O desenvolvimento e invasão de tumores cerebrais primários são diretamente influenciados pela matriz extracelular. Considerando que lisil oxidase (LOX) e os demais membros da família das lisil oxidases (LOXL1, LOXL2, LOXL3 e LOXL4) apresentam complexidade tanto estrutural quanto funcional e estão envolvidos em processos biológicos vitais, como motilidade celular, sinalização celular e regulação gênica, a desregulação da expressão destas proteínas pode levar à gênese e progressão tumoral. O presente trabalho teve como objetivos avaliar os níveis de expressão dos genes que codificam todos os membros da família das lisil oxidases em astrocitomas de diferentes graus de malignidade e correlacionar com a expressão de BMP1 e HIF1A, mutação de IDH1 e tempo de sobrevida total dos pacientes. Adicionalmente, as expressões das proteínas codificadas por estes genes foram também realizadas, além de um estudo funcional in vitro do papel de LOX em astrocitomas. A análise da expressão dos genes foi realizada por PCR quantitativa em tempo real numa série de 153 astrocitomas e 22 amostras de tecido cerebral não neoplásico. A expressão proteica foi conduzida por imuno-histoquímica em amostras de astrocitomas. O silenciamento da expressão de LOX foi realizado em linhagens celulares de glioblastoma humano U87MG e A172 transfectadas com o siRNA para os ensaios funcionais. A expressão de todos os genes (LOX, LOXL1, LOXL2, LOXL3, LOXL4, BMP1 e HIF1A) aumentou com o grau de malignidade dos astrocitomas, com maiores níveis nos casos de glioblastoma. Foi encontrada uma correlação positiva nos valores de expressão dos genes principalmente nos glioblastomas. Somente a expressão de LOXL3 teve um impacto na sobrevida dos casos com GBM. Pacientes com maior expressão apresentaram maior sobrevida em relação aos com menor expressão de LOXL3. Os casos de astrocitoma grau II com mutação de IDH1 apresentaram menor expressão de LOXL1 e de LOXL4 quando comparados com os casos sem mutação. Os casos de GBM com mutação de IDH1, por sua vez, apresentaram menores níveis de expressão de LOX e de LOXL1 do que os casos sem IDH1 mutado. Os níveis de expressão das proteínas da família das lisil oxidases também estavam maiores nas amostras de glioblastoma, com localização nuclear e citoplamastica das células, além de marcação do endotélio. Interessantemente, um caso de glioblastoma com mutação de IDH1 apresentou menor expressão de LOX, inclusive nas células endoteliais. Nas análises funcionais, o silenciamento de LOX por siRNA e o tratamento com o inibidor BAPN das linhagens celulares U87MG e A172 afetaram a capacidade de migração. Além sito, a menor expressão de LOX afetou a capacidade de invasão e crescimento independente de ancoragem das células. Em conjunto, esses resultados corroboram o papel de LOX em processo importantes da tumorigênese dos astrocitomas. Adicionalmente, a expressão de LOX é influenciada pelo status de mutação de IDH1. Portanto, este trabalho fornece novas informações para as possíveis intervenções terapêuticas para o tratamento dos pacientes com astrocitomas / The development and invasion of primary brain tumors are directly influenced by the extracellular matrix. Considering that lysyl oxidase (LOX) and other lysyl oxidase family members (LOXL1, LOXL2, LOXL3 e LOXL4) have both structural and functional complexity and that they are involved in vital biological processes such as cell motility, cell signaling and gene regulation, a deregulation of these proteins can lead to the genesis and tumor progression. This study aimed to evaluate the expression levels of genes that code for the lysyl oxidase family members in astrocytomas of different malignant grades and to correlate to the expression of BMP1 and HIF1A, IDH1 mutation and overall patients\' survival. Moreover, protein expression coded by these genes was also analyzed, besides an in vitro functional study of LOX role in astrocytomas. Gene expression analysis was performed by quantitative real-time PCR in a series of 153 astrocytomas and 22 samples of non-neoplastic brain. Protein expression was analyzed by immunohistochemistry in astrocytoma samples. LOX knockdown was performed in cells of human glioblastoma U87MG and A172 transfected with siRNA. Expression levels of all genes (LOX, LOXL1, LOXL2, LOXL3, LOXL4, BMP1 e HIF1A) increased with the malignant grade of astrocytomas, glioblastomas presenting the higher levels. Positive correlations of gene expression values were observed specially in glioblastomas. Only LOXL3 expression impacted in the overall survival of glioblastoma cases. Patients with higher expression presented longer survival time than those with lower LOXL3 expression. Astrocytoma grade II cases with IDH1 mutation presented lower LOXL1 and LOXL4 expression when compared to those cases with wild type IDH1. On the other hand, GBM cases with IDH1-mutated presented lower LOX and LOXL1 expression than GBM cases without IDH1 mutation. Protein expression levels of lysyl oxidase family members were also higher in glioblastoma samples, with both nuclear and cytoplasmic localization, and also endothelium staining. Interestingly, a glioblastoma case with IDH1-mutated had lower LOX expression, including endothelial cells. For functional analysis, LOX knockdown by siRNA and treatment with inhibitor BAPN of U87MG and A172 cell lines affected migration behavior. Furthermore, lower LOX expression affected invasion capacity and anchorage independent growth. Altogether, these results corroborate LOX role in important processes of astrocytoma tumorigenesis. Additionally, LOX expression is influenced by IDH1 mutational status in glioblastomas. Therefore, our work provides new insights for possible therapeutic interventions for patients with astrocytomas

[ES] Los tumores malignos del cerebro y del sistema nervioso central representan el 21% de los tumores en niños y son la segunda causa principal de muerte por cáncer pediátrico después de la leucemia. Los gliomas de alto grado siguen siendo incurables y poseen altas tasas de mortalidad: una supervivencia a cinco años del 4,7 % para pacientes con glioblastoma multiforme (GBM) y menos de un año en pacientes con glioma pontino intrínseco difuso (DIPG). Los bajos niveles de penetración del fármaco a través de la barrera hematoencefálica y la baja tasa de supervivencia asociada resaltan en la necesidad de nuevas propuestas de tratamiento para esta necesidad clínica no cubierta. La administración intranasal ofrece un enfoque no invasivo prometedor que elude el metabolismo hepato-gastrointestinal y la barrera hematoencefálica, constituyendo una ruta directa de la nariz al cerebro. Los poliglutamatos (PGA) representan excelentes candidatos para la administración de agentes terapéuticos en el cerebro debido a su biodegradabilidad y multivalencia, lo que permite la unión covalente de fármacos y grupos directores que puedan contribuir a cruzar las diferentes barreras biológicas existentes desde la nariz hasta el cerebro. La conjugación covalente del agente terapéutico a la cadena polimérica ofrece una estabilidad prolongada en la circulación sanguínea y un mayor control sobre la liberación del fármaco en el microambiente tumoral. Esta tesis se centra en el diseño racional y desarrollo de nuevos conjugados PGA-fármaco y una plataforma intranasal segura y eficiente para la administración dirigida y la liberación del fármaco en el cerebro como tratamiento del glioma pediátrico. Se desarrolló una familia de conjugados de poliglutamatos que incorporan palbociclib, un inhibidor de CDK, utilizando diferentes espaciadores sensibles a estímulos, así como diferente carga de fármacos. Estudiamos el efecto de la carga y conjugación de un fármaco sobre la conformación en solución de PGAs lineales y en forma de estrella. Con dispersión de rayos X de ángulo pequeño y dicroísmo circular, demostramos la evolución del conjugado polipeptídico al aumentar la carga de fármaco. Además, establecimos el vínculo entre la conformación de los conjugados y su actividad biológica en células GBM y DIPG derivadas de pacientes. Nuestros hallazgos ilustran la necesidad de una comprensión profunda de las propiedades fisicoquímicas de los nanosistemas estudiados que pueden ayudar a predecir su resultado biológico. Para el cribado rápido y la validación de la formulación intranasal, establecimos con éxito una plataforma de detección ex vivo basada en células de difusión verticales de Franz con mucosa nasal de oveja. Varios sistemas basados en PGA lineal y en forma de estrella modificadas con diferentes ligandos (incluyendo el ácido docosahexaenoico, ácido hialurónico (HA), odorranalectina), así como sistemas entrecruzados del PGA en forma de estrella y la mezcla física con un HA entrecruzado (HA-CP®). A continuación, se realizó estudios de biodistribución mediante administración intranasal de los candidatos seleccionados, seguidos de la cuantificación ex vivo con la técnica IVIS y el ensayo de fluorescencia estándar después de la homogeneización de órganos y los estudios histológicos del cerebro. Los datos obtenidos mostraron claramente la presencia de las diferentes plataformas en el cerebro e, su internalización en células del bulbo olfativo y una buena difusión en el cerebro a través de diferentes áreas, llegando incluso a detectarse en el hipocampo. Los candidatos seleccionados se han escalado y en la actualidad su evaluación biológica en un modelo in vivo está en curso. En conclusión, se ha diseñado, desarrollado y validado diferentes plataformas basadas en PGA capaces de llegar al cerebro a través de la administración intranasal, constituyendo una base prometedora para el tratamiento de múltiples trastornos relacionados con el cerebro. / [CA] Els tumors malignes del cervell i del sistema nerviós central representen el 21% dels tumors en nens i són la segona causa principal de mort per càncer pediàtric després de la leucèmia. Els gliomes d'alt grau que continuen sent incurables amb taxes de mortalitat altes: una supervivència a cinc anys del 4,7 % per a pacients amb glioblastoma multiforme (GBM) i menys d'un any en pacients amb glioma pontí intrínsec difús (DIPG). Els nivells baixos de penetració del fàrmac a través de la barrera hematoencefàlica i la baixa taxa de supervivència associada ressalten en la necessitat de noves propostes de tractament per a una necessitat clínica no satisfeta. L'administració intranasal ofereix un enfocament no invasiu prometedor que eludeix el metabolisme hepatogastrointestinal i la barrera hematoencefàlica, cosa que permet l'administració directa del nas al cervell. Els poliglutamats (PGA) representen excel·lents candidats per a l'administració al cervell a causa de la seva biodegradabilitat i multivalència, la qual cosa dóna suport a la introducció covalent de fàrmacs i grups objectiu i pot facilitar la transició del nas al cervell. La conjugació covalent de l'agent terapèutic a la cadena polimèrica ofereix una estabilitat prolongada en la circulació sanguínia i un major control sobre l'alliberament del fàrmac al microambient tumoral. Aquesta tesi se centra en el desenvolupament de nous conjugats PGA-fàrmac dissenyats racionalment i una plataforma intranasal segura i eficient per a l'administració dirigida i l'alliberament del fàrmac al cervell com a tractament del glioma pediàtric. Es va desenvolupar una família de conjugats de poliglutamat que incorporen l'inhibidor de CDK palbociclib, utilitzant diferents enllaçadors sensibles a estímuls i nivells variables de càrrega de fàrmacs. Estudiem l'efecte del fàrmac hidrofòbic conjugat sobre la conformació en solució de PGA lineals i en forma d'estrella. Amb dispersió de raigs X d'angle petit i dicroisme circular, demostrem l'evolució del sistema conjugat en augmentar la càrrega de fàrmac. A més, establim el vincle entre la conformació dels conjugats i la seva activitat biològica provada en cèl·lules GBM i DIPG derivades de pacients. Les nostres troballes van il·lustrar la necessitat d'una comprensió profunda de les propietats fisicoquímiques dels nanosistemes estudiats que poden ajudar a predir-ne el resultat biològic. Per a la detecció ràpida de la formulació intranasal basada en els portadors de PGA, vam establir amb èxit una plataforma de detecció ex vivo basada en cèl·lules de difusió verticals de Franz amb mucosa d'ovella. Diversos sistemes basats en PGA lineal i en forma d'estrella amb fraccions conjugades (inclòs l'àcid docosahexaenoic, àcid hialurònic (HA), odorranalectina), així com partícules reticulades (a través de cicloaddició azida-alquí o enllaços disulfur) i la barreja física amb un HA es van provar els polímers creuats (HA-CP®). Els estudis de biodistribució després de l'administració intranasal es van fer en candidats seleccionats, seguits de la quantificació ex vivo amb la tècnica IVIS i l'assaig de fluorescència estàndard després de l'homogeneïtzació d'òrgans i els estudis histològics del cervell. Les dades obtingudes van mostrar clarament el transport del nas al cervell, la internalització cel·lular dels conjugats al bulb olfactiu i, el que és més interessant, una bona difusió del cervell a través de diferents àrees, arribant fins i tot a l'hipocamp. Els sistemes seleccionats es van ampliar i la preparació per a l'avaluació biològica en un model in vivo està en curs. En general, vam ser capaços de dissenyar, desenvolupar i validar diferents plataformes basades en PGA capaces d'arribar al cervell mitjançant l'administració intranasal, la qual cosa podria ser la base per al tractament de múltiples trastorns relacionats amb el cervell. / [EN] Malignant brain and central nervous system tumors account for ~21% of tumors in children and represent the second leading cause of pediatric cancer deaths after leukemia. Standard chemotherapy allows a ~95% five-year survival rate for patients with low-grade gliomas. Unfortunately, high-grade gliomas remain generally incurable and suffer from high mortality rates - a 4.7% five-year survival for glioblastoma multiform (GBM) patients and less than one year in patients with diffuse intrinsic pontine glioma (DIPG). Low levels of drug delivery through the blood-brain barrier (BBB) and the associated poor survival rate highlight the necessity for novel treatment approaches for an unmet clinical need. Intranasal administration offers a promising non-invasive approach that circumvents hepato-gastrointestinal metabolism and the BBB, thereby enabling direct nose-to-brain delivery. Polyglutamates (PGAs), represent excellent candidates for brain delivery due to their biodegradability and multivalency, which supports the covalent introduction of drugs and targeting moieties to facilitate the nose-to-brain transition. Covalent conjugation of therapeutic agents to the polymeric main chain offers prolonged stability in the blood circulation and increased control over the drug release in the tumor microenvironment if adequately designed considering endogenous triggers, including acidic pH, an increased reductive environment, or the overexpression of specific proteases. This thesis focuses on developing novel, rationally-designed PGA-drug conjugates as a safe and efficient intranasal platform for targeted delivery and drug release in the brain as a pediatric glioma treatment. We developed a family of PGA-drug conjugates incorporating the CDK inhibitor palbociclib that employed different stimuli-responsive linkers and varying drug loading levels and studied the effect of drug loading on the solution conformation of linear and star-shaped PGAs. We demonstrated system evolution upon increased drug loading with SAXS and CD. Moreover, we established a link between conjugate conformation and their biological activity, as evaluated in patient-derived GBM and DIPG cells. Our findings illustrated the necessity of a deep understanding of the physico-chemical properties of studied nanosystems that can aid in predicting their biological outcome. For the development of an intranasal formulation, we successfully established an ex vivo screening platform based on vertical Franz diffusion cells with sheep mucosa. We evaluated several systems based on linear and star-shaped PGA with conjugated moieties (including docosahexaenoic acid, hyaluronic acid, odorranalectin), crosslinked particles (via azide-alkyne cycloaddition or disulfide bonds), and physical mixtures with a hyaluronic acid cross polymer (HA-CP®). After intranasal administration with selected candidates, we performed biodistribution studies, followed by ex vivo quantification with the IVIS spectrum in vivo imaging system and fluorescent assays after organ homogenization and brain histological studies. The obtained data demonstrated nose-to-brain transportation, internalization of conjugates in the olfactory bulb and robust diffusion through different brain areas, even reaching the hippocampus. We also scaled up selected systems and preparations for biological evaluation in an in vivo model that is currently ongoing. Overall, we were able to design, develop, and validate different PGA-based platforms capable of arriving at the brain via intranasal, which might be the base for the treatment of multiple brain-related disorders. / Melnyk, T. (2024). Drug Dellivery to the Brain Using Polymer Therapeutics as an Intranasal Platform for Pediatric Glioblastoma Treatment [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/203498

Vaken hjärnkirurgi med testning av tal- och språkfunktioner ger viktiga fördelar vid operation av lågmaligna gliom. Denna metod ökar möjligheten för radikal resektion av tumören samtidigt som viktiga funktioner som till exempel språk bevaras. Optimering av det intraoperativa språktestningsförfarandet ökar möjligheten för mer sensitiv språktestning. Det finns idag ingen konsensus, varken internationellt eller nationellt, kring hur man bör testa språkliga funktioner intraoperativt. Syftet med föreliggande arbete är att undersöka om det är möjligt att vidareutveckla de metoder som idag används för elicitering av spontantal och testning av läsning under vakenkirurgi av lågmaligna gliom på Akademiska sjukhuset i Uppsala. För att undersöka detta genomfördes en litteraturgenomgång vilken tillsammans med auskultationer vid operation och intervjuer med yrkesverksamma logopeder och neurokirurger har utmynnat i ett förslag på ett intraoperativt högläsningstest innehållande meningar med nonord, meningar utan nonord samt enskilda ord. Nivåindelningen av testmaterialet möjliggör för testledaren att individuellt anpassa språktestning utifrån patientens förmåga. I samband med färdigställandet av testet genomfördes en pilottestning av materialet. Det föreslagna lästestet förväntas vara mer heltäckande än det som används idag då det möjliggör testning av flera läsfunktioner så som de beskrivs i ”dual route-modellen”, en psykolingvistisk teori om läsning. Dessutom har ett förslag på riktlinjer kring intraoperativt elicitering av spontantal formulerats. / Awake surgery is the gold standard for treatment of low-grade gliomas nearby eloquent language areas in the brain. This method increases the possibility of radical resection while cognitive functions such as language are preserved, as the method allows for online testing of cognitive functions. Improvement of the intraoperative language tests could increase the possibility of more sensitive testing and thereby decrease the risk of postoperative language impairments. There is no international or national agreement on how intraoperative language testing should be performed. The aim of this study is to explore the possibility of developing the methods of language testing which are used today at Akademiska sjukhuset in Uppsala, focusing on reading and spontaneous speech. Therefore the authors have conducted a review of current literature. The literature review, observations of surgery and interviews with speech pathologists and neurosurgeons resulted in the development of an intraoperative reading aloud test with three tasks consisting of sentences with nonwords, sentences without nonwords and single words. The classification of the test material into different levels will allow clinicians to adapt the reading test based on each individual case. A pilot trial of the reading test was preformed. The proposed test is expected to be more comprehensive in comparison to the tests that are currently used, as it enables testing of the different reading routes described in the dual route model, a psycholinguistic reading model. Additionally, guidelines for eliciting spontaneous speech have been defined.

No description available.

Les modèles de croissance tumorale fondés sur l'équation de réaction-diffusion Fisher Kolmogorov FK ont montré des résultats probants dans la reproduction et la prédiction de l'invasion de cellules tumorales du cerveau pour les gliomes. Nous utilisons différentes formulations du modèle FK pour évaluer la nécessité de l’imagerie de diffusion pour construire des modèles spécifiques de Gliomes de Bas Grade GBG, l'étude de l'infiltration de cellules tumorales après une résection chirurgicale de la tumeur, et définir une métrique pour quantifier l’évolution de GBG. L'imagerie en tenseur de diffusion ITD a été suggérée pour modéliser la diffusion anisotrope des cellules tumorales dans la matière blanche du cerveau. Les ITD acquises en basse résolution impactent la précision des résultats des modèles de croissance. Nous utilisons une formulation FK pour décrire l'évolution de la frontière visible de la tumeur pour étudier l'impact du remplacement de l'ITD patient par une hypothèse de diffusion isotrope ou une ITD de référence anisotrope en haute résolution formée par la moyenne des ITD de plusieurs patients. Nous quantifions l'impact du remplacement de l'ITD acquise sur un patient à aide de simulations de croissance tumorales synthétiques et des prévisions d'évolution de la tumeur d'un cas clinique. Cette étude suggère que la modélisation de la croissance du gliome à base de motilité différentielle de tissus donne des résultats un peu moins précis qu'à l'aide d'une ITD. S'abstenir d'utiliser une ITD serait suffisant lors de la modélisation de GBG. Par conséquent, toutes ces options d'ITD sont valides dans une formulation FK pour modéliser la croissance de GBG dans le but d'aider les cliniciens dans la planification du traitement. Après la résection d’une tumeur cérébrale, ils veulent savoir quel serait le meilleur traitement de suivi pour chaque patient : une chimiothérapie pour des tumeurs diffuses ou bien une deuxième résection après un laps de temps donné pour les tumeurs massives. Nous proposons une méthode pour tirer profit de modèles de croissance de gliome FK sur les cas post-opératoires montrant des distorsions du cerveau pour estimer l'infiltration des cellules tumorales au-delà des frontières visibles dans les IRM FLAIR. Notre méthode répond à 2 défis de modélisation : celui du mouvement du parenchyme cérébral après la chirurgie avec une technique de recalage non-linéaire et celui de la segmentation incomplète de la tumeur post-opératoire en combinant 2 cartes d'infiltration : une ayant été simulée à partir d'une image pré-opératoire et l’autre à partir d'une image post-opératoire. Nous avons utilisé les données de 2 patients ayant des GBG afin de démontrer l'efficacité de la méthode. Celle-ci pourrait aider les cliniciens à anticiper la récurrence de la tumeur après une résection et à caractériser l’étendue de l'infiltration non visible par la radiologie pour planifier la thérapie. Pour les GBG visibles par une IRM FLAIR/T2, il y a un débat important au sein du groupe de travail RANO Response Assessment in Neuro-Oncology sur la sélection d'un seuil pertinent des métriques basées sur l’évolution de la taille de la tumeur pour déterminer si la maladie est évolutive ME. Nous proposons une approche pour évaluer la ME du GBG en utilisant des estimations de la vitesse de croissance de la tumeur à partir d'une formulation FK qui prend en compte les irrégularités de forme de la tumeur, les différences de vitesse de croissance entre la matière grise et la matière blanche, et les changements volumétriques. En utilisant les IRM FLAIR de 9 patients, nous comparons les estimations de ME de notre approche proposée avec celles calculées en utilisant les estimations manuelles de la vitesse de croissance tumorale 1D, 2D et 3D et celles calculées en utilisant un ensemble de critères basés sur la taille critères RECIST, Macdonald et RANO. Notre approche est prometteuse pour évaluer la ME du GBG à partir d'un nombre limité d'examens par IRM. / Tumor growth models based on the Fisher Kolmogorov (FK) reaction-diffusion equation have shown convincing results in reproducing and predicting the invasion patterns of glioma brain tumors. In this thesis we use different FK model formulations to i) assess the need of patient-specific DTIs when modeling LGGs, ii) study cancer cell infiltration after tumor resections, and iii) define a metric to determine progressive disease for low-grade glimoas (LGG).Diffusion tensor images (DTIs) have been suggested to model the anisotropic diffusion of tumor cells in brain white matter. However, patient specific DTIs are expensive and often acquired with low resolution, which compromises the accuracy of the tumor growth models' results. We used a FK formulation to describe the evolution of the visible boundary of the tumor to investigate the impact of replacing the patient DTI by i) an isotropic diffusion map or ii) an anisotropic high-resolution DTI atlas formed by averaging the DTIs of multiple patients. We quantify the impact of replacing the patient DTI using synthetic tumor growth simulations and tumor evolution predictions on a clinical case. This study suggests that modeling glioma growth with tissue based differential motility (not using a DTI) yields slightly less accurate results than using a DTI. However, refraining from using a DTI would be sufficient in situations when modeling LGGs. Therefore, any of these DTI options are valid to use in a FK formulation to model LGG growth with the purpose of aiding clinicians in therapy planning.After a brain resection medical professionals want to know what the best type of follow-up treatment would be for a particular patient, i.e., chemotherapy for diffuse tumors or a second resection after a given amount of time for bulky tumors. We propose a thorough method to leverage FK reaction-diffusion glioma growth models on post-operative cases showing brain distortions to estimate tumor cell infiltration beyond the visible boundaries in FLAIR MRIs. Our method addresses two modeling challenges: i) the challenge of brain parenchyma movement after surgery with a non-linear registration technique and ii) the challenge of incomplete post-operative tumor segmentations by combining two infiltration maps, where one was simulated from a pre-operative image and one estimated from a post-operative image. We used the data of two patients with LGG to demonstrate the effectiveness of the proposed three-step method. We believe that our proposed method could help clinicians anticipate tumor regrowth after a resection and better characterize the radiological non-visible infiltrative extent of a tumor to plan therapy.For LGGs captured on FLAIR/T2 MRIs, there is a substantial amount debate on selecting a definite threshold for size-based metrics to determine progressive disease (PD) and it is still an open item for the Response Assessment in Neuro-Oncology (RANO) Working Group. We propose an approach to assess PD of LGG using tumor growth speed estimates from a FK formulation that takes into consideration irregularities in tumor shape, differences in growth speed between gray matter and white matter, and volumetric changes. Using the FLAIR MRIs of nine patients we compare the PD estimates of our proposed approach to i) the ones calculated using 1D, 2D, and 3D manual tumor growth speed estimates and ii) the ones calculated using a set of well-established size-based criteria (RECIST, Macdonald, and RANO). We conclude from our comparison results that our proposed approach is promising for assessing PD of LGG from a limited number of MRI scans. It is our hope that this model's tumor growth speed estimates could one day be used as another parameter in clinical therapy planning.

L’étude des nystagmus du nourrisson est difficile pour des raisons méthodologiques. La plupart des études sont donc réalisées chez des adultes et a posteriori. Ces études chez l’adulte ont permis la révision des classifications des nystagmus infantiles, avec la consolidation des concepts de syndrome du nystagmus précoce et de nystagmus de type latent. La séméiologie et l’étiopathogénie des nystagmus du nourrisson – notamment des formes comportant un élément vertical et des formes transitoires – reste donc à étudier. Dans ce but : 1. Nous avons développé de nouvelles techniques d’enregistrement des mouvements oculaires chez les nourrissons, utilisables dans le cadre d’une consultation. Elles incluent de nouveaux stimuli, un appareil d’enregistrement des mouvements oculaires spécialement conçu pour le nourrisson et de nouvelles méthodes d’analyse statistique du signal. Nous avons vérifié la faisabilité de ces enregistrements chez 28 nourrissons atteints de nystagmus. 2. Nous avons étudié systématiquement 32 cas de nystagmus de type spasmus nutans, classiquement considéré comme une entité bénigne idiopathique, avec un examen clinique complet, une imagerie cérébrale, une électrophysiologie visuelle et des enregistrements oculo-moteurs. Dans 53,1% des cas, le spasmus nutans était le symptôme d’une autre maladie : neurologique (34,3%), notamment des gliomes du chiasma (21,9%), ou rétinienne (12,5%). Une atteinte des voies visuelles antérieures est probablement en cause dans la physiopathologie des spasmus nutans. 3. Huit cas de nystagmus ayant conduit au diagnostic de gliome des voies optiques (GVO) ont été également enregistrés et étudiés. L’âge d’apparition du nystagmus allait de 2,5 à 10 mois. Le GVO était toujours chiasmatique et constituait une sous-population spécifique. Le nystagmus était toujours de type spasmus nutans. Les enregistrements oculo-moteurs montraient : une fréquence entre 2,7 et 5 Hz, une morphologie sinusoïdale du nystagmus, une dissociation et une dysconjugaison particulière, avec une opposition de phase (180°) entre les oscillations des deux yeux dans le plan horizontal mais une correspondance de phase dans le plan vertical, à l’origine d’un mouvement semblable à un mouvement de convection. Rarement et brièvement, le rapport de phase changeait. Ces caractéristiques orientent vers des oscillations dans le système des vergences, possiblement la conséquence d’une atteinte des afférences sensorielles des centres du contrôle vergentiel dans le tronc cérébral, secondaire au GVO et survenant pendant la période sensible du développement visuel. 4. Cinq cas de nystagmus upbeat chez des nourrissons avec des rétines et une imagerie cérébrale normale ont été étudiés. Le nystagmus était observé en décubitus et électivement déclenché par des rotations de la tête en position allongée. Dans tous les cas, une résolution spontanée était observée après quelques mois d’évolution. Les caractéristiques de ce type de nystagmus suggèrent une participation du système otolithique, suivie d’une recalibration secondaire des circuits vestibulo-oculaires. En conclusion, le développement de techniques d’enregistrement des mouvements oculaires adaptées aux nourrissons aide à la compréhension de l’étiopathogénie de variétés mal décrites de nystagmus, notamment dans les cas comportant un élément vertical et dans les cas transitoires. Les processus de maturation des voies visuelles antérieures et des centres de contrôle de l’oculomotricité semblent jouer un rôle central dans les mécanismes de ces nystagmus. / Studying infantile nystagmus during infancy is difficult for methodological reasons. Most such studies have been performed in adults and a posteriori. These studies in adults allowed for an improvement in the existing classifications, with now robust knowledge about the two most frequent varieties of infantile nystagmus: infantile nystagmus syndrome and fusion maldevelopment nystagmus syndrome. The characteristics and pathophysiology of nystagmus in infants–notably varieties of nystagmus with a vertical component and transitory nystagmus– need further study. For this reason: 1. We developed new techniques for the recording of eye movements in infants in the setting of a clinic. They include new stimuli, the use of specially-designed infrared photo-oculography eyetrackers and new statistical analysis paradigms. We assessed these techniques in a population of 28 infants with a nystagmus. 2. We systematically studied 32 cases of spasmus nutans, classically considered an idiopathic entity, with comprehensive clinical examination, brain imaging, electrophysiology, nystagmus recording. In 53.1% of cases, it led to the diagnosis of another condition: a neurological disease (34.3%), including cases of chiasmal gliomas (21.9%), or a retinal dysfunction (12.5%). Anterior visual pathway dysfunction is likely involved in the pathophysiology of spasmus nutans. 3. Eight cases of nystagmus having led to a diagnosis of optic pathway glioma (OPG) were also recorded and studied. Age at nystagmus onset was 2.5-10 months. The associated OPG always involved the chiasm, and represent a specific subpopulation of OPG. Clinically, the nystagmus was always classified as spasmus nutans type. Oculographic recordings showed frequencies of 2.7-5 Hz, sinusoidal waveforms, dissociation and a special type of disconjugacy, with a 180° horizontal phase shift and no vertical phase shift, exhibiting a “convection-like” movement pattern. Rarely and for short periods of time, the phase shift could change. These characteristics point towards oscillations in the vergence system, which could possibly result from the specific disruption of the vergence centres afferences in the brainstem, induced by the OPG during the sensitive period of visual development. 4. Five cases of upbeat nystagmus in infants with normal retinas and normal brain imaging were studied. The nystagmus mostly occurred in supine position and could be triggered by head rotations in the supine position. All resolved spontaneously. The characteristics of this nystagmus suggest an involvement of the otolithic system, with a secondary recalibration of the vestibulo-ocular pathways. In conclusion, the development of infant-friendly devices for eye-movements recording helps providing new insights on the pathophysiology of poorly described varieties of nystagmus, including nystagmus with a vertical component and transitory nystagmus. The maturation process of both the anterior visual pathways and the oculomotor pathways appears to be central in the mechanisms of these nystagmus.

Die hier verwendete Methode der dynamischen Magnetresonanztomographie (dMRT) erlaubte bei Hirntumorpatienten erstmals, gleichzeitig neben dem regionalen zerebralen Blutvolumen (rCBV) und dem regionalen zerebralen Blutfluß weitere Parameter, wie Permeabilitäten, die interstitiellen Volumina und das Zellvolumen, zu bestimmen. Anhand dieser Parameter sollte erstens geprüft werden, inwieweit diese zu einer besseren Malignitätseinstufung von Hirntumoren beitragen. Zweitens sollte geklärt werden, inwiefern sich die untersuchten Tumorgruppen voneinander unterscheiden lassen. Drittens war es Ziel, ein in-vivo-Grading für die Gliome zu entwickeln. Es wurden 60 Patienten mit verschiedenen Tumoren, wie Gliome, Metastasen, Meningeome und Lymphome, untersucht. Die aus der dMRT-Untersuchung erhaltenen Daten wurden mit einem pharmakokinetischen Modell ausgewertet. Für jeden Patienten wurden die oben genannten Parameter in Form von Bildern dargestellt und quantitativ berechnet. Für die Tumordifferenzierung eignete sich das mittlere rCBV am besten: Innerhalb der Gliome konnte signifikant zwischen den Grad-II- und Grad-III-Gliomen und den Grad-II- und Grad-IV-Gliomen unterschieden werden. Weiterhin konnten die Meningeome signifikant von den anderen untersuchten Tumorentitäten abgegerenzt werden. Das in-vivo-Grading der Gliome erlaubte in 71 % der Fälle eine korrekte Zuordnung zum WHO-Grad. Die Parameterbilder lieferten neben Informationen für die Tumordifferenzierung auch beispielsweise Hinweise auf den heterogenen Tumoraufbau. Des weiteren ermöglichten sie, Narbengewebe gegenüber Tumorgewebe abzugrenzen und Folgen einer Strahlentherapie zu beobachten. Schließlich waren Aussagen über die Gefäßarchitektur und das Wachstum unterschiedlicher Tumorgruppen möglich. Die mit der hier verwendeten Methode der dMRT erhaltenen Parameter boten mehrere Vorteile: Eine Differenzierung einzelner Tumorgruppen war möglich. Für die Gliome konnte mittels des in-vivo-Gradings eine quantitative Malignitätseinschätzung erfolgen. Die gewonnenen Informationen über den heterogenen Tumoraufbau erlauben bessere Biopsieergebnisse. Zusätzlich wurden Hinweise auf die Tumorpathophysiologie erhalten und es erschien möglich Veränderungen nach Therapie zu beobachten. / A method of dynamic magnet resonance imaging (dMRI) was used, which allowed for the first time to determine simultaneously several parameters in patients with brain tumors. These parameters were the regional cerebral blood volume (rCBV), the regional cerebral blood flow, and in addition, permeabilities, interstitial volumes, and the cell volume. First, it should be determined to what extent these parameters allow a better classification of the malignancy of brain tumors. Second, it should be evaluated how far it is possible to differentiate the examined tumor groups from each other. Third, a method for an in-vivo-grading specifically for gliomas should be developed. Altogether 60 patients with different tumors such as gliomas, metastasis, meningiomas, and lymphomas were examined. The data of the dMRI examination were evaluated using a pharmacokinetic model. For every patient, the parameters mentioned above were shown in maps and calculated quantitatively. The mean rCBV resulted in the best tumor differentiation: within the group of gliomas it was possible to differentiate significantly between grade-II- and grade-III-gliomas and grade-II- and grade-IV-gliomas. Furthermore, meningiomas were differentiated significantly from the other tumors. In respect to the group of gliomas, the tumor grades determined by the developed in-vivo-grading corresponded with the WHO grade of each glioma in 71 % of the cases. The parameter maps were not only usefull for tumor differentiation, but also yielded information concerning the heterogenous tumor structure. Additionally, these maps allowed to differentiate scar tissue from tumor tissue and effects of a radiotherapy could be observed. Finally, information about the vessel architecture and the growth of different tumor groups could be obtained. The parameters determined by the dMRI method used here offered several advantages: it was possible to differentiate between single tumor groups. For the gliomas, a quantitative malignancy classification resulted from the in-vivo-grading. The information concerning the structure of the heterogeneity of the tumor allows for better biopsy results. Additionally, information was also obtained concerning the pathophysiology of the tumors and it seemed possible to observe changes after a therapy.

Kar Wing To. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 200-223). / Abstracts in English and Chinese. / Acknowledgements --- p.i / List of Abbreviations --- p.ii / Abstract --- p.v / 撮要 --- p.viii / List of Tables --- p.x / List of Figures --- p.xi / Contents --- p.xv / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- The General Characteristics of Glial Cells --- p.1 / Chapter 1.1.1 --- Astrocytes --- p.1 / Chapter 1.1.2 --- Oligodendrocytes --- p.5 / Chapter 1.1.3 --- Microglial --- p.6 / Chapter 1.2 --- Brain Injury and Astrocyte Proliferation --- p.6 / Chapter 1.3 --- Reactive Astrogliosis and Glial Scar Formation --- p.9 / Chapter 1.4 --- Astrocytes and Immune Response --- p.10 / Chapter 1.5 --- Cytokines --- p.10 / Chapter 1.5.1 --- Cytokines and the Central Nervous System (CNS) --- p.12 / Chapter 1.5.2 --- Cytokines and brain injury --- p.13 / Chapter 1.5.3 --- Cytokines-activated astrocytes in brain injury --- p.13 / Chapter 1.5.4 --- Tumour Necrosis Factor-a --- p.14 / Chapter 1.5.4.1 --- Types of TNF-α receptor and their sturctures --- p.16 / Chapter 1.5.4.2 --- Binding to TNF-α --- p.17 / Chapter 1.5.4.3 --- Different Roles of the TNF-a Receptor Subtypes --- p.17 / Chapter 1.5.4.4 --- Role of TNF-α and Brain Injury --- p.19 / Chapter 1.5.4.5 --- TNF-α Stimulates Proliferation of Astrocytes and C6 Glioma Cells --- p.23 / Chapter 1.5.5 --- Interleukin-1 (IL-1) --- p.26 / Chapter 1.5.5.1 --- Interleukin-1 and Brain Injury --- p.27 / Chapter 1.5.6 --- Interleukin-6 (IL-6) --- p.28 / Chapter 1.5.6.1 --- Interleukin-6 and brain injury --- p.29 / Chapter 1.5.7 --- γ-Interferon (γ-IFN) --- p.30 / Chapter 1.5.7.1 --- γ-Interferon and Brain Injury --- p.30 / Chapter 1.6 --- Ion Channels and Astrocytes --- p.31 / Chapter 1.6.1 --- Roles of Sodium Channels in Astrocytes --- p.33 / Chapter 1.6.2 --- Role of Potassium Channels in Astrocytes --- p.33 / Chapter 1.6.3 --- Importance of Calcium Ion Channels in Astrocytes --- p.34 / Chapter 1.6.3.1 --- Function of Cellular and Nuclear Calcium --- p.34 / Chapter 1.6.3.2 --- Nuclear Calcium in Cell Proliferation --- p.36 / Chapter 1.6.3.3 --- Nuclear Calcium in Gene Transcription --- p.36 / Chapter 1.6.3.4 --- Nuclear Calcium in Apoptosis --- p.38 / Chapter 1.6.3.5 --- Spatial and Temporal Changes of Calcium-Calcium Oscillation --- p.39 / Chapter 1.6.3.6 --- Calcium Signalling in Glial Cells --- p.39 / Chapter 1.6.3.7 --- Calcium Channels in Astrocytes --- p.41 / Chapter 1.6.3.8 --- Relationship Between [Ca2+]i and Brain Injury --- p.43 / Chapter 1.6.3.9 --- TNF-α and Astrocyte [Ca2+]i --- p.45 / Chapter 1.6.3.10 --- Calcium-Sensing Receptor (CaSR) --- p.46 / Chapter 1.7 --- Protein Kinase C (PKC) Pathways --- p.49 / Chapter 1.7.1 --- PKC and Brain Injury --- p.50 / Chapter 1.7.2 --- Role of Protein Kinase C Activity in TNF-α Gene Expression in Astrocytes --- p.51 / Chapter 1.7.3 --- PKC and Calcium in Astrocytes --- p.52 / Chapter 1.8 --- Intermediate Early Gene (IEGs) --- p.54 / Chapter 1.8.1 --- IEGs Expression and Brain Injury --- p.54 / Chapter 1.8.2 --- IEGs Expression and Calcium --- p.55 / Chapter 1.9 --- The Rat C6 Clioma Cells --- p.56 / Chapter 1.10 --- The Aim of This Project --- p.58 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.61 / Chapter 2.1.1 --- Sources of the Chemicals --- p.61 / Chapter 2.1.2 --- Materials Preparation --- p.65 / Chapter 2.1.2.1 --- Rat C6 Glioma Cell Line --- p.65 / Chapter 2.1.2.2 --- C6 Glioma Cell Culture --- p.65 / Chapter 2.1.2.2.1 --- Complete Dulbecco's Modified Eagle Medium (CDMEM) --- p.65 / Chapter 2.1.2.2.2 --- Serum-free Dulbecco's Modified Eagle Medium --- p.66 / Chapter 2.1.2.3 --- Phosphate Buffered Saline (PBS) --- p.66 / Chapter 2.1.2.4 --- Recombinant Cytokines --- p.67 / Chapter 2.1.2.5 --- Antibodies --- p.67 / Chapter 2.1.2.5.1 --- Anti-TNF-Receptor 1 (TNF-R1) Antibody --- p.67 / Chapter 2.1.2.5.2 --- Anti-TNF-Receptor 2 (TNF-R2) Antibody --- p.67 / Chapter 2.1.2.6 --- Chemicals for Signal Transduction Study --- p.68 / Chapter 2.1.2.6.1 --- Calcium Ionophore and Calcium Channel Blocker --- p.68 / Chapter 2.1.2.6.2 --- Calcium-Inducing Agents --- p.68 / Chapter 2.1.2.6.3 --- Modulators of Protein Kinase C (PKC) --- p.69 / Chapter 2.1.2.7 --- Reagents for Cell Proliferation --- p.69 / Chapter 2.1.2.8 --- Reagents for Calcium Level Measurement --- p.70 / Chapter 2.1.2.9 --- Reagents for RNA Extraction and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.71 / Chapter 2.1.2.10 --- Sense and Antisense Used --- p.72 / Chapter 2.1.2.11 --- Reagents for Electrophoresis --- p.74 / Chapter 2.2 --- Methods --- p.74 / Chapter 2.2.1 --- Maintenance of the C6 Cell Line --- p.74 / Chapter 2.2.2 --- Cell Preparation for Assays --- p.75 / Chapter 2.2.3 --- Determination of Cell Proliferation --- p.76 / Chapter 2.2.3.1 --- Determination of Cell Proliferation by [3H]- Thymidine Incorporation --- p.76 / Chapter 2.2.3.2 --- Measurement of Cell Viability Using Neutral Red Assay --- p.77 / Chapter 2.2.3.3 --- Measurement of Cell Proliferation by MTT Assay --- p.77 / Chapter 2.2.3.4 --- Protein Assay --- p.78 / Chapter 2.2.3.5 --- Data Analysis --- p.79 / Chapter 2.2.3.5.1 --- The Measurement of Cell Proliferation by [3H]- Thymidine Incorporation --- p.79 / Chapter 2.2.3.5.2 --- The Measurement of Cell growth in Neutral Red and MTT Assays --- p.79 / Chapter 2.2.3.5.3 --- The Measurement of Cell Proliferationin Protein Assay --- p.79 / Chapter 2.2.4 --- Determination of Intracellular Calcium Changes --- p.80 / Chapter 2.2.4.1 --- Confocal Microscopy --- p.80 / Chapter 2.2.4.1.1 --- Procedures for Detecting Cell Activity by CLSM --- p.81 / Chapter 2.2.4.1.2 --- Precautions of CLSM --- p.82 / Chapter 2.2.5 --- Determination of Gene Expression by Reverse- Transcription Polymerase Chain Reaction (RT-PCR) --- p.83 / Chapter 2.2.5.1 --- RNA Preparation --- p.83 / Chapter 2.2.5.1.1 --- RNA Extraction --- p.83 / Chapter 2.2.5.1.2 --- Measurement of RNA Yield --- p.84 / Chapter 2.2.5.2 --- Reverse Transcription (RT) --- p.84 / Chapter 2.2.5.3 --- Polymerase Chain Reaction (PCR) --- p.85 / Chapter 2.2.5.4 --- Separation of PCR Products by Agarose Gel Electrophoresis --- p.85 / Chapter 2.2.5.5 --- Quantification of Band Density --- p.86 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1 --- Effects of Different Drugs on C6 Cell Proliferation --- p.87 / Chapter 3.1.1 --- Effects of Cytokines on C6 Cell Proliferation --- p.87 / Chapter 3.1.1.1 --- Effect of TNF-α on C6 Proliferation --- p.88 / Chapter 3.1.1.2 --- Effects of Other Cytokines on C6 Cell Proliferation --- p.92 / Chapter 3.1.2 --- The Signalling Pathway of TNF-α induced C6 Cell Proliferation --- p.92 / Chapter 3.1.2.1 --- The Involvement of Calcium Ions in TNF-α-induced C6Cell Proliferation --- p.95 / Chapter 3.1.2.2 --- The Involvement of Protein Kinase C in TNF-α- induced C6 Cell Proliferation --- p.96 / Chapter 3.1.3 --- Effects of Anti-TNF Receptor Subtype Antibodies on C6 Cell Proliferation --- p.102 / Chapter 3.2 --- The Effect of in Tumour Necrosis Factor-α on Changesin Intracellular Calcium Concentration --- p.102 / Chapter 3.2.1 --- Release of Intracellular Calcium in TNF-α-Treated C6 Cells --- p.104 / Chapter 3.2.2 --- Effects of Calcium Ionophore and Calcium Channel Blocker on TNF-α-induced [Ca2+]i Release --- p.107 / Chapter 3.2.3 --- Effects of Other Cytokines on the Change in [Ca2+]i --- p.109 / Chapter 3.2.4 --- The Role of PKC in [Ca2+]i release in C6 Glioma Cells --- p.109 / Chapter 3.2.4.1 --- Effects of PKC Activators and Inhibitors on the Changes in [Ca2+]i --- p.114 / Chapter 3.3 --- Determination of Gene Expression by RT-PCR --- p.114 / Chapter 3.3.1 --- Studies on TNF Receptors Gene Expression --- p.117 / Chapter 3.3.1.1 --- Effect of TNF-α on TNF Receptors Expression --- p.117 / Chapter 3.3.1.2 --- Effects of Other Cytokines on the TNF Receptors Expression --- p.119 / Chapter 3.3.1.3 --- Effects of Anti-TNF Receptor Subtype Antibodies on the TNF-a-induced Receptors Expression --- p.121 / Chapter 3.3.1.4 --- Effect of Calcium Ions on TNF Receptors Expression --- p.121 / Chapter 3.3.1.4.1 --- Effect of Calcium Ionophore on TNF Receptors Expression --- p.126 / Chapter 3.3.1.4.2 --- Effect of TNF-α Combination with A23187 on the Expression of TNF Receptors --- p.128 / Chapter 3.3.1.4.3 --- Effects of Calcium Ionophore and Channel Blocker on TNF Receptors Expression --- p.130 / Chapter 3.3.1.4.4 --- Effects of Calcium-Inducing Agents on TNF Receptors Gene Expressions --- p.130 / Chapter 3.3.1.5 --- Effects of PKC Activator and Inhibitor on TNF-α- induced TNF Receptors Expressions --- p.135 / Chapter 3.3.1.6 --- Effect of PKC and Ro31-8220 on IL-l-induced TNF Receptors Expressions --- p.138 / Chapter 3.3.2 --- Expression of Calcium-sensing Receptor upon Different Drug Treatments --- p.138 / Chapter 3.3.2.1 --- Effect of TNF-α on the Calcium-sensing Receptor Expression --- p.141 / Chapter 3.3.2.2 --- Effects of Other Cytokines on CaSR Expression --- p.143 / Chapter 3.3.2.3 --- Effect of A23187 on CaSR Expression --- p.143 / Chapter 3.3.2.4 --- Effect of TNF-α and A23187 Combined Treatment on CaSR Expression --- p.146 / Chapter 3.3.2.5 --- Effects of Calcium-inducing Agents on CaSR Expression --- p.149 / Chapter 3.3.2.6 --- Effects of PKC Activator and PKC Inhibitor on CaSR Expression --- p.149 / Chapter 3.3.3 --- Expression of PKC Isoforms upon Treatment with Different Drugs --- p.153 / Chapter 3.3.3.1 --- Effect of TNF-α on the PKC Isoforms Expression --- p.155 / Chapter 3.3.3.2 --- Effect of A23187 on the PKC Isoforms Expression --- p.155 / Chapter 3.3.3.3 --- Effect of TNF-α and Calcium Ionophore Combined Treatment on PKC Isoforms Expression --- p.157 / Chapter 3.3.3.4 --- Effects of Calcium-inducing Agents on PKC Isoforms Expression --- p.159 / Chapter 3.3.4 --- Expression of the Transcription Factors c-fos and c-junin Drug Treatments --- p.161 / Chapter 3.3.4.1 --- Effect of TNF-a on c-fos and c-jun Expression --- p.163 / Chapter 3.3.4.2 --- Effect of A23187 on c-fos and c-jun Expression --- p.163 / Chapter 3.3.4.3 --- Effect of TNF-a in Combination with A23187 on c- fos and c-jun Expression --- p.165 / Chapter 3.3.4.4 --- Effects of Calcium-inducing Agents on c-fos and c- jun Expression --- p.167 / Chapter 3.3.5 --- Effects of Different Drugs on Endogenous TNF-α Expression --- p.167 / Chapter 3.3.5.1 --- Effect of TNF-α on Endogenous TNF-α Expression --- p.169 / Chapter 3.3.5.2 --- Effect of A23187 on Endogenous TNF-α Expression --- p.169 / Chapter 3.3.5.3 --- Effect of TNF-α in Combination with A23187 on the Expression of Endogenous TNF-α --- p.172 / Chapter 3.3.5.4 --- Effects of Calcium-inducing Agents on Endogenous TNF-α Expression --- p.172 / Chapter 3.3.6 --- Effect of Different Drugs on LL-1 Expression --- p.175 / Chapter 3.3.6.1 --- Effect of TNF-a on IL-lα Expression --- p.177 / Chapter 3.3.6.2 --- Effect of A23187 on the IL-lα Expression --- p.177 / Chapter 3.3.6.3 --- Effect of Calcium Ionophore and TNF-α Combined Treatment on IL-1α Expression --- p.179 / Chapter 3.3.6.4 --- Effects of Calcium-inducing Agents on IL-lα Expression --- p.179 / Chapter 3.3.6.5 --- Effect of PKC Activator on the IL-1α Expression --- p.181 / Chapter CHAPTER 4 --- DISCUSSIONS AND CONCLUSIONS / Chapter 4.1 --- "Effects of Cytokines, Ca2+ and PKC and Anti-TNF-α Antibodies on C6 Glioma Cells Proliferation" --- p.184 / Chapter 4.2 --- The Role of Calcium in TNF-α-induced Signal Transduction Pathways --- p.186 / Chapter 4.3 --- Gene Expressions in C6 Cells after TNF-a Stimulation --- p.187 / Chapter 4.3.1 --- "Expression of TNF-α, TNF-Receptors and IL-1" --- p.187 / Chapter 4.3.2 --- CaSR Expression --- p.190 / Chapter 4.3.3 --- PKC Isoforms Expressions --- p.192 / Chapter 4.3.4 --- Expression of c-fos and c-jun --- p.193 / Chapter 4.4 --- Conclusion --- p.194 / REFERENCES --- p.200

Les gliomes sont des tumeurs cérébrales infiltrantes difficilement curables, notamment à cause de la difficulté à visualiser toutes les infiltrations au bloc opératoire. Dans cette thèse, nous réalisons une étude clinique de spectroscopie de fluorescence de la protoporphyrine IX (PpIX) dans les gliomes de 10 patients selon l’hypothèse que les spectres collectés proviennent de la contribution de 2 états de la PpIX dont les proportions varient suivant la densité en cellules tumorales. Après avoir présenté le développement du système interventionnel proposant une excitation multi-longueurs d’onde, nous présentons son utilisation sur fantômes de PpIX mimant les propriétés des gliomes. Ceci permet tout d’abord d’obtenir les spectres émis par les 2 états séparément puis de proposer un modèle d’ajustement des spectres comme une combinaison linéaire des 2 spectres de référence sur la bande spectrale 608-637 nm. Ensuite, nous présentons la mise en place de l’étude clinique, notamment l’analyse de risques, avant d’appliquer ce système in vivo. Les mesures in vivo détectent de la fluorescence dans des tissus où le microscope chirurgical n’en détecte pas, ce qui pourrait s’expliquer par un changement d’état de la PpIX entre le cœur des gliomes et leurs infiltrations. L’intérêt de l’excitation multi-longueurs d’onde est démontré par la décroissance de la corrélation des spectres acquis aux trois excitations suivant la densité en cellules tumorale. Enfin, nous soulevons des pistes d’étude de l’identification peropératoire des zones de fonctionnalité cérébrale à l’aide d’une caméra optique ainsi que l’étude du temps de vie de fluorescence et de la fluorescence deux photons de la PpIX sur fantômes / Gliomas are infiltrative tumors of the brain which are yet hardly curable, notably because of the difficulty to precisely delimitate their margins during surgery. Intraoperative 5-ALA induced protoporphyrin IX (PpIX) fluorescence microscopy has shown its relevance to assist neurosurgeons but lacks sensitivity. In this thesis, we perform a spectroscopic clinical trial on 10 patients with the assumption that collected fluorescence is a linear combination of the contribution of two states of PpIX which proportions vary with the density of tumor cells. This work starts with the development of the intraoperative, portable and real time fluorescence spectroscopic device that provides multi-wavelength excitation. Then, we show its use on PpIX phantoms with tissues mimicking properties. This first enables to obtain a reference emitted spectrum for each state apart and then permits the development of a fitting model to adjust any emitted spectrum as a linear combination of the references in the spectral band 608-637 nm. Next, we present the steps led to get approvals for the clinical trial, especially the risk analysis. In vivo data analysis is then presented, showing that we detect fluorescence where current microscopes cannot, which could exhibit a change in PpIX state from glioma center to its margins. Besides, the relevance of multi-wavelength excitation is highlighted as the correlation between the three measured spectra of a same sample decreases with the density of tumor cells. Finally, the complementary need to intraoperatively identify cerebral functional areas is tackled with optical measurements as a perspective and other properties of PpIX on phantoms are also raised

La v-ATPase est une pompe à protons. Elle permet l’acidification d’organelles, ce qui est indispensable à de nombreux processus cellulaires. Cette enzyme est composée de 14 sous-unités différentes, organisées en deux domaines, le domaine catalytique V1 et le domaine membranaire V0. La sous-unité a du domaine V0 est essentielle au transport des protons. Il en existe 4 isoformes (a1 à a4) et des variants d’épissage (a1-I à a1-IV pour a1) permettant à la v-ATPase d’être adressée vers différents compartiments et donc d’être impliquée dans différents processus. Deux projets visant à étudier cette sous-unité ont été réalisés.En plus de son rôle dans le transport des protons, il a été montré que le domaine V0 de la v-ATPase est impliqué dans des évènements de trafic membranaire, tel que l’exocytose de vésicules de sécrétion. Ce rôle semble nécessiter des interactions avec les protéines SNAREs. J’ai montré, pendant la première partie de ma thèse, que les sous-unités flag-a1-I et flag-a1-IV sont toutes deux adressées aux granules de sécrétion de cellules neurosécrétrices et interagissent avec les protéines SNAREs VAMP2 et syntaxine-1. De façon intéressante la syntaxine-1 semble interagir préférentiellement avec la sous-unité a1-I qui dans les neurones est l’isoforme adressée aux terminaisons nerveuses. Les sous-unités a1-IV ne diffèrent d’a1-I que par l’ajout de 7 acides aminés dans sa moitié N-terminale. Le domaine d’interaction de la sous-unité a avec la syntaxine-1 semble donc être localisé dans cette région.Dans la deuxième partie de ma thèse, j’ai mis en évidence et étudié l’expression de la sous-unité rénale a4 dans des gliomes humains. Ces tumeurs sont les tumeurs cérébrales les plus fréquentes et sont en général associées à un mauvais pronostic. L’OMS distingue, en fonction de paramètres histologiques, les astrocytomes (de grade I à IV), les oligodendrogliomes et les gliomes mixtes (chacun de grade II ou III). Cette classification est controversée, notamment à cause de son manque de reproductibilité, et la prise en compte de marqueurs moléculaires semble s’imposer comme une solution pour la renforcer.J’ai quantifié par RT-PCR quantitative l’expression du gène ATP6V0A4 (codant la sous-unité a4) dans 188 prélèvements de gliomes humains. Nous avons ainsi montré que l’expression de la sous-unité a4 peut être utilisée comme marqueur diagnostique des oligodendrogliomes anaplasiques (35 % l’expriment). Dans un prélèvement, la présence de la codélétion 1p/19q et l’expression de a4, tout deux marqueurs indépendants des oligodendrogliomes, permettra le renforcement du diagnostique oligodendrogliome anaplasique. De plus a4 est fréquemment exprimée par les astrocytomes pilocytiques (70%), où elle est associée à la duplication en tandem de la région chromosomique 7q34 située à proximité directe du gène ATP6V0A4. Enfin une observation prometteuse est que l’expression de a4 pourrait être un marqueur de mauvais pronostic pour les patients atteints d’oligodendrogliome anaplasique ne présentant pas la co-délétion 1p/19q, observation qui devra être confirmée sur une plus grande cohorte de patients. / Vacuolar type H+-ATPase is a proton pump, which acidifies numerous organelles, crucial for many cellular processes. This enzyme is composed of 14 different subunits organized in two domains, a catalytic V1 domain and a V0 membrane domain. The a-subunit of V0 is essential for proton transport. There are 4 isoforms of a (a1 to a4) and splicing variants (a1-I to a1-IV for the a1 subunit). v-ATPases containing different a-subunit isoforms are localized in different compartments allowing v-ATPase to participate in different processes. The a-subunits were studied in this work in two distinct projects.Besides its role in proton pumping, V0 domain of v-ATPase is implicated in organelles trafficking events, like vesicles exocytosis. This role seems to require interactions of V0 with SNARE proteins. During my thesis work, I showed that flag-a1-I and flag-a1-IV are both targeted to secretion granules in PC12 neurosecretory cells. These subunits interact with the SNARE proteins VAMP2 and syntaxin-1. Interestingly, syntaxin-1 seems to preferentially interact with the a1-I subunit, isoform which in neurons is sorted to nerve terminals. The only difference between a1-I and a1-IV subunits is the addition of 7 amino acids in the N-terminal half of a1-IV. So syntaxin-1 probably interacts with a1-I at this location. In a second project, I studied the expression of the renal a4-subunit in human gliomas. These tumors are the most frequent brain tumors and are generally associated with a poor prognosis. Based on histological parameters,WHO distinguishes, astrocytomas (grade I to IV), oligodendrogliomas and oligoastrogliomas (each of grade II or III). This classification suffers of a lack of reproducibility, which could be overcome by the identification of specific molecular markers.In the present work, by real time quantitative PCR, ATP6V0A4 gene (encoding the renal a4) expression was quantified in 188 human glioma biopsies. We established a4 expression as a new marker of grade III oligodendrogliomas (35 % express it), independent of the 1p/19q codeletion, an established marker of oligodendrogliomas. Moreover, a4 is expressed in 70% of pilocytic astrocytomas, in which it is associated with the tandem duplication of 7q34, localized at direct proximity of the ATP6V0A4 gene. Of promising interest is the observation that a4 expression could be considered as a bad prognostic marker for patients with 1p/19q non-deleted oligodendrogliomas, an observation that should be confirmed on larger cohorts of patients.