Gli1 und Gli3 oder gar keinen Effekt spricht. Weiterhin konnte gezeigt werden, dass die Luciferaseaktivität und damit die Aktivität von Gli1 in Zellen der Zelllinie T98G und von vier Primärkulturen nicht durch Cyclopamin beeinflusst wird. Lediglich eine Probe der Primärkulturen reagierte mit einer Abnahme der Luciferaseaktivität. Außerdem konnte Cyclopamin die ATP-Produktion sowohl in Zellen von T98G als auch in Zellen der Zelllinie, deren Gli-Aktivität durch Cyclopamin vermindert wurde, senken. Dies sprach für eine Smo unabhängige Wirkung des Cyclopamins. Da Cyclopamin ein potenzielles Pharmakon für die Antitumortherapie ist, bedarf dieser Umstand näherer Untersuchungen

Les gliomes ont des conséquences dévastatrices. La morbidité et la mortalité sont élevées. Les gliomes représentent un groupe hétérogène complexe d'entités pathologiques et aucune cause n'a été identifiée pour la majorité des gliomes. Les données épidémiologiques varient d'une étude à l'autre. Le nombre de chaque sous-type histologique est trop petit, même pour un grand centre de neurochirurgie, pour permettre une bonne recherche sur chaque sous-type de gliome. Les spécificités oncologiques et cliniques (épilepsie, troubles cognitifs, troubles moteurs, etc.) nécessitent une prise en charge et une analyse spécifique. De plus, il est important de recueillir et d'enregistrer tous les nouveaux cas et le suivi sur une grande région ou un pays entier pour permettre des études fondamentales et cliniques de qualité. En effet, les études en population sont la seule façon de connaitre l'impact en pratique des différentes thérapeutiques effectuées. Les sociétés françaises impliquées en neuro-oncologie (Société Française de Neurochirurgie, Société Française de Neuropathologie, Association des Neuro-Oncologues d'Expression Française) ont récemment créé le Recensement national histologique des tumeurs primitives du système nerveux central (RnhTPSNC) ou French Brain Tumor DataBase (FBTDB) en anglais. L'objectif principal du RnhTPSNC est d'enregistrer de manière prospective tous les cas incidents de tumeur primitive du système nerveux central (TPSNC), en France, pour lesquels le diagnostic histologique est confirmé (1-3). Les objectifs à long terme du RnhTPSNC sont de créer un registre histologique et un réseau national pour : (1) réaliser des études épidémiologiques, (2) mettre en place une base de données pour favoriser toute étude clinique ou fondamentale à grande échelle, (3) permettre l'évaluation des pratiques médicales d'une région ou du pays tout entier, (4) harmoniser et optimiser la prise en charge médicale des patients atteints de TPSNC. La présente étudiante en thèse, Sonia Zouaoui, concentrera son travail sur les gliomes. D'abord, elle devra recueillir les données des patients, puis analyser les facteurs pronostiques, la survie et les prises en charges oncologiques. Deuxièmement, elle participera à l'étude de la répartition géographique des principaux types de gliomes et à la recherche de facteurs de causaux. Troisièmement, elle procédera à un inventaire du matériel cryopréservé disponible pour la réalisation d'études translationnelles<br>Gliomas have devastating consequences. Morbidity and mortality are high. Gliomas represent a complex heterogeneous group of pathologic entities and no underlying cause has been identified for the majority of them. Epidemiologic data vary from study to study. The number of each histological subtype is too small, even for a big neurosurgical center, to allow a good research on each subtype of glioma. Oncological and clinical specificities (epilepsy, cognitive disorders, motor impairments, etc) require a specific care and analysis. Indeed, we need to collect and record all new cases and follow up in large area, to allow good basic and clinical studies. Furthermore, population study is the only way to know what clinicians do to the patients, and make possible evaluating the medical care. The French societies involved in Neuro-Oncology (Société Française de Neurochirurgie, Société Française de Neuropathologie, Association des Neuro-Oncologues d'Expression Française) have recently created the French Brain Tumor DataBase (FBTDB). The main objective of the FBTDB is to prospectively record all primary central nervous system tumors (PCNST), in France, for which histological diagnosis is available (1-3). The long-term goals of the FBTDB are to create a histological national registry and a national network to (1) perform epidemiological studies, (2) implement a new database and use it for setting up both clinical and basic research protocols, (3) allow the evaluation of the medical practices of an area or of the entire country, and (4) harmonize the healthcare of patients affected by PCNST at the higher level. The present PhD student, Sonia Zouaoui, will focus her work on gliomas. First, she will collect data, and will analyze prognostic factors, survival and oncological patterns of care for patients with newly diagnosed glioma in France. Secondly, she will participate in the study of geographical distribution of the main types of glioma and in search of causal factors. Thirdly, she will conduct an inventory of cryopreserved material available for translational research

Les gliomes, et particulièrement les glioblastomes, sont, de par leur pronostic défavorable, un véritable problème de santé publique. La difficulté à les classifier correctement et le manque d'efficacité des thérapies anti-cancéreuses reflètent la méconnaissance de ces tumeurs. Afin d'améliorer le diagnostic, et de mieux comprendre le processus conduisant à la formation des gliomes et donc de trouver de nouveaux traitements, nous avons choisi d'étudier l'immuno-réactivité des sérums de patients. Nous avons ainsi pu découvrir une centaine de protéines dont le statut immunitaire change entre la condition normale et celle pathologique. En identifiant et étudiant ces protéines, nous pouvons mettre en lumière certains mécanismes importants pour la cancérogenèse et définir des clibles clés pour la thérapie. Parmi cette centaine de protéines, nous avons étudié l'immuno-réactivité d'une dizaine qui nous ont permis de définir deux types d'antigènes : ceux dont le statut immunitaire change avec l'apparition de la tumeur et ceux dont le statut change selon la sensibilité de la tumeur vis-à-vis des traitements. Nous avons alors choisi 3 de ces protéines pour les analyser plus en détail : eef1a1, crhsp24, mark3. Ces trois natigènes se trouvent être surexprimés dans les gliomes. L'inhibition de leur expression par si RNA induit une baisse de la prolifération des cellules tumorales, indiquant qu'ils sont impliqués dans la régulation de ce mécanisme. Cette fonction essentielle fait de ces protéines de potentielles cibles thérapeutiques. Les premiers tests de vaccination contre mark3 montrent que l'étude de cet antigène est intéressante même si les résultats ne sont pas ceux espérés. Nous montrons ici que des auto-anticorps associés à la présence des tumeurs gliales peuvent être trouvés dans le sérum. Ces anticorps ont clairement un intérêt diagnostique mais peuvent aussi avoir un intérêt pronostique. Leur analyse biologique peut amener à les considérer comme des cibles thérapeutiques potentielles<br>Gliomas, particulary glioblastoma, are public health problem because of their dark pronostic. Difficulties to classify and lack of anti-tumor therapy efficiency are due to ignorance about these tumors. To improve diagnosis and also to understand the process leading to glioma formation and so to find new treatment, we choose to study patient sera immuno-reactivity. So we discovered about a hundred of proteins which immune status change between normal and pathological condition. Identifying and studying these proteins, we highlight important mechanisms for cancer and define key targets for therapy. Among these proteins, we studied immuno-reactivity to ten which leads us to define two types of antigens : those which immune status changes with tumor appearance and those who change depending on the sensibility to treatment of the tumor. We then choose three of these proteins for detail analysis : eef1a1, crhsp24, mark3. These three antigens are overexpressed in gliomas. Expression inhibition by siRNA induced a diminution of tumoral cell proliferation, indicating that they are implicated in this mechanism regulation. This essential function makes these potential therapeutic targets. First tests of vaccination toward mark3 show that studying this antigen is of particular interest despite results are not those wished. We show here that auto-antibody associated to glioma presence can be found in sera. These antibodies clearly have diagnosis interest but also can have prognostic interest. Their biological analysis could lead to consider them like potential therapeutic targets

Biomarkers for the classification, clinical management and prognosis of pediatric brain tumors (ependymoma and high grade glioma, (HGG)) are lacking. To address this, biomarkers were developed and explored in view of classification, prognostication, target identification and prediction of the efficacy of treatment for patients with such tumors.We show that overexpression of neuronal markers distinguishes supratentorial from infratentorial ependymoma, and among the former higher immunoexpression of neurofilament 70 (NEFL) is correlated with better progression free survival (PFS). Tenascin-C (TNC) is significantly overexpressed in infratentorial ependymoma. A multi-institutional European ependymoma collaboration group was established and analyses were performed in a pediatric cohort of 250 patients, where immunohistochemistry (IHC) for TNC showed to be a robust marker of poor overall survival (OS) and PFS, particularly among children under 3 years, this being further validated in an independent cohort. Techniques and scoring performed in different laboratories were highly reproducible. IHC for NEFL and TNC could be used for prognostication of pediatric ependymoma.The analysis of putative predictive markers for the response to targeted therapies in pediatric HGG in the setting of a clinical trial with the anti-EGFR agent erlotinib was performed by IHC and fluorescent in situ hybridization. The frequent loss of PTEN in diffuse intrinsic pontine glioma (DIPG) and the confirmation of the biological singularity of the certain subgroups (expressing EGFR, displaying oligodendroglial differentiation) which seem to be associated with better response to erlotinib have helped our group to establish the design of the next Phase III protocol for this disease at our institution. We report mutations in PI3KCA constituting the first identification of oncogene mutations in some DIPG, which further highlight their biological heterogeneity. Further studies are needed to define the interaction between PTEN loss, EGFR overexpression, oligodendroglial differentiation, PI3KCA mutations and other recent findings such as PDGFRA/MET gains/amplification and TP53 mutations in these heterogeneous lesions and their relationship to the outcome of patients under new targeted therapies for this largely fatal disease.This thesis has allowed us to explore the molecular pathology in the context of biology and clinical setting of pediatric brain tumors.

Il est largement documenté que les patrons épigénétiques sont altérés dans les cancers. Pour autant, l’étendue et la nature précise de ces altérations, tout comme leur impact sur l’expression des gènes, restent encore peu appréciés. Mon projet de thèse est bâti sur ce constat, et s’inscrit en particulier dans la recherche des causes et conséquences des altérations épigénétiques dans les gliomes. Ces tumeurs du système nerveux central représentent en effet un excellent modèle, car elles présentent des défauts de méthylation de l’ADN permettant de discriminer deux populations de tumeurs avec des caractéristiques cliniques différentes. Notre stratégie, basée sur des analyses moléculaires exhaustives, s’est appuyée sur une cohorte de 70 échantillons tumoraux, classés sur la base de leur statut IDH, et de six lignées de cellules souches de glioblastomes (CSG).Ces travaux ont tout d’abord permis de relativiser la contribution de la méthylation de l’ADN dans les dérégulations transcriptionnelles observées dans les gliomes. Il apparait en effet que ce sont plutôt les altérations au niveau de la chromatine bivalente, et plus spécifiquement de la marque H3K27me3, qui sont la cause principale de ces dérégulations transcriptionnelles. Spécifiquement, nos données supportent un modèle selon lequel l’altération dans le contrôle de la marque H3K27me3, et plus spécifiquement dans les interactions entre le complexe PRC2 et la machinerie de transcription spécifique au cerveau, est la cause principale des altérations transcriptionnelles dans les gliomes.Cette étude révèle également que les gènes à homéodomaine, et en particulier les gènes HOX, constituent une catégorie à part dans les gliomes les plus agressifs (IDHwt). Leur signature moléculaire, associant gain d’expression et gain de méthylation de l’ADN, est en effet atypique. Nos données révèlent que cette altération est généralisée aux quatre clusters HOX, et que la réactivation de ces gènes est liée à la perte drastique et spécifique de la marque H3K27me3 sur ces régions. Cette étude conduit également à proposer un modèle original selon lequel l’hypométhylation globale de l’ADN est un élément déclencheur de l’expression ectopique détectée au niveau de nombreux gènes, et dont l’altération des gènes HOX aurait, via un effet domino, un rôle central. L’observation de l’altération de la marque H3K27me3 dans les gliomes, et en particulier aux clusters HOX, nous a également amené à nous interroger sur le rôle des ARN non codants dans ces mécanismes. En ce sens, un transcrit non codant encore peu caractérisé, nommé HOXA-AS2 (situé en antisens au niveau du cluster HOXA), a été identifié. Ce transcrit est significativement et spécifiquement surexprimé dans les gliomes IDHwt. Des approches de sous-expression dans des lignées bien caractérisées de CSG suggèrent un rôle central de HOXA-AS2 dans la biologie de ces cellules. Il contribuerait ainsi au caractère pathologique des CSG en inhibant les voies de l’inflammation et en favorisant la capacité des cellules à proliférer.Dans son ensemble, ce travail revisite le lien entre altérations épigénétiques et défauts d’expression dans les cancers et met en évidence qu’une altération dans le contrôle de la marque H3K27me3 est la principale cause des défauts d’expression des gènes<br>Epigenetic alterations are a well-known signature of cancer cells. However, the causes of these defects, as well as their consequence on gene expression, remain elusive. My thesis project specifically lies in this thematic, and focuses on the causes and consequences of epigenetic alterations in gliomas. These brain tumors can be divided into two subsets, based on IDH mutation status, that are characterized by different methylation profiles. Interestingly, the mutation of IDH is also associated with a better prognosis. Our strategy, based on exhaustive molecular analyses, relies on the study of 70 glioma samples, classified according to their IDH status, and of six glioblastoma stem cell (GSC) lines.We found that most transcriptional alterations in tumor samples were DNA methylation-independent. Instead, altered histone H3 trimethylation at lysine 27 (H3K27me3) was the predominant molecular defect at deregulated genes. Our results also suggest that the presence of a bivalent chromatin signature at CpG island promoters in stem cells predisposes not only to hypermethylation, as widely documented, but more generally to all types of transcriptional alterations in transformed cells. In addition, the gene expression strength in healthy brain cells influences the choice between DNA methylation- and H3K27me3-associated silencing in glioma. Highly expressed genes were more likely to be repressed by H3K27me3 than by DNA methylation. Our findings support a model in which altered H3K27me3 dynamics, more specifically defects in the interplay between Polycomb protein complexes and the brain-specific transcriptional machinery, is the main cause of transcriptional alteration in glioma cells. Also, our study revealed that homeodomain genes, and in particular HOX genes, are characterized by an atypical defect in aggressive gliomas (IDHwt), associating a gain of expression with an aberrant gain of methylation. We determined that this alteration affect all the four HOX clusters, and that the reactivation of these genes is likely a consequence of the aberrant loss of H3K27me3 that specifically affect these clusters. This study allows to propose a model whereby global DNA hypomethylation triggers ectopic expression of numerous genes through a cascade of events, in which HOX gene alteration would have a central role.The observation that H3K27me3 is deregulated in gliomas, and particularly on HOX genes, also lead us to investigate for the role of non-coding RNA in these mechanisms. We have identified HOXA-AS2, a yet poorly characterized long non-coding RNA located at HOXA locus, that is specifically and significantly overexpressed in IDHwt gliomas. The inhibition of HOXA-AS2 in well-characterized CSG lines suggests that this transcript play a central role in the biology of these cells. Thus, it would contribute to the aggressiveness of CSG by inhibiting inflammatory pathways and promoting cell proliferation. Altogether, these works revisit the relationship between epigenetic alterations and aberrant transcription, and present the control of H3K27me3 as the main cause of transcriptionnel defects in cancer

Les gliomes diffus de bas grade (DLGG) sont des tumeurs gliales de grade II qui affectent principalement les jeunes adultes. Elles sont caractérisées par une croissance lente et une activité mitotique réduite. Cependant, ces tumeurs diffusent et envahissent le cerveau sain via les vaisseaux sanguins et les fibres nerveuses. Après plusieurs années de croissance lente, ces tumeurs peuvent évoluer vers des glioblastomes, des tumeurs cérébrales très agressives dont la survie médiane moyenne est alors de 12 à 15 mois après le diagnostic. La caractérisation cellulaire des DLGG est encore limitée ce qui nuit à la recherche d’un traitement à un stade précoce. Dans ma thèse de doctorat, je me suis focalisée sur la caractérisation de 'hétérogénéité cellulaire des DLGG mutés pour IDH1. En effectuant une analyse d'immunofluorescence sur des astrocytomes et oligodendrogliomes de grade II, j'ai identifié deux sous-populations cellulaires largement non chevauchantes et exprimant respectivement les facteurs de transcription SOX9 et OLIG1. Ces cellules s’apparentent à des cellules de type astrocytaire et oligodendrocytaire et expriment des marqueurs moléculaires distincts. Les cellules SOX9 expriment APOE, KCNN3, CRYAB et ID4, tandis que les cellules OLIG1 expriment préfentiellement PDGFRA, SOX8, MASH1 et SOX4. Par ailleurs, j’ai montré que les cellules SOX9 présentent une activation particulière des voies de signalisation, notamment Notch, BMP et leurs cibles en aval. Pour étudier le rôle de la voie de signalisation Notch dans la formation de ces 2 sous-populations tumorales, j'ai purifié par tri magnétique les cellules tumorales à partir d'échantillons de gliomes fraîchement réséqués et j'ai surexprimé le domaine intracellulaire Notch (NICD), une forme active de Notch. J’ai ainsi montré que cette activation augmentait l’expression des marqueurs cellulaires associés aux cellules SOX9+ et une baisse de ceux associés aux cellules OLIG1+. J'ai ensuite étendu ces analyses à une lignée cellulaire anaplasique dérivée d'un patient et mutée pour IDH1. Ces résultats indiquent un rôle clé de la signalisation Notch dans la régulation de la plasticité des cellules tumorales. Des expériences similaires pour étudier l'activation de la signalisation BMP (bone morphogenetic protein) n'ont pas montré d'effet notable sur la plasticité. Néanmoins, le traitement des cellules par des membres de la famille BMP a fortement augmenté l’expression de CRYAB, un marqueur associé à SOX9, et a diminué l’expression de OLIG1 et OLIG2. En conclusion, j'ai identifié deux sous-populations tumorales non chevauchantes dans des gliomes diffus de bas grade et j'ai démontré le rôle déterminant de la voie de signalisation Notch dans leur formation. Ces résultats permettront de mieux comprendre l'hétérogénéité tumorale dans les DLGG et de concevoir de nouvelles stratégies thérapeutiques contre ces tumeurs<br>Diffuse Low-Grade Gliomas (DLGG) are WHO grade II glial tumors affecting younger adults. They are characterized as silent, slow growing tumors with fewer mitotic activities. However, they diffuse and invade the healthy brain via blood vessels and nerve fibers. These, over a period of years develop to malignant Glioblastoma, aggressive brain tumors where patients have an average medial survival of 12-15 months after diagnosis. Ill-defined phenotypic and cellular diversity of DLGG poses serious limitation to treatment and prevention at the early stage.In my PhD thesis, I aimed to address this limitation by characterizing the cellular heterogeneity in IDH1-mutated DLGG. By performing immunofluorescence analysis on grade II astrocytoma and oligodendroglioma, I have identified two largely non-overlapping cellular subpopulations expressing SOX9 and OLIG1 transcription factors, which represent astrocyte-like and oligodendrocyte-like cells, respectively. Upon further investigation, I have shown that these subpopulations express distinct molecular markers. Sox9 cells are associated with APOE, KCNN3, CRYAB and ID4, while Olig1 cells showed strong correlation with the expression of PDGFRA, SOX8, MASH1, and SOX4. In addition, the sox9 cells show a particular activation of signaling pathways including Notch, BMP and their downstream targets.To ascertain the role of Notch signaling in regulating the formation of these tumoral subpopulations, I used magnetic sorting of tumor cells from freshly resected glioma samples and overexpressed Notch Intracellular Domain (NICD), an active form of Notch. Increased Notch activation resulted in an upregulation of Sox9- and downregulation of Olig1-associated cell markers. I have then extended these analyses on one anaplastic IDH1 mutated patient derived cell line which reproduced similar gene expression profile confirming the robustness of the role of Notch signaling in regulating the plasticity of the cells. Parallel experiments performed by activation of Bone Morphogenetic Protein (BMP) signaling on IDH1 mutated cell line did not show a prominent effect on the plasticity. Nevertheless, BMP signal activation highly upregulated CRYAB, a SOX9 related marker and downregulated OLIG1 and OLIG2.In conclusion, I have identified two non-overlapping tumor subpopulations in diffuse low-grade gliomas and demonstrated the deterministic role of Notch signaling pathway in their formation. I believe that these findings would aid in better understanding tumoral heterogeneity in DLGG and be extended in designing new therapeutic strategies against these tumors

L'objectif de cette thèse concerne la possibilité d'améliorer le diagnostic et la thérapie des gliomes par le ciblage des intégrines à l’aide du RGD et par le développement d'agents multimodaux de type alcoxyamine. L’étude de l’internalisation du RGD révèle une régulation par la densité cellulaire, paramètre histologique dans la catégorisation des gliomes. Dans notre modèle, la densité cellulaire impacte la contribution de l’endocytose clathrine-dépendante et le métabolisme mais n’influence pas le rôle du cytosquelette. La régulation de l’internalisation des peptides RGD par la densité cellulaire reste à mieux comprendre afin de perfectionner les agents utilisant ce ciblage pour l’imagerie et le diagnostic des gliomes. Dans le même temps, les propriétés multimodales des alcoxyamines ont été évaluées àdes fins théranostiques. Ces molécules s’homolysent spontanément pour libérer un nitroxyde et un radical alkylant cytotoxique pouvant en plus induire une réactivation immunitaire antitumorale. Le nitroxyde est un agent de contraste pour l’IRM rehaussée par effet Overhauser. Le fort rehaussement du signal observé à proximité du nitroxyde assure un suivi en temps réel de l’apparition de l’agent alkylant. L’adaptation des alcoxyamines pour une homolyse conditionnelle dans le gliome permettrait une action thérapeutique avec un contrôle spatial et un suivi temporel du composé cytotoxique. L’acheminement de molécules d’intérêt vers la cible est rendu difficile par la présence de barrières physiologiques. Dans ce travail, la progression de nanoparticules par la voie intratrachéale peut se substituer à celle intraveineuse avec une augmentation du temps de rétention dans le gliome<br>The aim of this thesis is to improve the diagnostic and the therapy of glioma through both the integrin targeting by RGD and the development of Alkoxyamine as multimodal agent. The RGD internalization is regulated by the cellular density, a histologic parameterfor the glioma classification. In our model, the cellular density increases the contribution of both the clathrin-mediated endocytosis and the metabolism but not the one of the cytoskeletal. A better knowledge about the RGD internalization regulation by the cell density could help the MRI probe development for glioma diagnosis. Properties of alkoxyamine as multimodal agent were evaluated to perform theranostic. The spontaneous alkoxyamine homolysis give a nitroxide radical and a cytotoxic alkylating agent that could induce immune reactivation against the tumor. This nitroxide is an Overhauser enhanced MRI contrast agent. The strong signal enhancement in the nitroxide vicinity gives information in real-time about the release of the alkyl radical. Alkoxyamine adaptation for a conditional homolysis through specific glioma proteolysis activity could induce a localized alkyl therapeutic effect with a real-time monitoring. Physiological barriers limit the drug accumulation in the targeted sites. In this study, the intratracheal instillation of nanoparticles can substitute the intravenous administrationincreasing their intratumoral retention time

Bakgrund: Primär malign hjärntumör medför en tung symtombörda som yttrar sig i en stor variation av fysiska, kognitiva och neurologiska symtom som berör patientens funktionsförmåga och psykiska välmående. Utöver den emotionella och existentiella påfrestningen av att leva med en cancersjukdom ingår hos hjärntumörsjuka patienter även en sviktande kognitiv komponent. Syfte: Syftet med denna litteraturöversikt var att belysa psykologiska påfrestningar för vuxna patienter med primär malign hjärntumör. Metod: Studien utfördes som en allmän litteraturöversikt där tiovetenskapliga kvalitetsgranskades och analyserades för att sedan delas in i tre stycken övergripande teman. Resultat: Det första temat, Osäkerheten i prognosen, belyser den ovisshet som uppstod till följd en oförutsägbar framtid och behovet av mer information gällandes behandlingsmöjligheter och vilka symtom som kan väntas drabba dem. Temat Psykosociala konsekvenser beskriver hur patienter kände att de förlora sig själva i sjukdomen på grund av minnespåverkan, personlighetsförändringaroch en oförmåga att upprätthålla den livsstil som de tidigare haft. Gemensamt bland de sjuka var en rädsla för att vara en börda för dem i patientens närhet och en oro om att förlora sin självständighet. Den existentiella konfrontationen talar om de oundvikliga tankarna om döden och om hoppets betydelse.<br>Background: Primary malignant brain tumor carries a heavy symptom burden that presents itself in a big variation of physical, cognitive and neurological symptoms that affects the patients functioning and psychological wellbeing. Along with the emotional and existential stress of living with cancer, patients diagnosed with brain cancer also suffer from cognitive dysfunction. Purpose: The aim of this study was to illustrate the psychological strain of adult patients living with primary malignant brain tumor. Method: The study was executed as a general literature review based on ten scientific articles. The articles were quality-tested and analyzed to later be sorted into three main themes. Results: The first theme, The uncertainty in the prognosis, illuminate the uncertainty that occurred due to an unpredictable future and the need of information concerning treatment options and what symptoms to expect. The theme The psychosocial consequences, describes how the patients felt as though they had lost themselves to the disease due to memory loss, personality disorders and the inability to maintain the lifestyle they previously had. The patients shared a fear of being a burden to the people around them and a concern of losing their independency. The existential confrontation speaks about the inevitable thoughts of death and the importance of hope.

L’incidence des gliomes est estimée à 6.6 pour 100 000 habitants. Les survies varient selon le sous-type de gliomes, avec des taux de survie à 5 ans d’environ 48% pour les astrocytomes diffus selon la classification de l’Organisation Mondiale de la Santé (OMS), 28% pour les astrocytomes anaplasiques, 80% pour les oligodendrogliomes, 52% pour les oligodendrogliomes anaplasiques et 5% pour les glioblastomes, tumeurs cérébrales malignes les plus fréquentes.Une meilleure compréhension des mécanismes et de la biologie de ces tumeurs et de nouvelles pistes thérapeutiques sont essentielles afin d’identifier de nouvelles thérapies pouvant améliorer le pronostic des patients. La classification OMS 2016 des tumeurs du système nerveux central a, pour la première fois, intégré les données de biologie moléculaires aux données histopathologiques, afin d’améliorer la distinction des différents sous-groupes de tumeurs et d’orienter au mieux les choix thérapeutiques pour chaque sous-groupe.Nous nous sommes intéressés dans ce travail à l’apport de l’approche en protéomique par imagerie par matrix-assisted laser desorption/ionization spectrométrie de masse MALDI (MALDI-MSI) couplée à l’analyse en microprotéomique dans les gliomes dans le cadre de l’étude clinique GLIOMIC (NCT02473484) qui a pour but de réaliser une classification moléculaire des gliomes en intégrant les données cliniques et celles obtenues par ces nouvelles approches.La faisabilité de la technique a d’abord été validée sur une série de gliomes anaplasiques. Dans cette première analyse, nous avons pu démontrer que, bien que l’approche protéomique confirmait également l’hétérogénéité tumorale, les analyses histologiques et protéomiques divergent et apportent des informations complémentaires. L’imagerie moléculaire protéomique a mis en évidence trois différents groupes d’expression de protéines : un groupe de protéines associé au cancer, un groupe de protéines impliquées dans l’inflammation et un groupe de protéines impliquées dans la différentiation des cellules nerveuses et la croissance des neurites.Nous nous sommes ensuite intéressés aux glioblastomes. Les premiers résultats ont également confirmés l’existence des 3 régions d’intérêt définies sur le plan moléculaires, apportant de nouvelles informations par rapport aux données histopathologiques. Ces résultats doivent être confirmés dans une cohorte plus large de patients.En conclusion, l’intégration de ces biomarqueurs protéomiques, aux données cliniques, histopathologiques et de biologie moléculaire, pourrait permettre d’améliorer les connaissances sur les gliomes, leur classification et l’identification de nouvelles cibles thérapeutiques potentielles<br>The annual incidence of gliomas is estimated at 6.6 per 100,000. Suvival varies profoundly by type of glioma, with 5-year survival rates of 48% for World Health Organization (WHO) grade II diffuse astrocytoma, 28% for WHO grade III anaplastic astrocytomas, 80% for WHO grade II oligodendroglioma, 52% for WHO grade III anaplastic oligodendroglioma and 5% for WHO grade IV glioblastoma, the most frequent primary malignant brain tumor. A better understanding of the molecular pathogenesis and the biology of these tumors is required to design better therapies which can ultimately improve the prognosis of patients. The WHO 2016 classification of central nervous system tumors has for the first time integrated molecular data with the histopathological data, in order to improve the classification of the different subgroups of central nervous system tumors and to allow to derive more specific therapeutic strategies for each of the different subgroups.In the present work, we aimed at evaluating the value of a proteomic approach using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry coupled with microproteomic analysis in gliomas through the GLIOMIC clinical study (NCT02473484), we aimed at obtaining a molecular classification of glioblastomas by integrating clinical data to the ones obtained by such technologies. The feasibility of this approach was first demonstrated in a cohort of anaplastic gliomas. In this first analysis, we showed that although proteomic analysis confirmed the heterogeneity of brain tumors already observed with the histological analysis, the two approaches may lead to different and complementary information. Three different groups of proteins of interest were identified: one involved in neoplasia, one related to glioma with inflammation, and one involved neurogenesis. Then, analyses of glioblastomas confirmed the three proteomic patterns of interest already observed in the anaplastic gliomas, which represents new information as compared to histopathological analysis alone. These results have to be confirmed in a larger cohort of patients.We conclude that MALDI mass spectrometry coupled with microproteomic analysis may provide new diagnostic information and may aid in the identification of new biomarkers. The integration of these proteomic biomarkers into the clinical data, histopathological data and data from molecular biology may improve the knowledge on gliomas, their classification and development of new targeted therapies

Apesar das evidências apontarem à participação da amilóide sérica A (SAA) em processos que favorecem a carcinogênese e metástase, associado à proposta da SAA como um marcador de progressão tumoral, não há ainda nenhum estudo avaliando a atividade direta da SAA em células tumorais. Este estudo examinou o efeito direto da SAA em duas linhagens de glioma humano. Gliomas são tumores cerebrais primários mais comuns em adultos e as linhagens deste estudo, A172 e T98G, representam carcinomas humanos caracterizados por um comportamento biológico altamente agressivo e quase sempre fatais, sendo classificados como grau IV. As linhagens A172 e T98G possuem algumas diferenças importantes em relação à invasividade; elas são respectivamente menos invasiva e mais invasiva. Para este estudo, avaliamos o efeito de SAA sobre a síntese de compostos representativos de algumas das diferentes classes de substâncias que estão envolvidas na progressão do tumor; entre elas estão a citocina IL-8, IL-6, TNF-&#945;, a molécula mensageira NO, as metaloproteases, MMP2 e MMP9 e o gene RECK. Além disso, nos perguntamos se SAA pode estar envolvida nos processos de proliferação, migração e invasão celular. SAA induziu a produção de NO em ambas as linhagens de glioma. Em relação a IL-8 observamos que sua produção basal é bastante diferente dependendo da linhagem, sendo pouco produzida pela linhagem A172 que foi a única que respondeu à SAA. IL-6 e TNF-&#945; não foram produzidas pelos gliomas quando estimulados com SAA. O tratamento das células com SAA aumentou a expressão das MMP-2 e MMP-9 e diminuiu a de RECK em ambas linhagens. SAA mostrou ser um estímulo mitogênico para os gliomas T98G e A172. SAA aumentou a migração e invasão da linhagem T98G e inibiu estes mesmos parâmetros nas células A172. SAA é constitutivamente expressa e produzida por ambas linhagens, sendo que a isoforma SAA1 é a predominante. A expressão gênica de todas as isoformas, SAA1, SAA2, SAA4, e a síntese protéica das SAA foram aumentadas pela adição de IFN-&#947;. Nossos resultados com base em ensaios in vitro suportam uma contribuição direta da SAA para a progressão e metástase do tumor, dependendo do tipo de célula e concentração da SAA. O fato de que IFN-&#947; é um indutor de SAA nos gliomas aponta que SAA pode exercer tanto um efeito intrácrino quanto autócrino ou parácrino nos tumores.<br>In spite of the evidences sustaining the participation of SAA in processes that favor carcinogenesis and metastasis, and the proposal of SAA as a marker of tumor progression, no studies have yet addressed a potential direct activity of SAA on tumor cells. This study examined the direct effect of SAA on two human glioma lineages. Gliomas are primary brain tumors more common in adults and the lineages of this study, A172 and T98G, represent human carcinomas characterized by a highly aggressive biological behaviour and almost always fatal, classified as grade IV. A172 and T98G have some important differences in the invasiveness, they are less invasive and more invasive, respectively. For this study, we evaluated the effect of SAA on the synthesis of compounds representing different classes of substances that are somehow involved in tumor progression, among them we can cite the cytokine IL-8, the messenger molecule NO, the metalloproteinases MMP2 and MMP9 and RECK gene. Furthermore, we wonder if SAA was involved in the processes of proliferation, migration and cell invasion. SAA stimulated the production of IL-8 in lineage A172, while T98G produced high amounts of IL-8 that were not modified by SAA addition. SAA did not stimulate the production of IL-6 and TNF-&#945;. SAA induced the production of NO, increased the expression of MMP-2 and MPP-9 and decreased the regulator of the expression of the MMPs; gene RECK. Moreover, we observed that SAA was a mitogenic stimulus, but it had a dual effect on migration and invasiveness behavior depending on cell lineage. For T98G SAA increased migration and invasion, and for A172 SAA inhibited migration and invasion. SAA was constitutively expressed and produced by both strains, and the isoform SAA1 predominated. The gene expression of all isoforms, SAA1, SAA2, SAA4, and protein synthesis of SAA were increased by the addition of INF-&#947;. Our findings based on in vitro assays support a direct contribution of SAA to tumor development, progression and metastasis depending on the cell type and concentration of SAA. Besides the role of SAA on tumor growth during an acute phase, the fact that SAA was expressed in tumor cells suggests an intracrine or an autocrine action of SAA.

La caractérisation moléculaire multidimensionnelle des tumeurs et des tumeurs gliales en particulier est une étape importante pour l’identification de biomarqueurs (diagnostique, pronostique, théranostique et/ou de prédisposition), pour l’identification de cibles thérapeutiques et pour une meilleure compréhension de l’oncogénèse moléculaire.Nos travaux ont permis de confirmer et de consolider certaines données de la littérature comme par exemple : (i) la valeur pronostique favorable de la codélétion 1p/19q, (ii) la valeur pronostique favorable de la mutation IDH, (iii) le caractère mutuellement exclusive des mutations TP53 et de la codélétion 1p/19q et (iv) la rareté des altérations génétiques du PDGFRA dans les gliomes de bas grade (GDBG). De manière plus originale, nous avons identifié plusieurs sous-groupes génomiques de GDBG pertinents sur le plan clinico-biologique, notamment au sein des GDBG non 1p/19q codélétés : (i) 19q-délété ; (ii) 11p-délété, (iii) 7-gagné, (iv) 19-gagné et (v) inclassés. La perte du bras chromosomique 19q annule la valeur pronostique favorable de la mutation IDH dans les GDBG non 1p/19q codélétés. Nous avons également identifié des mutations géniques originales dans les GDBG (i.e. mutation TEP1 et RNF40) qui renforcent le rôle des télomères et du remodelage de la chromatine au sein des GDBG.Enfin, nous nous sommes concentrés sur la caractérisation des GDBG 11p-délétés qui sont de phénotype majoritairement astrocytaire et de moins bon pronostic. Ces GDBG surexpriment des gènes des cellules immunitaires (les GIM -Glioma infiltrating microglia-, les macrophages de type 1, les macrophages de type 2) et sont infiltrés par des cellules macrophagiques et microgliales. Ce microenvironnement dérégulé peut constituer une cible thérapeutique au sein des GDBG 11p-délétés. En conclusion, nos travaux participent à la dissection clinico-moléculaire des GDBG et à préciser la biologie d’un sous-type de GDBG caractérisé la perte du bras chromosomique 11p<br>Multildimensional molecular characterization of tumors and more specifically of gliomas is of pivotal importance to identify: (i) new biomarkers (i.e. diagnostic, prognostic, theranostic or predisposing), (ii) new therapeutic targets and (iii) to improve our understanding of molecular oncogenesis.Our work has confirmed and consolidated previous data published in the literature, for example that: (i) 1p/19q co-deletion is associated with better prognosis, (ii) IDH mutation is associated with better prognosis, (iii) TP53 mutations and 1p/19q codeletion are mutually exclusive and (iv) PDGFRA is rarely altered, at genomic level, in low-grade gliomas (LGG).More originally, we have identified several genomic groups, with clinical and biological relevances, in LGG and more specifically in LGG without 1p/19q co-deletion: (i) 19q-deleted, (ii) 11p-deleted, (iii) 7-gained, (iv) 19-gained and (v) unclassified. Interestingly, 19q deletion abrogates the positive prognostic value of IDH mutation in LGG without 1p/19q codeletion.We have also identified new recurrent somatic gene mutations in LGG (i.e. TEP1 and RNF40 mutations), supporting the critical role of telomeres and chromatin remodelling in LGG.Finally, we have characterized further 11p-deleted LGG that exhibit mostly astrocytic phenotype and poor prognosis. This subgroup includes LGG overexpressing genes of inflammatory/immune cells (GIM -Glioma infiltrating microglia-, M1 macrophages and M2 macrophages) and infiltrated by macrophagic/microglial cells. This peculiar microenvironment detected in 11p-deleted LGG might be used as a therapeutic target. In conclusion, our work participates to characterize clinico-biological portrait of LGG and to describe a singular genomic subgroup of LGG characterized by 11p loss

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<br>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

Les gliomes sont les tumeurs cérébrales les plus fréquentes chez l’adulte. Il s’agit d’un groupe de tumeurs hétérogène pouvant être classées dans différents sous-groupes selon des critères moléculaires et histopathologiques, donnant une indication sur leur agressivité. Les p21-activated kinase (PAK) sont des sérine-thréonine kinases effectrices des GTPases Rac et Cdc42 et font ainsi partie de nombreuses voies de signalisation. Elles régulent de nombreuses voies d’intérêt dans la biologie des cancers, comme les voies Mek/Erk, PI3K/Akt et Wnt/b-caténine. PAK1 est fréquemment surexprimé et/ou suractivé dans plusieurs cancers, notamment du sein, des ovaires de la prostate et du cerveau, alors que PAK3 induit la sortie du cycle cellulaire et la différenciation des cellules au cours de la neurogenèse dans plusieurs modèles animaux. Durant ma thèse, j’ai pu observer que l’expression de PAK3 était de bon pronostic pour les patients atteints de gliomes, et était plus élevée dans un sous-type de tumeurs caractérisé par la codélétion 1p/19q. In vitro, l’expression de PAK3 était plus élevée dans une lignée de cellules de gliome de signature proneurale, non tumorigène après xenogreffe chez la souris. Au cours de la différenciation des cellules de gliome, l’augmentation de l’expression de PAK3 est associée avec des marqueurs de lignage neural et neuronal. L’inhibition de l’expression de PAK3 entraîne l’augmentation de la capacité d’auto-renouvellement et de la tumorigénicité d’une lignée de cellules de gliome, et favorise la différenciation gliale des cellules<br>Gliomas are the most common and lethal adult primary brain tumors. Their complex heterogeneity is evidenced by numerous genomic studies showing distinct molecular entities in glioma. P21-activated-kinases (PAK) are serine threonine kinases involved in multiple signal transduction pathways as downstream effectors of Rac and Cdc42. They regulate several key cancer-relevant pathways like cell division and movement. PAK1 and PAK3 are highly expressed in the brain; PAK1 is frequently overexpressed and/or over-activated in several human cancers whereas PAK3 is involved in neural differentiation and the developmental proneural pathway. The role of these two kinases in brain tumor pathophysiology is unknown. We have observed that PAK3 expression was associated with a longer survival for patients with glioma and was higher in 1p/19q gliomas. In vitro, PAK3 was highly expressed in a glioma cell line with a proneural signature that did not induce tumor after xenograft. Its increasing expression upon a set of differentiation paradigms was correlated with those of neural and/or neuronal markers in glioma cell lines. Inhibition of PAK3 expression increased cell renewal and tumorigenicity. It impaired cell differentiation, promoting the glial lineage

Les gliomes malins sont caractérisés par une prolifération importante, une migration diffuse des astrocytes tumoraux dans le parenchyme cérébral et un taux important de néo-angiogenèse. La gastrine appartient à la famille des peptides apparentés à la cholécystokinine et cette dernière est présente en abondance dans le cerveau. De plus la gastrine est capable de modifier le comportement biologique d’un certain nombre de tumeurs. Le groupe de recherche au sein duquel j’ai réalisé mon travail de thèse fut le premier à suggérer le rôle potentiel de la gastrine au niveau des taux de prolifération et de migration des astrocytes tumoraux. Nous avons précisé dans le présent travail divers effets biologiques provoqués par la gastrine au niveau de gliomes et de gliosarcome expérimentaux.<p>Nous avons au préalable tenté de caractériser par une technique de RT-PCR l’expression d’ARN pour divers récepteurs à la gastrine au sein de tumeurs du système nerveux central et périphérique (comprenant des gliomes, des méningiomes et des schwannomes), au sein de gliomes et d’un gliosarcome expérimentaux, et au sein de cellules endothéliales humaines de veines ombilicales HUVEC et de manchons vasculaires obtenus par microdissection au laser d’un glioblastome humain. Nous avons également développé un modèle de neurochirurgie expérimentale chez le rat consistant en la résection microchirurgicale de la tumeur cérébrale après un bilan iconographique par IRM. Nous avons ainsi montré que l’administration de gastrine dans le foyer opératoire après résection tumorale augmente significativement la période de survie de rats immunodéficients porteurs du modèle de gliome humain U373 et de rats conventionnels porteurs du modèle C6 de rat. In vitro, nous avons montré grâce au test colorimétrique MTT que la gastrine induit une diminution significative du taux global de croissance de ces deux modèles avec une accumulation des astrocytes tumoraux dans la phase G1 de leur cycle cellulaire. Par la technique de Western blotting nous avons également montré que la gastrine induit une diminution significative des taux protéiques du complexe cycline D3-Cdk4 dans les deux modèles expérimentaux. Nous avons montré que la gastrine est capable de réduire significativement l’invasion des modèles C6 de rat, U373 humain et de gliosarcome 9L de rat au travers d’une matrice de collagène et de réduire l’invasion des cellules U373 en chambre de Boyden. La gastrine modifie également significativement la motilité des cellules C6 et U373 et l’organisation de leur cytosquelette d’actine.<p>Nous avons découvert que la gastrine administrée en intracérébral dans le foyer tumoral U373 augmente significativement le taux d’angiogenèse au sein de la tumeur. Nous avons alors investigué l’effet de la gastrine et des antagonistes des récepteurs à cholécystokinine sur le taux d’angiogenèse in vitro en utilisant le modèle des cellules HUVEC cultivées sur Matrigel. L’effet pro-angiogénique in vitro et in vivo de la gastrine est significativement contrecarré par le produit L365,260, un antagoniste relativement spécifique du récepteur CCK-B de la gastrine. La gastrine est chémoattractante sur les cellules HUVEC et augmente significativement leur sécrétion d’IL-8. Toutefois l’effet pro-angiogénique de la gastrine serait en partie dépendant de la modification du taux d’expression des sélectines par les cellules HUVEC, et non de la sécrétion d’IL-8. Nous avons réalisé une revue de la littérature pour tenter de comprendre pourquoi les astrocytes tumoraux migrants sont résistants à la chimiothérapie conventionnelle. A la fin du chapitre Discussion, dans le sous-chapitre intitulé « Quels sont les espoirs thérapeutiques dans le cas des gliomes dits diffus? », nous tentons d’analyser les implications thérapeutiques potentielles qu’il serait possible de tirer du présent travail. <p><p><p><br>Doctorat en sciences médicales<br>info:eu-repo/semantics/nonPublished

Cette thèse présente des travaux en lien avec l’utilisation de données cliniques dans la construction de modèles appliqués à l’oncologie. Les modèles actuels visant à intégrer plusieurs mécanismes biologiques liés à la croissance tumorale comportent trop de paramètres et ne sont pas calibrables sur des cas cliniques. A l’inverse, les modèles plus simples ne parviennent pas à prédire précisément l’évolution tumorale pour chaque patient. La multitude et la variété des données acquises par les médecins sont de nouvelles sources d’information qui peuvent permettre de rendre les estimations des modèles plus précises. A travers deux projets différents, nous avons intégré des données dans le processus de modélisation afin d’en tirer le maximum d’information. Dans la première partie, des données d’imagerie et de génétique de patients atteints de gliomes sont combinées à l’aide de méthodes d’apprentissage automatique. L’objectif est de différencier les patients qui rechutent rapidement au traitement de ceux qui ont une rechute plus lente. Les résultats montrent que la stratification obtenue est plus efficace que celles utilisées actuellement par les cliniciens. Cela permettrait donc d’adapter le traitement de manière plus spécifique pour chaque patient. Dans la seconde partie, l’utilisation des données est cette fois destinée à corriger un modèle simple de croissance tumorale. Même si ce modèle est efficace pour prédire le volume d’une tumeur, sa simplicité ne permet pas de rendre compte de l’évolution de forme. Or pouvoir anticiper la future forme d’une tumeur peut permettre au clinicien de mieux planifier une éventuelle chirurgie. Les techniques d’assimilation de données permettent d’adapter le modèle et de reconstruire l’environnement de la tumeur qui engendre ces changements de forme. La prédiction sur des cas de métastases cérébrales est alors plus précise<br>This thesis deals with the use of clinical data in the construction of models applied to oncology. Existing models which take into account many biological mechanisms of tumor growth have too many parameters and cannot be calibrated on clinical cases. On the contrary, too simple models are not able to precisely predict tumor evolution for each patient. The diversity of data acquired by clinicians is a source of information that can make model estimations more precise. Through two different projets, we integrated data in the modeling process in order to extract more information from it. In the first part, clinical imaging and biopsy data are combined with machine learning methods. Our aim is to distinguish fast recurrent patients from slow ones. Results show that the obtained stratification is more efficient than the stratification used by cliniciens. It could help physicians to adapt treatment in a patient-specific way. In the second part, data is used to correct a simple tumor growth model. Even though this model is efficient to predict the volume of a tumor, its simplicity prevents it from accounting for shape evolution. Yet, an estimation of the tumor shape enables clinician to better plan surgery. Data assimilation methods aim at adapting the model and rebuilding the tumor environment which is responsible for these shape changes. The prediction of the growth of brain metastases is then more accurate

Les gliomes, tumeurs cérébrales primaires du système nerveux central, sont souvent de pronostic défavorable, d'autant plus que l'absence de critères indiscutables pour les identifier rend leur diagnostic et leur prise en charge particulièrement difficiles. L’analyse conjointe, d’une cohorte française de 64 patients porteurs de gliomes et d’une cohorte internationale de 671 patients issus du TCGA, a permis de mettre en évidence deux groupes pronostiques constitués par un panel d’expression différentielle de 26 gènes (p = 0,007). Cette stratification en deux groupes pronostiques a été confirmée quels que soient le grade et le groupe moléculaire de la tumeur (p &lt; 0,0001). Nous avons établi une nouvelle stratégie diagnostique à partir de la classification moléculaire des gliomes en intégrant deux biomarqueurs pronostiques CHI3L1 et NTRK2. L’analyse multivariée confirme que ces biomarqueurs sont indépendants du statut IDH et du grade tumoral. Si nous avons mis en évidence par l’analyse protéique de CHI3L1 une concordance avec les transcrits, les résultats divergent pour TrkB. Ainsi, une expression élevée de TrkB et son corécepteur p75NTR serait liée à l’agressivité tumorale indépendamment du statut IDH. Enfin, TrkB et p75NTR sont présents aussi bien dans les exosomes issus du plasma de témoins sains et de patients atteints de gliomes mais leur expression augmente en fonction de l’agressivité de la tumeur<br>Gliomas, primary brain tumours of the central nervous system, are often of poor prognosis.The absence of clear criteria to identify them makes their diagnosis and management particularly difficult. The combined analysis of a cohort of 64 glioma patients and an international cohort of 671 patients from the TCGA revealed two prognostic groups of a differential expression panel of 26 genes (p = 0.007). This stratification into two prognostic groups was confirmed independently of the grade and molecular group of the tumor (p &lt;0.0001). We have established a new diagnostic strategy based on the molecular classification of gliomas by integrating two prognostic biomarkers CHI3L1 and NTRK2. Multivariate analysis confirms that these biomarkers are independent of IDH status and tumor grade.While we have demonstrated by the protein analysis of CHI3L1 concordance with the transcripts, the results are different for TrkB. Therefore, a high expression of TrkB and its p75NTR co-receptor would be associated with tumor aggressiveness regardless of IDH status. Lastly, TrkB and p75NTR are present in exosomes from plasma of healthy controls and glioma patients, but their expression increases with the aggressiveness of tumor

Introduction Les gliomes de bas grade sont des tumeurs cérébrales de progression lente qui affectent l’adulte jeune. Ce mode d’évolution laisse le temps aux réseaux neuraux de se réorganiser de façon massive ce qui permet d’expliquer pourquoi les patients ne présentent habituellement aucun déficit neurologique au diagnostic bien que la tumeur concerne des zones dites «éloquentes». Ces lésions sont donc un sujet d’étude particulièrement intéressant dans la compréhension des mécanismes de plasticité cérébrale.Ces patients bénéficient, comme traitement optimal, d’une chirurgie en condition éveillée permettant une résection la plus importante possible tout en préservant les fonctions neurologiques du patient pour qu’il conserve la meilleure qualité de vie possible.L'imagerie fonctionnelle de repos est un outil robuste en IRM pour étudier la connectivité fonctionnelle et la plasticité cérébrale. Elle est basée sur l’analyse du signal BOLD et présente plusieurs avantages : 1) la possibilité d’être réalisée chez des patients non coopérants 2) la possibilité d’analyser l’ensemble des réseaux neuraux simultanément.Dans ce travail nous souhaitions mesurer les fluctuations de connectivité fonctionnelle durant la période péri-opératoire d’une chirurgie éveillée pour gliome diffus de bas grade afin d’évaluer la plasticité fonctionnelle engendrée par la résection de la tumeur.Dans un second temps, nous avons tenté d'expliquer ces données fonctionnelles péri-opératoires à l’aide de l’imagerie multimodale en analysant l’évolution péri-opératoire de la connectivité anatomique et des paramètres hémodynamiques.Méthodes L’analyse principale portait sur une cohorte de 82 patients porteurs de gliomes diffus de bas grade et opérés en chirurgie éveillée. Pour chaque patient une IRM avec séquences fonctionnelles de repos était réalisée à trois temps : pré-opératoire, post-opératoire immédiat et lors du suivi à 3 mois. Toutes les IRM étaient effectuées pour chaque patient sur la même machine au cours du suivi, soit un système IRM 3.0 Tesla (Skyra, Siemens), soit un système IRM 1.5 Tesla (Avanto, Siemens). Après des étapes classiques de prétraitement, les données fonctionnelles étaient traitées à l’aide du logiciel CONN v16.a.La connectivité anatomique a secondairement été analysée par imagerie de diffusion anisotropique en IRM en se concentrant sur le corps calleux.Enfin les conséquences hémodynamiques de la chirurgie étaient évaluées d’une part via des séquences de perfusion en IRM et d’autre part par une analyse innovante du signal BOLD.RésultatsNous avons constaté de façon surprenante, durant la période post-opératoire immédiate, une altération significative transitoire globale quasi-exclusive de la connectivité interhémisphérique entre régions miroirs, nommée connectivité homotopique.Des modifications de connectivité anatomique concernant le corps calleux et des modifications hémodynamiques régionales et globales ont également été constatées de façon concomitante en période post-opératoire immédiate et à plus long terme après la chirurgie sans qu’un lien direct avec nos données fonctionnelles n’ait pu être mis en évidence.L’analyse des données hémodynamiques a enfin mis une lumière une région intéressante : le striatum. Cette structure pourrait être une région centrale dans le maintien de la connectivité homotopique et son atteinte alors mener aux modifications fonctionnelles observées.Conclusion La rupture d’homotopie fonctionnelle transitoire que nous constatons en période post-opératoire immédiate est probablement d’origine multifactorielle. La prise en compte des données anatomiques et hémodynamiques, dans l’interprétation des résultats fonctionnelles en IRM, est indispensable tant en période post-opératoire immédiate que à plus long terme après la chirurgie. Des travaux d’analyse de la vasoréactivité cérébrale d’une part et de modélisation d’autre part pourraient aider à mieux comprendre les différents phénomènes intriqués<br>IntroductionDiffuse low-grade gliomas (DLGG) are slow-growing brain tumors occurring in young adults. This slow progression induces extensive neuroplasticity and explains why patients most of the time do not show any obvious neurological deficit at the time of diagnosis although tumors are located in ‘eloquent’ areas. Therefore DLGG provide an interesting model in understanding mechanisms of neuroplasticity.Awake surgery with direct cortical and subcortical electrostimulation mapping is recommended as first-line treatment of DLGG, allowing to maximize tumoral resection and limiting postoperative neurological deficit, maintaining patients quality of life.Resting-state fMRI, based on BOLD signal analysis, is used to study functional connectivity and neural plasticity. This technique allows robust evaluation of neural networks without performing a task. Consequently, it bypasses the impact of confusion, sedation or neurological deficits on task execution. In this thesis, we aimed to investigate perioperative functional connectivity modifications in order to evaluate neural plasticity after awake surgery.Subsequently we explained the functional results using multimodal MRI imaging to analyze anatomic connectivity and hemodynamic parameters.Methods82 patients with DLGG who underwent awake surgical resection were included in the principal study. MRI acquisitions were performed successively before, within 36 h after and three months post-surgery. All scans were executed on the same MRI magnet for each patient, i.e. either a 3.0 T magnet (Skyra, Siemens) or a 1.5 T magnet (Avanto, Siemens). First, data were preprossed using a standardized classical pipeline and analyzed with the CONN toolbox v16.a.Second, anatomic connectivity was evaluated using diffusion tensor imaging of the corpus callosum.Finally hemodynamic changes induced by surgery were assessed with traditional perfusion imaging as well as using an innovative analysis of the BOLD signal’ s temporal shift.ResultsSurprisingly, it was found that specifically a diffuse transient postoperative interhemispheric disconnectivity occurred between homologous regions, known as homotopic connectivity.In parallel, immediate and long-term postoperative alterations in the anatomic connectivity of the corpus callosum were observed. Immediate and long-term postoperative modifications were also found regarding both regional and global hemodynamics characteristics. Yet, no significant link between the homotopic connectivity findings and the anatomical and hemodynamic changes could have been established at this point.Nevertheless, the hemodynamic analysis allowed the identification of a a specific brain region : the striatum. It was hypothesized that it acts as a central region for the maintenance of homotopic connectivity, explaining simultaneously the decreased post-surgical homotopic connectivity observed.ConclusionThe highlighted transient postoperative functional homotopy is probably due to multifactorial causes To start entangling these causes, the use of anatomic and hemodynamic imaging data analyses seems crucial to interpret functional connectivity data both immediate and long-term postoperative.Cerebral vasoreactivity and modelling studies provide thereby a very promising tool to better understand the interrelated processes underlying postoperative functional connectivity modifications

La protéine kinase C alpha (PKCα) est une sérine/thréonine kinase ubiquitaire. Elle intervient dans la régulation de différentes fonctions cellulaires en interagissant avec de nombreuses protéines. Parmi ces dernières, nous avons réussi à identifier Trm61, la sous-unité catalytique de la m1A58 ARNt méthyltransférase qui joue un rôle essentiel dans la stabilité de l'ARNtiMet. Les études de localisation de PKCα et des deux sous-unités Trm6 et Trm61 ont permis de démonter que ces deux sous-unités ne partagent pas toujours les mêmes compartiments cellulaires : si la sous-unité Trm6 est toujours nucléaire, la Trm61 est pancellulaire et se co-localise avec PKCα dans le cytoplasme. Nous avons apporté la preuve que l'augmentation de l'expression de PKCα entraîne une diminution de Trm61, alors que la diminution de l'expression de PKCα s'accompagne d'une augmentation aussi bien de Trm61 que d'ARNtiMet et se traduit par une importante augmentation de la prolifération à forte densité cellulaire. Ce travail a permis également de démontrer que la sous-unité Trm61 est essentielle pour la survie des cellules C6. La surexpression de Trm6 et/ou de Trm61 a permis de pointer la Trm6 comme le déterminant essentiel du niveau de la m1A58 ARNt méthyltransférase fonctionnelle et de suggérer un rôle secondaire de Trm61 cytoplasmique dans la régulation de la prolifération de façon indépendante de l'action du complexe Trm6-Trm61. De façon intéressante, les gliomes de bas grade présentent des taux plus élevés d'ARNm PKCα que les glioblastomes et inversement pour les taux des ARNm TRM6 et TRM61, apportant un argument en faveur de la relevance de nos observations dans la tumorigenèse gliale humaine<br>Protein kinase C alpha (PKCα) is a ubiquitous serine/threonine kinase. It is involved in the regulation of various cellular functions by interacting with many intracellular proteins. Among these, we were able to identify Trm61, the catalytic subunit of the tRNA m1A58 methyltransferase which plays an essential role in the stability of the tRNAiMet. Localization studies of PKCα, Trm6 and Trm61 demonstrated that these two subunits do not always share the same subcellular compartment: while Trm6 is strictly nuclear, Trm61 is both in the nucleus and in the cytoplasm where it co-localizes with PKCα. We also provided the evidence that the increased expression of PKCα induces a decrease in that of Trm61, while reduced PKCα expression is accompanied by an increase in both Trm61 and tRNAiMet levels. These changes in expression are accompanied by a significant increase in cell proliferation at high-density. This work has also shown that Trm61 subunit is essential for the survival of the C6 glioma cell line. Our results suggest that Trm6 is the essential determinant of functional tRNA m1A58 methyltransferase level and we discuss the possibility of a secondary role for cytoplasmic Trm61 in the regulation of the proliferation independently of Trm6-Trm61 action. Interestingly, human grade II and III gliomas expressed higher levels of PKCα mRNA than glioblastomas and inversely for TRM6 and TRM61 mRNA levels, arguing for a relevance of our observations for human gliomagenesis

Le travail de thèse est dédié à la caractérisation de fusions spécifiques oncogéniques entre les gènes FGFR et TACC dans les gliomes. Nous avons analysé 907 gliomes pour la présence du gène de fusion FGFR3-TACC3. Nous avons montré que les fusions FGFR3-TACC3 ne touchent que les gliomes IDH wild-type (3%), sont mutuellement exclusives avec l'amplification de EGFR et avec la forme tronquée EGFRvIII et inversement, sont associées à l'amplification de CDK4 et de MDM2 et à la délétion du 10q. Les fusions FGFR3-TACC3 sont associées à une expression intense et diffuse de FGFR3 en immunohistochimie (IHC) et l'IHC pour FGFR3 est un marqueur prédictif très sensible de la présence des fusions FGFR3-TACC3. Les patients porteurs d'une fusion FGFR3-TACC3 ont une survie globale significativement plus longue comparés aux patients avec gliome IDH wild-type. Nous avons traité deux patients porteurs d'un gène de fusion FGFR3-TACC3 avec un inhibiteur tyrosine-kinase (TK) spécifique pour FGFR et nous avons observé une stabilisation de maladie et une réponse mineur chez un patient. Dans la deuxième section nous avons optimisé une nouvelle séquence de spectroscopie différentielle-MEGA-PRESS-pour la détection de l'oncometabolite 2-hydroxyglutarate (2 HG) qui s'accumule de manière spécifique dans les gliomes IDH mutés. Nous avons analysé de façon prospective une cohorte de 25 patients avant chirurgie pour probable gliome de grade II et grade III. Nous avons trouvé que la MEGA-PRESS est hautement spécifique (100%) et sensible (80%) dans la prédiction de la présence de la mutation IDH. Son taux est corrélé aux concentrations de 2 HG mesurés sur tissu congelé par spectrométrie de masse (GC-MS/MS)<br>This work is devoted to the characterization of a specific oncogenic fusion between FGFR and TACC genes in gliomas. Overall, we screened 907 gliomas for FGFR3-TACC3 fusions. We found that FGFR3-TACC3 fusions exclusively affect IDH wild-type gliomas (3%), and are mutually exclusive with the EGFR amplification and the EGFR vIII variant, whereas it co-occurs with CDK4 amplification, MDM2 amplification and 10q loss. FGFR3–TACC3 fusions were associated with strong and homogeneous FGFR3 immunostaining. We show that FGFR3 immunostaining is a sensitive predictor of the presence of FGFR3-TACC3 fusions. FGFR3-TACC3 glioma patients had a longer overall survival than those patients with IDH wild-type glioma. We treated two patients with FGFR3–TACC3 rearrangements with a specific FGFR-TK inhibitor and we observed a clinical improvement in both and a minor response in one patient. In the second section, we developed a non-invasive diagnostic tool by 1H-magnetic resonance spectroscopy in IDH mutant gliomas. We optimized a uniquely different spectroscopy sequence called MEGA-PRESS for the detection of the oncometabolite 2-hydroxyglutarate (2 HG) that specifically accumulates in IDH mutant gliomas. We analysed a prospective cohort of 25 patients before surgery for suspected grade II and grade III gliomas and we assessed specificity and sensitivity, correlation with 2 HG concentrations in the tumor and associations with grade and genomic background. We found that MEGA-PRESS is highly specific (100%) and sensitive (80%) for the prediction of IDH mutation and correlated with 2 HG levels measured by gas chromatography-tandem mass spectrometry (GC-MS/MS) in frozen tissue

Un tiers de la population européenne est touché par au moins un trouble du cerveau. En effet, la maladie d’Alzheimer, et les gliomes, représentent respectivement le syndrome de démence et les tumeurs cérébrales les plus fréquentes chez l’homme. Plusieurs études épidémiologiques ont montré l’existence d’une corrélation inverse entre le risque de développer une maladie neurodégénérative et un cancer cérébral. Ceci suggère la présence de dénominateurs moléculaires communs entre ces pathologies. Dans les deux cas, un dysfonctionnement mitochondrial est rapporté, représentant une caractéristique partagée par ces deux troubles neurologiques. La protéine kinase mitochondriale PINK1 responsable, lorsqu’elle est mutée, d’une forme précoce et familiale de Parkinson, est particulièrement impliquée dans les processus de maintien de l’homéostasie mitochondriale. Par conséquent, les mécanismes moléculaires régulant PINK1 ainsi que leurs impacts au cours des désordres mitochondriaux répertoriés dans la maladie d’Alzheimer et les tumeurs cérébrales, ont suscité un intérêt central, lors de ma thèse. Au cours de ce travail, nous avons examiné certaines des fonctions mitochondriales de PinK1, associées au maintien de l’homéostasie mitochondriale dans un contexte « Alzheimerisé ». Nous mettons en évidence le rôle de la γ-secretase dans la physiologie mitochondriale en contrôlant la régulation transcriptionnelle de PINK1 par l’AICD, le fragment généré conjointement avec le peptide amyloïde toxique Aβ, à partir du précurseur βAPP. Nous montrons de surcroît, l’initiation de cette régulation par la parkine<br>One third of the European populations is affected by a brain disorder. Thus, Alzheimer’s disease and gliomas represent the most frequent human brain dementia syndrome and tumor type, respectively. Several epidemiological studies have shown an inverse relationship between the risk of developing a neurodegenerative disease and a brain tumor, suggesting the existence of common molecular denominators between these pathologies. Interestingly, both pathologies are characterized by a mitochondrial dysfunction. The mitochondrial kinase associated to autosomal recessive Parkinson’s disease, PINK1, is particularly implicated in the control of mitochondrial homeostasis. The main objective of my thesis was to study the molecular mechanisms underlying PINK1 gene regulation and their link with the mitochondrial dysfunction observed in either Alzheimer’s disease or gliomas. Thus, during my thesis we have examined the ability of PINK1 to control mitochondria homeostasis in an Alzheimer’s pathological context. We demonstrate that AICD, a cleavage product of the trans-membrane protein βAPP by γ-secretase, impacts mitochondrial physiology via its ability of positively controlling PINK1 transcription. In addition, we show that the signaling cascade linking γ-secretase and PINK1 is initiated by parkin transcriptional regulation of presenilins, the main component of γ-secretase catalytic complex. Finally, we also establish that the tumor suppressor p53 can negatively regulate PINK1 transcription in vitro and in vivo suggesting that the misregulated autophagic response associated to brain tumors development may be caused by defective p53-PINK1 interplay

Orientador: Liana Maria Cardoso Verinaud, Roger Chammas<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia<br>Made available in DSpace on 2018-08-13T19:24:03Z (GMT). No. of bitstreams: 1 Ikemori_RafaelYamashita_M.pdf: 4516080 bytes, checksum: 6a332c77058e884f308075bb69496592 (MD5) Previous issue date: 2009<br>Resumo: Gliomas são tumores primários do sistema nervoso central e o glioblastoma multiforme é sua forma clínica mais comum e de pior prognóstico. Na tentativa de entender sua biologia, a linhagem NG97 foi estabelecida, demonstrando características de glioblastoma com atipia nuclear e elevadas taxas de mitose. Recentemente, descobriu-se que esta é uma linhagem híbrida humano-murina derivada da fusão de células de astrocitoma humano e estroma murino que provavelmente ocorreu no processo de estabelecimento desta linhagem, a qual foi posteriormente denominada NG97ht. Esta linhagem apresenta crescimento de massas tumorais quando inoculada em camundongos imunodeficientes, demonstrando características histopatológicas de pseudopaliçada, comuns a glioblastomas. Estas são regiões hipercelulares que margeiam ambientes necróticos e postula-se que sejam células migrantes de ambientes necróticos/hipóxicos. Além disso, estas áreas têm como característica a expressão de moléculas relacionadas à adaptação a hipóxia, como o fator induzido por hipóxia (HIF), atuando na sobrevivência celular pela indução de diferentes genes. É visto que em hipóxia há aumento da produção de galectina-3, a qual está envolvida em diversos processos celulares e que é somente expressa nestas regiões de pseudopaliçada, não sendo detectada em suas áreas tumorais adjacentes. A galectina-3 é uma lectina que possui ligação a beta-galactosídeos e se relaciona com o aumento da mobilidade, adesão, crescimento e progressão tumoral. Além disso, estudos indicam que em alguns tipos tumorais, a metilação do promotor de galectina-3 é responsável pela modulação de sua expressão. Nossos resultados apresentados neste trabalho demonstraram que a hipóxia é capaz de modular positivamente a expressão de galectina-3, tanto em câmara de hipóxia quanto em cloreto cobaltoso, composto químico capaz de mimetizar a hipóxia, apresentando aumento de expressão de galectina-3 em meio completo ou privado de soro fetal bovino, mimetizando ambientes necróticos com pouco oxigênio e nutrientes. Além disso, foi demonstrado que a regulação da expressão gênica de galectina-3 in vitro e in vivo não é realizada pela metilação de seu promotor. Ensaios utilizando a técnica de interferência por RNA demonstraram que o knockdown de galectina-3, em situação in vitro com privação de oxigênio e nutrientes, induziu aumento das taxas de morte celular. Estes dados podem indicar também que a galectina-3 protege as células tumorais dentro de ambientes necróticos em glioblastomas, criando as áreas de pseudopaliçada. Em conclusão, estes experimentos demonstram as propriedades da galectina-3 de proteção contra a morte em privação de oxigênio e nutrientes, comuns dentro de tumores, destacando sua importância como alvo para agentes anti-neoplásicos.<br>Abstract: Gliomas are primary Central Nervous System tumors. Among them, glioblastomas are the most common clinical forms and have the worst prognosis. In an attempt to understand glioma biology, the NG97 cell line was established. This cell line presents glioblastoma's characteristics, showing nuclear atipia and high growth rate. Recently, it was discovered that this is a human-murine hybrid cell line derived from the fusion between human astrocytoma and murine stroma cells that likely occurred in the process of cell line establishment. The cell line was therefore renamed NG97ht. This cell line grows as tumors in immunodeficient mice displaying histopathological characteristics of pseudopalisades commonly seen in glioblastomas. These areas are comprised by hypercellular regions in the edge of necrotic environments and are possibly constituted by actively migrating cells out of necrotic/hypoxic environments. Besides, these pseudopalisades show the expression of molecules related to adaptation to oxygen deprivation, like Hypoxia Inducible Factor (HIF), which is involved in cell survival through the induction of many genes. Also, it has been shown that under hypoxia, galectin-3 production is stimulated, a protein involved in diverse cellular processes and that is only present in these pseudopalisades in glioblastomas, not being detected in its adjacent areas. Galectin-3 is a lectin that binds to beta-galactosides and is related to increased motility, adhesion, tumor growth and progression. Also, studies describe that galectin-3 expression is related to its promoter methylation degree in some tumor types. Our results presented here demonstrated that assays performed in hypoxic chamber and in a chemical condition mimicking hypoxia (incubation with cobaltous chloride) showed galectin-3 induction in either complete medium or deprived of fetal bovine serum, mimicking tumor's necrotic areas deprived of oxygen and nutrients. Besides, it was demonstrated that galectin-3 modulation in vitro and in vivo is not due to promoter methylation. Tests related to galectin-3 knockdown in oxygen and nutrient deprivation demonstrated that this protein has a key role in protection against cell death. It is possible that these results may indicate that galectin-3 can also protect tumor cells inside glioblastoma's necrotic areas, acting as a survival factor in disadvantageous environments with low concentrations of oxygen and nutrients. In conclusion, these experiments demonstrate galectin-3 properties related to protection against cell death in environments deprived of oxygen and nutrients, commonly found inside tumors, highlighting its importance as a target to antineoplastic agents.<br>Mestrado<br>Imunologia<br>Mestre em Genética e Biologia Molecular

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)<br>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<br>Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil<br>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.<br>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.

BACKGROUND Few high-quality observational studies have been conducted to examine clinically relevant features of emotional distress and Major Depressive Disorder (MDD) in adults with primary cerebral glioma. Our knowledge of these important complications of glioma is currently poor. AIMS This thesis aims to answer a series of relevant clinical questions. I have studied: [1] the frequency, independent clinical associations and course of general emotional distress measured using the NCCN Distress Thermometer (DT); [2] the utility of three depression screening tools for identifying MDD; [3] the frequency, independent clinical associations and course of MDD in glioma; [4] current patterns of practice, and the apparent tolerability of antidepressant treatment of depression in glioma; and [5] barriers to the effective management of MDD in glioma. METHODS I conducted a prospective, twin-centre, observational cohort study. Adults with a new histological diagnosis of primary supratentorial glioma were enrolled and interviewed three times: shortly after starting radiotherapy (T1), three months later (T2) and six months later (T3). At each time point participants completed the DT, the Hospital Anxiety and Depression Scale (Depression subscale, HAD-D), the Patient Health Questionnaire-9 (PHQ-9) and the Structured Clinical Interview for DSMIV MDD (SCID). Barriers to depression management were studied using questionnaires completed by the patient and their named GP. RESULTS During a two-year recruitment period, 223 patients were eligible and 155 provided useable data (57.4% male, mean age = 54.2 years, 85.8% high-grade glioma, 78.1% radical radiotherapy, 55.5% chemotherapy). [1] High distress (DT score ≥ 4/10) was consistently a frequent complication, occurring in between 36.4% ± 7.6% of patients at T1 to 33.7% ± 10.2% at T3. In a logistic regression analysis, high distress at T1 was independently associated with MDD, functional impairment and younger age (χ2 for model = 39.882, p < 0.001, R Square = 0.312). Patients who reported high distress at T1 (median DT score = 8; IQR 7 - 9) remained highly distressed on follow-up (T2 median score = 8, IQR 6 - 8; T3 median score = 7, IQR 5 - 8). [2] As screening tools, the HAD-D and PHQ-9 showed good internal consistency (α = 0.769 - 0.862 at any time point). The HAD-D displayed the best operating characteristics on ROC curve analysis. At a threshold of 7+, sensitivity = 0.933, specificity = 0.907 and Positive Predictive Value (PPV) = 0.56. A threshold of 8+ displayed similar PPV, however. [3] The cross-sectional prevalence of MDD was 13.5% ± 5.4% at T1, 14.8% ± 6.7% at T2 and 6.8% ± 5.8% at T3. Inter-rater diagnostic agreement was good (κ = 0.81, 95% CI 0.60 – 1.00). MDD was independently associated with a past history of depression (OR = 3.8, 95%CI 1.5 - 9.8), and with current functional impairment (OR = 3.6, 95%CI 1.4 - 9.4). MDD persisted for at least three months in 9/17 patients who could be followed up. [4] The frequency of antidepressant prescription was 8.4% ± 4.4% at T1, 7.4% ± 4.9% at T2 and 12.6% ± 6.9% at T3. Citalopram was the most frequent antidepressant choice. Antidepressant tolerability appeared to be good among patients who could be followed up. [5] Barriers to the management of depression included 78.4% of GPs regarding major depression as a normal reaction to having glioma, and 39.2% expressing a belief that major depression did not always require treatment. In addition, most patients expressed a degree of resistance to any kind of future depression treatment. DISCUSSION This is the largest cohort study of depression in consecutively presenting adults with glioma, and the first to utilise criterion standard structured interview diagnoses in a longitudinal design. There is a degree of theoretical uncertainty about the nosological validity of MDD in glioma, although the clinical relevance of this uncertainty can be debated. Methodological limitations to the presented study include an absence of alternative potential psychiatric diagnoses to MDD, the likelihood of selection bias in recruitment, and considerable attrition. Due to these and other limitations, findings from this study are tentative and should ideally be replicated. Clinicians should have a high index of suspicion for identifying low mood in glioma patients, particularly those with functional impairment or previous depressive episodes. The HAD-D (suggested threshold 8+) can reasonably be used to screen for depression, if desired. Caution is required when prescribing antidepressants. Clinicians should be educated about the frequency and consequences of MDD in glioma. Researchers interested in psychological neuro-oncology could convene a meeting to guide future projects, particularly since multi-centre studies may be necessary to recruit sufficient sample sizes in future.

Os gliomas estão entre os mais freqüentes e fatais tumores do sistema nervoso central. Apesar dos grandes avanços da Medicina nas áreas diagnósticas e terapêuticas, o tratamento desses tumores ainda é, na grande maioria dos casos, paliativo, sendo a extirpação cirúrgica do tumor o único recurso com potencial curativo e, ainda assim, apenas para os gliomas de baixo grau. Neste trabalho, foram analisados os perfis de expressão gênica diferencial entre linhagens de glioma de rato com diferentes graus de tumorigenicidade (linhagens ST1 e P7), visando identificar genes com potencial de aplicação na doença humana como marcadores moleculares e/ou novos alvos terapêuticos. Numa primeira abordagem, foi realizado um rastreamento de genes potencialmente diferencialmente expressos entre as linhagens ST1 e P7 de glioma de rato através da hibridização de macroarrays de cDNA comerciais. Um conjunto de três membranas comerciais foi hibridizado com alvos radioativos gerados a partir de mRNA obtido destas duas linhagens, totalizando aproximadamente 3.000 genes. Nestes experimentos, foram identificados aproximadamente 200 genes como sendo possivelmente diferencialmente expressos entre as linhagens ST1 e P7. Os níveis de expressão relativa de cerca de 40 destes genes foram analisados por Northern blot, sendo que seis deles foram confirmados como sendo mais expressos na linhagem ST1 enquanto que 4 foram confirmados como sendo mais expressos na linhagem P7. Num segundo momento, foi realizado um rastreamento de expressão gênica em larga escala, através do uso de microarrays de DNA contendo sondas que permitem a análise de expressão de aproximadamente 24.000 transcritos de rato. Esta análise levou à identificação de uma lista contendo aproximadamente 1.300 genes candidatos a diferencialmente expressos, que apresentaram razões de expressão relativa entre 2 e >3.000. Desta lista, -700 genes foram identificados como provavelmente mais expressos na linhagem ST1, e cerca de 500 genes com expressão maior na linhagem P7. A etapa de subseqüente de validação da expressão diferencial foi realizada através de PCR quantitativo em tempo real (Q-PCR). A expressão de 49 genes foi quantificada em quatro preparações independentes de RNA originárias das linhagens ST1 e P7, sendo que 27 destes genes foram identificados como possivelmente diferencialmente expressos através dos microarrays, 10 dos quais haviam sido previamente confirmados por Northern Blot e 12 genes diversos. Destes genes, 21 foram confirmados por Q-PCR como sendo diferencialmente expressos entre as linhagens ST1 e P7, além daqueles 10 genes cuja expressão diferencial havia sido anteriormente confirmada por Northern. Ao todo, oito genes foram confirmados como sendo mais expressos na linhagem ST1, e 23 genes o foram como sendo mais expressos na linhagem P7. Vários dos genes confirmados como sendo diferencialmente expressos na linhagem ST1 estão, direta ou indiretamente, relacionados a parada de crescimento e supressão de fenótipo tumoral. Em contrapartida, diversos dos genes confirmados como sendo diferencialmente expressos na linhagem P7 codificam para receptores de fatores de crescimento peptídicos e seus respectivos ligantes. Em particular, foi detectado maior expressão, na linhagem P7, de receptores e Iigantes relacionados ao fatores de crescimento derivados de plaquetas (PDGFs), fatores de crescimento para fibroblastos (FGFs) e, também, do fatores de crescimento da família de pleiotrofina/midquina (PTN/MDK), e seus respectivos receptores. Sabidamente, vários destes genes estão envolvidos na neoangiogênese e na fechamento de alças autócrinas/parácrinas de estímulo da proliferação tumoral, em particular de gliomas humanos. Além disso, estes achados foram coerentes com o maior potencial tumorigênico (-2,5x) apresentado pela linhagem P7, quando injetadas s.c. no dorso de animais imunocomprometidos (p<O,01). As linhagens ST1 e P7 foram originalmente isoladas como sendo sublinhagens derivadas da linhagem C6, cuja origem é donal. Assim, uma possível interpretação para as diferenças marcantes dos perfis de expressão detectadas entre as linhagens ST1 e P7, é de que estas diferenças sejam, mais provavelmente, a conseqüência de algumas poucas alterações moleculares em genes-chave, e não de alterações extensas do genoma celular, uma vez que as duas linhagens têm, a priorí, o mesmo \"background\" genético. Assim, um número limitado de aberrações genéticas adicionais, particulares de cada linhagem, seria suficiente para uma modificação substancial dos perfis de expressão gênica, se os genes alterados forem capazes de modular a expressão de vários outros genes. Na busca de indícios que fortalecessem esta hipótese, foi quantificado, em seis linhagens humanas de glioma, a expressão dos mesmos genes cuja expressão foi originalmente quantificada nas linhagens ST1 e P7. Em seguida, foram realizados testes de correlação em pares (Teste de Pearson) entre os níveis de expressão relativos destes genes para as seis linhagens, buscando identificar conjuntos de genes que apresentassem expressão coordenada, que, por sua vez, sugeririam a existência de possíveis relações regulatórias entre os mesmos. Foi possível observar expressão significativamente correlacionada de seis genes (CHD7, FGF1, PDGFRA, PTN, PTPRZ1 e SCG2), sugerindo uma possível co-regulação recíproca entre diferentes vias de fatores de crescimento e/ou um novo fator remodelador de cromatina (CHD7). Pararelamente, o gene CHD7 foi, por sua vez, identificado como um novo fator remodelador de cromatina putativo, cujo cDNA completo pode ser amplificado, com êxito, através de RT-PCR longo. A expressão destes mesmos seis genes foi quantificada, por Q-PCR, em 64 amostras clínicas de glioma e cérebro humano não-tumoral. Com exceção do gene SCG2, os demais cinco (CHD7, FGF1, PDGFRA, PTN e PTPRZ1) são significativamente mais expressos em todos os graus de glioma, quando comparados com cérebro humano não-tumoral (p<0,05). Foi possível verificar, inclusive, que os níveis de expressão de vários destes genes estão altamente correlacionados nessas amostras. Em particular, observou-se correlação com alta significância entre os genes CHD7xPDGFRA (p=4,7x10-17), FGF1 xPTN (p=1, 1x10-19), do receptor PTPRZ1 contra todos os demais genes (100-6<p<10-9) e, especificamente, contra seu Iigante PTN (p=1,6x10-14). Como os loci dos genes PTN e PTPRZ1 se encontram relativamente próximos (-15 Mb) numa região do cromossomo 7 humano, a qual frequentemente se encontra amplificada em gliomas humanos (7q), é possível que estes dois genes, codificantes para um fator de crescimento e seu respectivo receptor, façam parte de um novo amplicon em gliomas humanos. Além disso, vários genes com maior expressão na linhagem P7 (ALK, FGFR1, RNF130 e ROS01) estão, também, significativamente mais expressos em certos graus de gliomas humanos, quando comparados com tecido cerebral humano não-tumoral. De um modo geral, estes dados indicam que foi possível obter, a partir de linhagens de glioma de rato, incursões aplicáveis para um entendimento mais amplo da biologia que envolve a patogênese da doença humana. Um destes dados extrapoláveis entre diferentes modelos inter-espécies é que a co-expressão de múltiplas vias autócrinas de crescimento parece ser um achado comum tanto em modelos experimentais de glioma em rato quanto em linhagens e amostras clínicas de gliomas humanos, e que estas vias estão, potencialmente, interconectadas ao nível transcricional. Além disso, foi possível verificar que um novo gene codificante para um fator de remodelamento de cromatina (CHD7), apresenta níveis de expressão relativos muito aumentados em amostras clínicas de glioma, quando comparado com cérebro humano não-tumoral. Analisados em conjunto, os resultados obtidos aqui têm o potencial para conduzir ao desenvolvimento racional de novas estratégias terapêuticas e diagnósticas/prognósticas para gliomas humanos. A análise do papel funcional destes genes em glioma, e das vias moduladas por seus produtos, se constituirá de um passo fundamental para o estabelecimento destes genes como potenciais novos alvos terapêuticos e/ou marcadores moleculares.<br>Gliomas are among the most frequent and fatal tumors of the central nervous system. Despite the recent and important advances in the diagnostic and therapeutic fields, treatment of these tumors still is, in the vast majority of the cases, palliative. Surgical ressection of the tumor remains as the sole potentially curative resource, but only for the low grade gliomas. In the present work, differential gene expression profiles were analyzed between rat glioma cell lines differing in tumorigenic potential (ST1 and P7 cell lines), aimed at identifying genes with potential to become novel molecular markers and therapeutic targets for the human disease. As a first approach, a commercial macroarray-based screening was undertaken in order to identify differentially expressed genes between ST1 and P7 rat glioma cell lines. Three different sets of commercial membranes comprising 3,000 genes were hybridized against radiolabeled targets that were synthesized from mRNA extracted from both cell lines. As a result, near 2,000 genes were identified as being possibly differentially expressed between ST1 and P7 celllines. The relative expression levels of 40 genes from this list were analyzed by Northern blot, and six genes were successfully confirmed as being expressed at higher levels in the ST1 cell line, whereas four genes confirmed heightened expression in P7 cells. As a second approach, a large-scale, Affymetrix microarray-based screening was performed, which allowed measuring the relative expression levels of about 24,000 rat transcripts. This analysis led to the identification of 1,300 candidate differentially expressed genes, for which the differential expression ratios ranged from 2 up to >3,000. From these, -700 genes displayed preferential expression in the ST1 cell line, while around 500 genes were more expressed in P7 cells. The following step, namely, validation of the differential expression, was achieved through Real-Time PCR (Q-PCR). Expression levels of 49 genes were measured in four independent RNA preparations from ST1 and P7 cell lines. About 27 of these genes were identified as putative differentially expressed , 10 of which had previously been confirmed by Northern blot as differentially expressed and 12 additional genes were also included. From this list, 21 genes were confirmed by Q-PCR as being diferentially expressed between the ST1 and P7 cell lines, in addition to those 10 whose differential expression was confirmed by Northern Slol. In total, eight genes were confirmed as being differentially expressed in the ST1 cell line, and 23 genes were confirmed as being expressed at higher leveis in the P7 cells. Many of the genes confirmed as being differentially expressed genes in the ST1 cell line are, directly or indirectly, related to growth arrest and suppression of the malignant phenotype. On the other hand, several of the genes displaying elevated expression in the P7 cell line code for growth factor ligands and receptors related to the PDGFs (platelet-derived growth factors), FGFs (fibroblast growth factors) and PTN/MDK (pleiotrophin/midkine) growth factor families. Many of these genes are already known as being related to neoangiogenesis and to the establishment of autocrine/paracrine loops in human gliomas. In addition, these findings are in keeping with the significantly higher (p<O.01) tumorigenic potential displayed by the P7 cellline (-2,5x), when both cell lines are injected s.c. in the dorsal region of immunodeficient nude mice. Moreover, ST1 and P7 celllines were originally isolated as clonal celllines from the C6 cell line which, in turn, is also acionai cell line. Therefore, a possible interpretation for the remarkably different gene expression profiles between ST1 and P7 cell lines is that those differences are, most Iikely, a consequence of a few molecular defects in key/master genes, rather than extensive aberrations of the cellular genome, given that those celllines have, a priori, similar genetic backgrounds. Thus, a limited number of genetic aberrations, characterisitic of each cell line, would be sufficient for a substantial modification of the gene expression profiles, provided that the altered genes are able to modulate the expression of each other. In an attempt to investigate theis point, the relative expression levels of the same genes were analyzed by Q-PCR, and pairwise correlation tests (Pearson correlation tests) were performed using expression data from the six human glioma cell lines, aimed at identifying gene sets that displayed coordinate expression whichwould suggest the existence of putative regulatory loops among them. It was possible to identify a c1uster of six genes (CHD7, FGF1, PDGFRA, PTN, PTPRZ1, SCG2) that displays coregulated expression, thus suggesting a possible reciprocal co-regulation among distinct growth factor pathways and a putative chromatin remodeling factor (CHD7). Furthermore, the CHD7 gene was identified as a novel, putative chromatin remodeling factor, and its full-Iength cDNA could be successfully amplified by means of long RTPCR. The expression levels of the same genes was quantified, by Q-PCR, in 64 clinicai samples, comprising gliomas and non-tumoral human brain tissue samples. Except for the SCG2 gene, the remaining five genes (CHD7, FGF1, PDGFRA, PTN and PTPRZ1) were expressed at significantly higher levels in glioma samples of all grades, when compared to non-tumoral human brain tissue samples (p<O.05). Likewise, we were able to verify that expression levels for many of these genes are strictly correlated in those samples. A particularly high levei of significance was found for the correlation of expression levels between CHD7xPDGFRA (p=4.7x10-17) and also for the FGF1xPTN gene pair (p=1.1x10-19). A strikingly high level of significance (p=1.6x10-14) was also found for the expression levels between the PTPRZ1 receptor and its PTN ligand. Given that the PTN and PTPRZ1 loei are relatively close to each other (-15 Mb) on the long arm of human chromosome 7, which, in turn, is frequently amplified in human gliomas, the genomic region including these two genes may well constitute a novel amplicon in human gliomas. In addition, several other genes with higher expression in the P7 cell line (ALK, FGFR1, RNF130 and ROB01) are also expressed at significantly higher levels in certain glioma grades, when compared to non-tumoral human brain tissue samples. Overall, these data indicate that starting from the rat glioma cell lines model, it was possible to obtain, insights applicable to a better understanding of the biology underlying the pathogenesis of human gliomas. Thus, co-expression of multiple autocrine loops seems to be a commonplace in the rat experimental glioma models as well as in the human glioma cell lines and in clinicai samples, where these pathways are potentially interconnected at the transcriptional leveI. Furthermore, we were able to detect increased mRNA expression levels of a novel putative chromatin remodelling factor (CHD7) in human glioma tissue samples, when compared to non-tumoral human brain tissue samples. Taken together, the results obtained in this work may potentially lead to the rational development of novel therapeutic, diagnostic and prognostic strategies for human gliomas. Functional analysis of these genes in glioma, and the pathways modulated by their products, will be a fundamental step for the establishment of these genes as potentially novel therapeutic targets and/or molecular markers.

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&#8594;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.<br>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.

Comme décrit dans la partie But du Travail, les tumeurs gliales sont particulièrement agressives d’un point de vue clinique. Le glioblastome, qui correspond au grade de malignité le plus élevé des gliomes, est associé à un pronostic très sombre car aucun patient atteint de ce cancer n’a pu être guéri à ce jour. Ces tumeurs envahissent de manière diffuse (par essaimage de cellules tumorales isolées) le parenchyme cérébral, ce qui empêche une résection chirurgicale complète de la tumeur. De plus, les cellules tumorales gliales d’origine astrocytaire sont souvent résistantes à l’apoptose et donc aux thérapies adjuvantes telles que la chimiothérapie et la radiothérapie. La galectine-1 est une petite protéine intervenant directement dans les processus migratoires des cellules gliales tumorales. Nous avons donc poursuivi la caractérisation des rôles biologiques que pourrait exercer la galectine-1 au sein des gliomes.<p>Nous avons tout d’abord montré que la galectine-1 est impliquée dans la chimiorésistance des gliomes. En effet, nous avons démontré que la diminution du taux d’expression de la galectine-1, au moyen d’un siRNA au sein d’un modèle de gliome expérimental, permet d’augmenter le bénéfice thérapeutique du témozolomide in vivo sans toutefois induire d’apoptose, d’autophagie ou de perméabilisation de la membrane des lysosomes. Nous avons également montré que la diminution du taux d’expression de la galectine-1 au sein de ce modèle de gliome expérimental affecte les processus d’angiogenèse in vivo et de « vasculogenic mimicry » in vitro. Nous avons identifié la protéine ORP150 comme l’une des principales cibles de l’effet pro-angiogénique de la galectine-1, sachant que la protéine ORP150 contrôle la maturation du facteur VEGF. Nous avons ensuite montré que le rôle de la galectine-1 dans la chimiorésistance des gliomes et dans l’angiogenèse est directement lié à l’implication de la galectine-1 dans le processus de réponse au stress du réticulum endoplasmique. Via ce processus, la galectine-1 modulerait l’expression d’un certain nombre de gènes tels que ATF3, DUSP5 et HERP, qui sont impliqués dans la chimiorésistance et des gènes tels que ORP150 et MDG1 qui sont impliqués dans l’angiogenèse. <p>Enfin, nous avons également montré que la galectine-1 régule l’expression du gène BEX2 et que celui-ci joue un rôle important dans la biologie des gliomes, notamment dans les processus d’angiogenèse et de migration cellulaire. <p>En conclusion, notre travail suggère que l’étiquette « biomarqueur » pourrait être attribuée à la galectine-1 pour qualifier l’agressivité biologique des gliomes malins et que la galectine-1 pourrait représenter une nouvelle cible thérapeutique dans le combat contre les gliomes malins en général, et le glioblastome en particulier.<p><br>Doctorat en Sciences biomédicales et pharmaceutiques<br>info:eu-repo/semantics/nonPublished

Glioblastoma multiforme (GBM) is the most common form of primary brain cancer and the current prognosis for patients is poor. New therapies are required that target the invasive cells that are characteristic of GBM. GBM is infiltrated by immune cells but, as with other cancers, immune evasion pathways minimise productive anti-tumour immunity. Natural killer (NK) cells are able to recognise and kill tumour cells and are being developed for the immunotherapy of other cancers. The aim of this work was to analyse the interaction between human NK cells and GBM cells in vivo and in vitro, as a prerequisite to future NK cell based immunotherapy of GBM. Analysis of the cell surface phenotype for GBM infiltrating NK and T cells revealed that the tumour microenvironment exerts localised immune evasion mechanisms which downregulate activation receptors and upregulate inhibitory receptors. The interaction of NK cells with patient-derived GBM stem cells, which are thought to be responsible for recurrent disease, was investigated in vitro. A high-throughput, multiplex flow cytometry-based screen of tumour cells revealed the expression of a number of cell surface molecules that regulate NK cell activation. Furthermore, GBM cells were more susceptible to NK cell lysis in vitro compared to a non-cancerous neural progenitor cell line, revealing specificity in the NK cell response. Furthermore, this screen identified potential mechanisms by which GBM might evade immune surveillance in vivo. Targeting these pathways and restoring functional immune surveillance provides a potential route for future immunotherapy of this disease. However, GBM patients often experience cerebral oedema and are treated with immunosuppressive corticosteroids, such as dexamethasone; this induces a similar immunosuppressed phenotype to that observed with the GBM infiltrating NK cells, and inhibits their lytic function. Gene expression profiling identified the transcription factor c-Myc as a key regulator of NK cell activation and as a hub for the immunosuppressive action of steroids and the immunosuppressive cytokine TGF-β. The demonstration that therapeutic steroids target the same pathway as TGF-β and induce immunosuppression has important implications for the use of steroids in patients undergoing immunotherapy.

Les gliomes malins sont les tumeurs les plus fréquentes du système nerveux central. Les glioblastomes représentant plus de 50% des gliomes, constituent la forme la plus agressive et sont particulièrement résistants à la radiochimiothérapie. Au sein de ces tumeurs réside une sous-population de cellules souches tumorales (CSG) qui pourrait être responsable de leurs initiation, progression et résistance aux traitements. Ces processus sont gouvernés par des voies de signalisation, pour la plupart activées de manière constitutive et dont l’étude est nécessaire afin de mieux comprendre les mécanismes impliqués dans la gliomagenèse. L’objectif de ces travaux de thèse consistait en l’exploration des voies de signalisation STAT3 et Hippo dans les gliomes dans le but d’identifier de nouveaux marqueurs pronostiques et de nouvelles cibles thérapeutiques potentielles. La première partie de ces travaux a montré que la phosphorylation S727 de STAT3 jouait un rôle important dans la radioresistance des CSG et que son inhibition pharmacologique induisait leur radiosensibilisation. Dans un second temps, ces travaux ont montré que deux effecteurs de la signalisation Hippo, YAP1 et TEAD3, sont associés à un mauvais pronostic et qu’ils seraient impliqués dans la prolifération cellulaire et le phénotype des CSG notamment par inhibition de la signature proneurale. Ainsi, ces travaux visent à proposer de nouvelles pistes thérapeutiques, d’une part l’inhibition de la pS727-STAT3 afin de potentialiser les effets de la radiothérapie et d’autre part, les effecteurs de la signalisation Hippo comme biomarqueurs pronostiques et potentielles cibles thérapeutiques<br>Malignant gliomas are the most common tumors of central nervous system. Glioblastomas represent more than 50% of all glioma and constitute the most aggressive form of the tumor which is particularly resistant to radiotherapy. The presence of the subpopulation of glioblastoma stem cells (GSC) could be involved in tumor initiation, progression and therapeutic resistance. Hence, these processes are governed by signaling pathways which are mostly constitutively activated and their study is necessary for a better understanding of gliomagenesis. The aim of this PhD thesis was to assess STAT3 and Hippo signaling pathways in glioma to identify new prognostic markers and potential therapeutic targets. The first part on this work showed that pS727 phosphorylation of STAT3 could be involved in radioresistance and its inhibition induced GCS radiosensitization. Additionally, this work showed that YAP1 and TEAD3, two effectors of Hippo signaling, are associated with poor patient survival and could be involved in GSC proliferation and phenotype maintenance by inhibiting proneural gene signature. Thereby, this work aims to offer new therapeutic avenues, on the one hand the inhibition of pS727-STAT3 for radiotherapy potentiation and on the other hand the effectors of Hippo signaling as prognostic biomarkers and potential therapeutic targets

Les gliomes sont les principales tumeurs primitives du cerveau affectant environ 4000 nouveaux patients par an en France. La moitié des gliomes est détectée au stade avancé de glioblastome (grade IV) tandis que 15% des tumeurs sont diagnostiquées au stade II de gliomes diffus dit de bas grade. Ces tumeurs affectent des patients jeunes et présentent des mutations caractéristiques, notamment une mutation pour l’enzyme IDH1 communément retrouvée dans les glioblastomes secondaires. Ces tumeurs de bas grade sont traitées par une chirurgie, idéalement en condition éveillée mais du fait de leur nature diffuse, la partie résiduelle progressera inexorablement vers un stade III ou IV avec une survie globale entre 5 ans et 15 ans après diagnostique. La progression tumorale est hautement variable et non prédictible d’un patient à l’autre. Des foyers de progression tumorale chez 20% des patients atteints de gliome diffus de bas grade ont été identifiés. Ces foyers montrent une densité cellulaire plus élevée ainsi qu’un Ki67 augmenté. Mon travail de thèse aura consisté à étudier les modifications cellulaires et moléculaires associées à ces foyers de progression tumorale. À partir du profil ARN des foyers et des territoires adjacents, j’ai pu mettre en évidence par des techniques haut-débit la baisse d’expression significative de gènes dans les foyers notamment de AGXT2L1/ETNPPL, carboxypeptidase E, EDNRB, SFRP2. J’ai émis l’hypothèse que SFRP2 et ETNPLL pourraient s’opposer à la prolifération cellulaire et que leur diminution dans les foyers ouvrirait la voie à la transformation tumorale. Une corrélation inverse entre la quantité d’ETNPPL enzyme et la survie de patients atteints d’hépatocarcinomes a été publiée. En limitant la quantité de précurseurs de phospholipides dans la cellule, ETNPPL pourrait agir comme un frein en s’opposant à la prolifération et de fait, sa diminution dans les foyers de transformation des gliomes pourrait lever cette inhibition. Mes travaux auront été innovants tant dans leur approche comparative des différents compartiments tumoraux pour chaque patient étudié et auront révélés ETNPPL comme corrélé à la gliomagenèse et potentielle cible thérapeutique<br>Gliomas are the main primary brain tumours affecting around 4000 new patients in France each year. Half of gliomas are detected in the advanced stage of glioblastoma (grade IV) while 15% of tumours are diagnosed in stage II (diffuse low-grade gliomas-DLGG). These tumors affect young patients and bear characteristic mutations, including a mutation for the enzyme IDH1 commonly found in secondary glioblastomas. These low-grade tumours are treated by surgery, ideally in awake condition but due to their diffuse nature, the residual part will progress inexorably to stage III or IV with overall survival between 5 and 15 years after diagnosis. Tumor progression is highly variable and unpredictable from one patient to another. Foci of tumor progression have been identified in 20% of patients with DLGG. These foci show a higher cell density and an increased Ki67. My thesis work consisted in studying the cellular and molecular changes associated with tumor progression. From the RNA profile of the foci and adjacent territories, I was able to highlight through high-throughput techniques significant decrease in gene expression in the foci, particularly of AGXT2L1/ETNPPL, carboxypeptidase E, EDNRB, SFRP2. I hypothesized that SFRP2 and ETNPLL could oppose cell proliferation and that their decrease would pave the way for tumor transformation. An inverse correlation between the amount of ETNPPL and the survival of patients with hepatocarcinoma has been published. By limiting the amount of phospholipid precursors in the cell, ETNPPL could act as a brake against proliferation and indeed, its decrease in glioma transformation foci could remove this inhibition. My PhD work will have been innovative in the comparative approach of the different tumors’ compartments for each patient studied and will have revealed ETNPPL as correlated to gliomagenesis and as potential therapeutic target

Les dernières années ont laissé place à de nombreuses avancées médicales, montrant de plus en plus d’intérêt à l’amélioration des modalités de soin et du cadre de vie des patients, en apportant plus d’efficacité et moins de risque. Dans ce contexte, trois études de recherche clinique ont été menées avec le potentiel microphonique cochléaire (PMC) pour évaluer les capacités de cette réponse cochléaire dans des applications médicales originales et proposer des outils de surveillance de grand intérêt pour la prise en charge des patients. La première étude s’est intéressée à la survenue de surdités suite aux exérèses de neurinome de l’acoustique, notamment les pertes auditives d’origine vasculaire. L’amplitude du PMC a détecté tous les événements chirurgicaux responsables de l’altération de la vascularisation cochléaire et a ainsi fourni une meilleure compréhension de l’origine des pertes auditives lors des chirurgies dans l’angle pontocérébelleux. Les deux autres études ont porté sur la fiabilité d’un monitorage non invasif de la pression intracrânienne (PIC) par la phase du PMC sur une longue période de suivi, chez des patients pour lesquels il est attendu une variation de la PIC. Le PMC a montré une bonne capacité à détecter les variations de la PIC au cours du temps, aussi bien lors d’une installation lente d’une PIC élevée (progression de gliome malin) que lors de l’apparition transitoire et aiguë d’une PIC augmentée (hypertension intracrânienne, hydrocéphalie).Plusieurs observations parfois inattendues ont été obtenues avec le PMC et ouvrent de nouvelles pistes d’intérêt et de réflexion sur les mécanismes de fonctionnement de la PIC ou de la cochlée. Parmi elles : une répercussion épisodique de l’embolisation des anévrysmes cérébraux sur la PIC, l’aptitude de la phase du PMC à prédire la survenue prochaine d’une crise de Menière et la possible prédiction préopératoire d’une fragilité cochléaire au fraisage du conduit auditif interne quand le signal IRM des fluides cochléaires du côté affecté (par le neurinome de l’acoustique) est hypointense<br>The last years, healthcare and living conditions of patients have been of growing interest in medical advances with the goal to bring more efficiency and less risk. In this context, three clinical researches have been conducted with cochlear microphonic potential (CMP) to assess the abilities of this cochlear response in unusual medical applications and propose monitoring tools of major interests for patients’ management.The first study is interested in the occurrence of deafness following vestibular schwannoma resection, in particular hearing loss due to vascular origin. The CMP amplitude detected all the surgical events responsible for the alteration of the cochlear vascularization and thus provided a better understanding of the origin of the hearing losses during surgeries in the cerebellopontine angle.The other two studies examined the reliability of non-invasive intracranial pressure (ICP) monitoring, by the CMP phase, over a long period to follow patients for whom a change in ICP is expected. The CMP has shown good ability to detect changes in ICP over time, both in a slow installation of a high ICP (progression of malignant glioma) and in the transient and acute onset of increased ICP (intracranial hypertension, hydrocephalus).Several observations, sometimes unexpected, have been obtained with the CMP and open up new track of interest and reflections on the mechanisms of ICP and cochlea functioning. These discoveries included: episodic repercussion of cerebral aneurysm embolization on ICP, ability of CMP phase to predict the next occurrence of a Meniere crisis, and preoperative prediction of cochlear fragility during the drilling of the internal auditory meatus when the MRI signal of the cochlear fluids on the affected side (vestibular schwannoma) is hypointense

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.

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 &mu;g 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.<br>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 &mu;g 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 sont les tumeurs cérébrales de l’adulte les plus fréquentes, dont l’étiologie reste encore largement inconnue. Plusieurs études épidémiologiques ont montré l’existence d’une corrélation entre maladies neurodégénératives et cancers cérébraux. Nous avons émis l’hypothèse qu’il existait des dénominateurs moléculaires communs entre ces pathologies. Je me suis particulièrement intéressé au rôle de la parkine (PK), une ubiquitine ligase responsable des formes génétiques de la maladie de Parkinson (MP). En effet, plusieurs arguments soutiennent l'implication de la PK dans les gliomes.Des études ont montré que la PK présente une expression altérée dans les cas de cancers du sein et de la prostate. La PK possède également une fonction de facteur de transcription. Elle est capable de se fixer à l’ADN de p53 et inhibe sa transcription. p53 est un suppresseur de tumeur fréquemment inactivé (50% des cancers). Une étude a mis en évidence l'existence de mutations somatiques de la PK spécifiques des cancers cérébraux. Mon projet s'est articulé autour de trois axes. 1- PK et Maladie d’Alzheimer. Elle active la transcription de la préséniline 1 et d’inhiber celle de la préséniline 2. 2- PK et MP. La PK par l’intermédiaire de p53 régule XBP-1, un facteur de transcription notamment activé par le stress du réticulum, qui à son tour régule l’expression de DJ-1. 3- PK et gliomes. Nous avons observé une diminution d’expression de la PK corrélé à l’augmentation d’expression de p53 dans des biopsies de gliomes. Nous avons alors montré que p53 est capable d’activer la synthèse de la PK, effet aboli par des mutations de p53 dans les gliomes<br>Gliomas are the most common form of brain tumor, the etiology of which remains unknown. Several epidemiological studies have shown the existence of a correlation between neurodegenerative diseases and brain tumor. We hypothesis that these two pathology share common molecular denominators. Here I study the role of parkin (PK) an ubiquitin ligase responsible of early onset Parkinson diseases. Several arguments support the involvement of PK in glioma. Studies have shown that PK expression is alterated in many types of cancers. PK is also a transcription factor which can bind to p53 DNA and inhibits its transcription. P53 is a tumor suppressor often find inactivate in cancers (50%). There is evidence of specific somatic mutations found in glioma. My work was organize according to three axes 1- PK and Alzheimer disease: PK activates préséniline 1 expression and inhibits préséniline 2. 2- PK through XBP-1 regulates p53, a transcription factor activated by reticulum stress, which in turn regulates the expression of DJ-1. 3- PK and Glioma: There is a decrease in parkin expression that can be correlated to p53 expression increase in glioma biopsies. I show that p53 is able to activate PK synthesis, a mechanism abolish by p53 mutations in tumors

Glioblastome représente la tumeur la plus courante du cerveau primaire avec une survie de moins de 2 ans. Ces tumeurs sont très infiltrantes et angiogéniques et contiennent une sous population de cellules souches cancéreuses. Nkx2.2 est un homéodomaine facteur de transcription, impliqué dans la formation d'oligodendrocytes au cours du développement. Nkx2.2 joue un rôle central dans la tumorogenèse de Ewing'sarcoma. L'utilisation de la QPCR et de la matrice du tissu de gliome, nous a permis de mettre en évidence la forte expression de Nkx2 dans le glioblastome. Nkx2.2 a également été détecté dans 3 cultures de cellules de gliomes où il est co-exprimé avec des marqueurs de cellules souches tels que CD133 et CD15. Il a été récemment proposé que la surexpression de Nkx2.2 pourrait induire à la différenciation oligodendrocytaire de la cellule du gliome tige-comme et au blocage de la formation des tumeurs dans la xénotransplantation (Cancer Res fév 2011 1; 71 (3): 1135-1145). Pour explorer cette possibilité, nous avons utilisé des rétrovirus pour surexprimer Nkx2.2 dans nos cultures cellulaires. De manière surprenante, nous avons trouvé que Nkx2.2, induit la prolifération des cellules souches du gliome et n'a en conséquent aucun effet de différenciation. Microarray analyses a confirmé que la surexpression de Nkx2.2 n'a en effet aucune influence sur la différenciation des oligodendrocytes. Cette analyse a également révélé que Nkx2.2 était capable d'induire une forte expression de YKL-40 40 dans le surnageant des cellules souches du gliome. YKL-40 est en fait, une glycoprotéine sécrétée et impliquée dans l'inflammation, l'angiogenèse et la prolifération. Elle est souvent associée à un mauvais pronostic dans plusieurs types de cancers. En outre, nous avons effectué une transplantation orthotopique afin d'explorer le rôle de Nkx2.2 dans la gliomagenèse in vivo et avons constaté que Nkx2.2 ne réduit pas l'agressivité du glioblastome.Dans l'autre partie de ma thèse, nous avons utilisé la gamme Taqman à basse densité et la validation des miRNA par la Qpcr afin de chercher ces derniers dans la culture cellulaire du glioblastome humain. Nous avons ensuite étudié le rôle des miARN dans la transcription de 3'UTR de Nkx2.2. Les résultats d'analyse de la mutagénèse dirigée (SDM) et de la double-luciférase ont montré que l'expression de Nkx2.2 est régulée par la diminution de mir-133b ainsi que celle de mir-202<br>Glioblastoma represent the most common primary brain tumor with an overall survival of less than 2 years. These tumors are highly infiltrative and angiogenic and contain a sub population of cancer stem cells. Nkx2.2 is a homeodomain transcription factor which is implicated in the formation of oligodendrocytes during development. Nkx2.2 is central in tumorogenesis of Ewing'sarcoma. Using QPCR and glioma tissue array, we found that Nkx2.2 is highly expressed in glioblastoma. Nkx2.2 was also detected in 3 glioma stem-like cell cultures (neurospheres) where it is co-expressed with stem cell markers such as CD133 and CD15. It was recently proposed that overexpression of Nkx2.2 could induce terminal oligodendrocytic differentiation of glioma stem-like cell and inhibit tumor formation in xenotransplantation (Cancer Res. 2011 Feb 1;71(3):1135-45).To explore this possibility further, we used retroviruses to overexpress Nkx2.2 in our cell cultures. Surprisingly, we found that Nkx2.2, induce glioma stem cell proliferation and had no oligodendrocyte differentiating effect. Microarray analyses confirmed that Nkx2.2 overexpression had no influence in oligodendrocyte differentiation. This analysis further revealed that Nkx2.2 was able to induce a strong expression of YKL40 protein in the supernatant of glioma stem cells and increase YKL-40 promoter activity. YKL-40 is a secreted glycoprotein which is involved in inflammation, angiogenesis and proliferation and which is often associated with a bad prognosis in several cancers. In addition, we performed orthotopic transplantation to explore the role of Nkx2.2 in gliomagenesis in vivo and found that Nkx2.2 did not reduce the aggressiveness of glioblastoma. In the other part of my thesis we used Taqman low-density arrays (TLDA) and individual miRNA QPCR validation to find the microRNA (miRNA) signature in human glioblastoma cell cultures. Then we investigated the role of miRNA in the 3'UTR of Nkx2.2 transcript. Site directed mutagenesis (SDM) and dual-Luciferase reporter assay results showed that the Nkx2.2 expression is downregulated by mir-133b and mir-202

El maneig dels gliomes de baix grau es basa en les característiques clíniques i radiològiques, incloent la classificació pronostica de Pignatti, que classifica als pacients com de baix o d'alt risc de recaiguda . Per determinar si les dades moleculars poden definir d’ una forma mes sensible el pronòstic dels pacient, hem examinat diverses alteracions moleculars en una cohort de 58 pacients. Hem registrat les dades clíniques i radiològiques que permeten obtenir la classificació pronòstica segons els criteris de Pignatti, i hem dut a terme l’estudi de les mutacions de IDH, les mutacions de TP53, la codeleció 1p / 19q, i la metilació del promotor de MGMT. Hem correlacionat les nostres troballes amb la supervivència lliure de progressió (SLP ) i la supervivència global (SG). L'edat mitjana dels pacients va ser de 45 anys; 69% de pacients es van classificar com de baix risc de recaiguda. Es van detectar mutacions de IDH en el 62% de casos, mutacions de TP53 en el 17%, codeletion 1p / 19q en el 46%, i metilació de MGMT en 40% dels pacients. Les anàlisis de supervivència es van realitzar en els 49 pacients que no presentaven captació de contrast a la resonància magnètica basal. En l'anàlisi univariant, les mutacions de IDH, la codeleció de 1p / 19q, i la combinació de les mutacions IDH amb la codeleció 1p / 19q es van associar amb una millor SLP (P = 0,006, P = 0,037 i P = 0,003, respectivament) i SG (P <0,001, P = 0,02 i P <0,001, respectivament). L'anàlisi multivariant va identificar la absència de mutacions de IDH com factor de risc en relació a la progressió ( [HR] = 3,1; P = 0,007) i a la mort (HR = 6,4; P <0.001). Podem concloure d’acord amb els nostres resultats, que l'estat mutacional de IDH es un marcador pronòstic independent a les variables clíniques, radiològiques i altres moleculars i per tant es planteja com a eina decisiva a l'hora de definir el tractament post-quirúrgic dels pacients amb gliomes de baix grau.<br>El manejo de los gliomas de bajo grado se basa en las características clínicas y radiológicas, incluyendo la clasificación pronóstica de Pignatti, que clasifica a los pacientes como de bajo o de alto riesgo de recaída. Para determinar si los datos moleculares pueden definir de una forma más sensible el pronóstico del paciente, hemos examinado varias alteraciones moleculares en una cohorte de 58 pacientes. Hemos registrado los datos clínicos y radiológicos que permiten obtener la clasificación pronóstica según los criterios de Pignatti, y hemos llevado a cabo el estudio de las mutaciones de IDH, las mutaciones de TP53, la codeleción 1p / 19q, y la metilación del promotor de MGMT. Hemos correlacionado nuestros hallazgos con la supervivencia libre de progresión (SLP) y la supervivencia global (SG). La edad media de los pacientes fue de 45 años; 69% de pacientes se clasificaron como de bajo riesgo de recaída. Se detectaron mutaciones de IDH en el 62% de casos, mutaciones de TP53 en el 17%, codeleción 1p / 19q en el 46%, y metilación de MGMT en 40% de los pacientes. Los análisis de supervivencia se realizaron en los 49 pacientes que no presentaban captación de contraste en la resonancia magnética basal. En el análisis univariante, las mutaciones de IDH, la codeleción de 1p / 19q, y la combinación de las mutaciones IDH con la codeleción 1p / 19q se asociaron con una mejor SLP (P = 0,006, P = 0,037 y P = 0,003, respectivamente) y SG (P <0,001, P = 0,02 y P <0,001, respectivamente). El análisis multivariante identificó la ausencia de mutaciones de IDH como factor de riesgo en relación a la progresión ([HR] = 3,1; P = 0,007) y la muerte (HR = 6,4; P <0.001). Podemos concluir de acuerdo con nuestros resultados, que el estado mutacional de IDH es un marcador pronóstico independiente de las variables clínicas, radiológicas y otras moleculares y por tanto se plantea como una herramienta decisiva a la hora de definir el tratamiento post- quirúrgico de los pacientes con gliomas de bajo grado.<br>Management of low-grade gliomas (LGG) is based on clinical and radiologic features, including the Pignatti prognostic scoring system, which classifies patients as low- or high-risk. To determine whether molecular data can offer advantages over these features, we have examined the prognostic impact of several molecular alterations in LGG. In a cohort of 58 patients with LGG, we have retrospectively analyzed clinical and molecular characteristics, including the Pignatti criteria, IDH mutations, TP53 mutations, the 1p/19q deletion, and MGMT methylation, and correlated our findings with progression-free survival (PFS) and overall survival (OS). Mean age of patients was 45 years; 69% were classified as low-risk by the Pignatti system. IDH mutations were detected in 62%, p53 mutations in 17%, the 1p/19q codeletion in 46%, and MGMT methylation in 40% of patients. Survival analyses were performed in the 49 patients without contrast enhancement. In the univariate analysis, IDH mutations, the 1p/19q codeletion, and the combination of IDH mutations with the 1p/19q codeletion were associated with both longer PFS (P = 0.006, P = 0.037, and P = 0.003, respectively) and longer OS (P < 0.001, P = 0.02, and P < 0.001, respectively). The multivariate analysis identified absence of IDH mutations as a factor for greater risk of progression (hazard ratio [HR] = 3.1; P = 0.007) and death (HR = 6.4; P < 0.001). We conclude that IDH mutations may be more effective than the Pignatti score in discriminating low- and high-risk patients with LGG.