Im ersten Teil meiner Arbeit habe ich den molekularen Mechanismus beschrieben, mit dem endogene neuronale Vorläuferzellen antitumorigen gegen Gliomstammzellen wirken. Unsere Forschungsgruppe hat in bereits veröffentlichten Arbeiten gezeigt, dass neuronale Vorläuferzellen zu experimentellen Gehirntumoren migrieren und Tumorzelltod induzieren können. In der nun vorliegenden Arbeit zeige ich, dass die neuronalen Vorläuferzellen nicht nur benefiziell gegen die Hauptpopulation der Tumorzellen wirken, sondern darüber hinaus auch die kleinere Population der sehr aggressiven Tumorstammzellen – mittels Sekretion von BMP7 – supprimieren. Insgesamt zeigt meine Arbeit, dass neuronale Vorläuferzellen die Pathogenität der Gliomstammzellen unterdrücken. Im zweiten Teil meiner Arbeit habe ich einen zellautonomen Mechanismus untersucht, der Gliomzellen in vitro und in vivo vermehrt expandieren lässt. Meine Ergebnisse zeigen, dass die Familie der ets-Transkriptionsfaktoren Gliomzellen zur Proliferation anregen, indem sie die Expresion eines Eisentransporters (dem Transferrin-Rezeptor-1) induzieren und damit die intrazelluläre Akkumulation von Eisenionen begünstigen. Die Veränderung des Redox-Gleichgewichts in den Gliomzellen regt die Tumore zu verstärkter Sekretion von Glutamat an. Dadurch werden die Gliome sehr zytotoxisch und induzieren Zelltod in den Zellen des tumorumgebenden Parenchyms. Das untergegangene Nervengewebe schafft damit den Platz, den der Tumor zur Expansion braucht. Insgesamt zeigt meine Arbeit, dass die ets1-induzierte CD71 Expression nicht nur das Tumorwachstum befördert, sondern auch den Platz zum Tumorwachstum schafft. / In my first part,Gliomas cells with stem-like properties (GSCs) control tumor growth and recurrence. Here, I showed that endogenous neural precursor cells (NPCs) perform an anti-tumor response by specifically targeting GSCs: In vitro, NPCs predominantly expressed BMP7; BMP7 was constitutively released from neurospheres and induced canonical BMP-signaling in GSCs. Exposure of human and murine GSCs to neurosphere-derived BMP7 increased GSC differentiation, attenuated GSC-marker expression, GSC self-renewal and the ability for tumor initiation.This anti-tumor response of NPCs protect the brain from gliomas by releasing BMP7, which acts as a paracrine tumor suppressor that represses proliferation, self-renewal and tumor-initiation of GSCs. In the 2nd part, Transferrin receptors (TfR) are overexpressed in brain tumors, but the pathological relevance has not been fully explored. Here, I showed that TfR is an important downstream effector of ets transcription factors that promotes glioma proliferation and increases glioma-evoked neuronal death. TfR mediates iron accumulation and reactive oxygen formation and thereby enhanced proliferation in clonal human glioma lines. TfR-induced oxidant accumulation modified cellular signaling by inactivating a protein tyrosine phosphatase (low-molecular-weight protein tyrosine phosphatase), activating mitogen-activated protein kinase and Akt and by inactivating p21/cdkn1a and pRB. Inactivation of these cell cycle regulators facilitated S-phase entry. Besides its effect on proliferation, TfR also boosted glutamate release, which caused NMDA mediated reduction of neuron cell mass. Overall my results indicate that TfR promotes glioma progression by two mechanisms, an increase in proliferation rate and glutamate production, the latter mechanism providing space for the progressing tumor mass.

Introduction. The inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC), are common in Western Europe (200-400 cases /100,000) and associated with substantial morbidity, although mortality is now low. There is presently a great unmet need for novel therapeutics in IBD as present agents are limited by lack of efficacy, toxicity and poor patient acceptance. Recent findings from genome-wide association studies (GWAS) have characterised the genetic architecture of CD and UC. Defects in innate and adaptive immunity have been clearly established, and substantial novel insights into disease pathogenesis have been gained. Over 30 genes / loci are now associated with CD; a number of these, along with a few specific loci, are also associated with UC. The hedgehog (HH) signalling pathway is critical to gastrointestinal development and plays key roles in intestinal and immune homeostasis. Furthermore, in addition to well described roles in tumorigenesis, it is evident that recapitulation of embryonic HH signals play critical roles in response to acute and chronic inflammatory challenge in diverse tissues. Aims. The main aims of the work presented in this thesis were to characterise the expression of key HH signalling components in the healthy and inflamed human intestine, establish whether germline variation in HH genes is associated with IBD and describe the in vitro responses of intestinal epithelial cells to pathogen associated molecular patterns. The WNT pathway, antagonised by HH in the intestine, and two HH target genes (NKX2.3 and CCL20) were also analysed for evidence of association with IBD. Methods. Expression of HH and WNT signalling components was described by immunohistochemistry and microarray analysis in healthy controls (HC), CD, UC, and non- IBD inflamed terminal ileal and colonic samples. Gene-wide haplotype-tagging studies were performed for GLI1 in Scottish, English and Swedish CD and UC, and Scottish early-onset colo-rectal cancer, IHH in Scottish IBD, NKX2.3 in Scottish and UK IBD, and CCL20 in Scottish, Swedish and Japanese IBD. Evidence for association of all HH (n=13) and WNT (n=27) signalling genes in CD was established by analysis of UK GWAS data and metaanalysis from UK, French/Belgium and N American studies. The effect of lipopolysaccharide (LPS) and muramyl dipeptide (MDP) on HH signalling was assessed in colonic epithelial cells (SW480). The effect of HH pathway agonists and antagonists on NFκB activity and cytokine expression was analysed in SW480 cells and peripheral blood mononuclear cells (HC and IBD patients) in vitro. Results. The expression of HH pathway ligand is present in the intestinal epithelium and the pathway response network in the lamina propria demonstrating the paracrine nature of HH signalling in the intestine. Immunohistochemical studies and microarray analysis demonstrates that HH pathway activity is decreased in all forms of colonic inflammation studied in man. Variation in Glioma-associated oncogene homolog 1 (GLI1), a key HH transcription factor located at 12q13 (IBD2), was associated with IBD (p<0.0001), UC (p<0.0001) and to a lesser extent CD (p=0.03) in Scotland, a finding replicated in English IBD and UC. This association was attributed to a non-synonymous SNP (rs2228226C→G) with pools odds ratio of 1.194 in meta-analysis of over 5000 individuals from Scotland, England and Sweden (p=0.0002). There was association of this SNP with early-onset colorectal cancer, but of borderline significance (p=0.05). The variant protein (Q1100E) is 50% less active than wild-type protein in vitro. IHH was not associated with CD or UC. Preliminary evidence was produced for association at SUFU (10q24; p=0.005), a GLI1- binding protein, and at the WNT3 / WNT9B locus (17q21; p=0.0005). MDP stimulation of colonic epithelial cells decreased HH pathway activity. Exogenous HH increased expression of CCL20. CCL20 promoter polymorphisms were associated with UC in Japanese patients (p=0.018) but not in Scotland or Sweden. NKX2.3 was associated with IBD in Scotland (UC>CD), but there was insufficient power for fine-mapping of causative variants. Conclusions. Multiple lines of evidence presented here demonstrate that the HH signalling pathway is involved in IBD pathogenesis. In key complementary in vivo studies (conceived by CWL; conducted in collaboration with the Gumucio lab in Ann Arbor) we have demonstrated that Gli1+/- mice develop early, severe colitis with high mortality in response to acute inflammatory challenge. Furthermore, lamina propria antigen presenting cells are identified as the key HH target cells. With HH agonists and antagonists in extensive preclinical and early clinical testing, these studies have real potential to translate into novel therapeutics for patients with IBD.

Notre étude est dédiée au développement d’un protocole d’évaluation du langage à visée clinique. Nous examinons les spécificités du traitement du langage oral à travers le filtre des fonctions langagières actualisées par la prosodie. Système composite par excellence, largement négligé tant dans l’étude du traitement du langage que dans les évaluations orthophoniques, la prosodie est cependant en étroite relation avec les composantes du langage et réalise par ce biais l’ensemble des fonctions structurantes du langage. Deux fonctions linguistiques de la prosodie sont ici testées en compréhension : une fonction syntaxique, sous-tendue par le marquage accentuel des frontières de syntagmes réalisé par un accent final et un accent initial, et une fonction pragmatique impliquant la fonction de focalisation initiée par un accent emphatique. Après avoir procédé à la normalisation de notre protocole auprès d’un groupe de sujets contrôles, notre évaluation a été intégrée à la prise en charge longitudinale de deux patients atteints de gliomes de grade II. Dans ce cadre spécifique, nous avons adapté nos épreuves au contexte de la chirurgie éveillée. Les capacités langagières des patients ont été testées en condition pré-opératoire afin d’observer l’impact cognitif de la tumeur et en condition post-opératoire afin d’évaluer les conséquences de l’exérèse. Les résultats soulèvent des questionnements concernant la spécialisation hémisphérique de la prosodie. Ils ont aussi permis de mesurer la sensibilité de notre protocole. L’étude exploratoire réalisée en condition peri-opératoire nous a permis de soulever les contraintes inhérentes à la méthode de stimulation électrique directe. / This study is dedicated to the development of an exploratory clinical protocol for language evaluation, where prosody is seen as the core system for assessing spoken language processing in a more comprehensive way than is usually the case in clinical studies. Indeed, prosody is usually largely neglected in studies on language processing, as well as in speech therapy and neuropsychological evaluations. However, prosody is tightly linked to the different components of language, and plays a central role in linguistic structuring. Two linguistic functions of prosody are tested here in language comprehension: 1) the syntactic function of prosody, where both final and initial accenting help delimit syntagmatic grouping ; 2) the pragmatic function of prosody, where prosodic focus brings salience to a specific part of discourse. The protocol was first normalized on a control group of healthy participants. We then ran the evaluation in a peri-surgical context on two patients suffering from grade II glioma. The protocol had to be adapted to the context of awake surgery. The language capacities of the patients were tested first in pre-surgical context in order to assess the cognitive consequences of the brain tumor, then in post-surgical context to evaluate the consequences of the tumor removal. Results raise questions regarding the hemispheric specialization of prosody, and more globally regarding the localization of language processing. The other interesting outcome of this research is to propose improvements for the evaluation of language capacities in a surgical context using direct brain electric stimulation.

L’angiogenèse est une composante majeure de l’agressivité des tumeurs malignes comme le glioblastome (GBM). Pourtant le traitement des patients par l’Avastin, un anticorps bloquant du VEGF ne leur confère qu’une augmentation limitée de la durée de survie sans progression. Les mécanismes de récurrence tumorale sont extrêmement complexes. Les glioblastomes sont en effet des tumeurs particulièrement hétérogènes sur le plan génétique, il existe très peu de marqueurs moléculaires d’expression fiables. La contribution à la récidive des potentiels angiogéniques, infiltrants, et souches est difficile à modéliser. Notre laboratoire s’intéresse à la caractérisation de nouveaux modulateurs de l’angiogenèse, dont certains pourraient contribuer à la croissance tumorale, indépendamment ou en aval du VEGF chez les patients traités par l’Avastin. Nous avons étudié l'expression de deux gènes candidats, surexprimés de façon significative dans les GBM et dont l'expression semble liée à l'angiogenèse tumorale : DPY19L1 et KIF20A. Nous avons identifié DPY19L1 comme marqueur pronostique du GBM. Ce gène est exprimé dans les cellules musculaires lisses, où il pourrait participer à la résistance de la tumeur aux anti-angiogéniques, en interagissant avec la voie thrombospondine/TGFβ. KIF20A quant à lui est exprimé dans les cellules souches tumorales et semble impliqué dans la vascularisation et la résistance tumorale. Dans un second temps, nous avons étudié la façon dont les GBM échappent aux traitements anti-angiogéniques, tel que l’Avastin, par la mise en place d'un système d'étude in vitro et in vivo, basé sur l’utilisation de cellules de patients atteints de GBM, ayant la capacité de pousser sous forme de neurosphères. Les cellules xénogreffées chez la souris immunodéfisciente permettent le développement d’une tumeur très invasive, co-optive et insensible aux traitements anti-angiogéniques. Ces tumeurs vont donc permettre d’étudier ce mode de vascularisation participant activement à la récidive de la tumeur chez les patients traités avec l’Avastin afin de développer des traitements contrecarrant ce mécanisme. Dans ce modèle, seul l’un des gènes candidats définis au début de ce travail, DPY19L1, semble participer à la croissance tumorale. / Angiogenesis is a major element driving malignancy of tumors like glioblastoma (GBM). However, Avastin,a neutralizing antibody directed against VEGF, provides only a limited therapeutical benefit in terms ofprogression free survival. The mechanisms of recurrence are complex due to extreme heterogeneity ofglioblastoma at the genetic and tissular levels. There is a lack of diagnosis and prognosis markers for GBM.The relative contribution of the angiogenic, infiltrative, and stem potentials to tumor relapse is difficult tomodel. Our laboratory aims at characterizing new modulators of tumor vascularization, some of whichcould contribute to the tumor growth and resistance, independently or downstream VEGF in patientstreated with Avastin. We have studied the expression of two candidate genes, significantly overexpressedin GBM and which expression seems to be linked to tumor vascularization: DPY19L1 and KIF20A. Weidentified DPY19L1 as a prognosis marker of GBM. This gene is expressed in smooth muscle cellsspecifically in tumoral tissue, where it could participate to tumor resistance to anti-angiogenics, byinteracting with the thrombospondin/TGFβ pathway. KIF20A is expressed in glioma stem cells and seemsto be implicated in the vascularization and tumor resistance. Next, we have studied the way by whichGBM resist to anti-angiogenics such as Avastin, by the development of an in vitro and in vivo modelsystem, based on GBM cells cultured as neurospheres. When xenografted in immunodeficient mice, thesecells induce the growth of very invasive, co-optive tumors which are insensitive to angiogenesis inhibitors.These tumors will allow investigating alternative modes of vascularization which are actively involved intumor recurrence in patients treated with Avastin, namely co-option and transdifferentiation and theirmolecular regulation. In this model, one of the candidate genes defined at the beginning of this study,DPY19L1, seems to be implied in tumor growth and specifically labels tumor cells with co-optive andtransdifferenciating properties.

abstract: Rapid intraoperative diagnosis of brain tumors is of great importance for planning treatment and guiding the surgeon about the extent of resection. Currently, the standard for the preliminary intraoperative tissue analysis is frozen section biopsy that has major limitations such as tissue freezing and cutting artifacts, sampling errors, lack of immediate interaction between the pathologist and the surgeon, and time consuming. Handheld, portable confocal laser endomicroscopy (CLE) is being explored in neurosurgery for its ability to image histopathological features of tissue at cellular resolution in real time during brain tumor surgery. Over the course of examination of the surgical tumor resection, hundreds to thousands of images may be collected. The high number of images requires significant time and storage load for subsequent reviewing, which motivated several research groups to employ deep convolutional neural networks (DCNNs) to improve its utility during surgery. DCNNs have proven to be useful in natural and medical image analysis tasks such as classification, object detection, and image segmentation. This thesis proposes using DCNNs for analyzing CLE images of brain tumors. Particularly, it explores the practicality of DCNNs in three main tasks. First, off-the shelf DCNNs were used to classify images into diagnostic and non-diagnostic. Further experiments showed that both ensemble modeling and transfer learning improved the classifier’s accuracy in evaluating the diagnostic quality of new images at test stage. Second, a weakly-supervised learning pipeline was developed for localizing key features of diagnostic CLE images from gliomas. Third, image style transfer was used to improve the diagnostic quality of CLE images from glioma tumors by transforming the histology patterns in CLE images of fluorescein sodium-stained tissue into the ones in conventional hematoxylin and eosin-stained tissue slides. These studies suggest that DCNNs are opted for analysis of CLE images. They may assist surgeons in sorting out the non-diagnostic images, highlighting the key regions and enhancing their appearance through pattern transformation in real time. With recent advances in deep learning such as generative adversarial networks and semi-supervised learning, new research directions need to be followed to discover more promises of DCNNs in CLE image analysis. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2019

<p>Malignant primary brain tumors, gliomas, often overexpress both platelet-derived growth factor (PDGF) ligands and receptors providing an autocrine and/or paracrine boost to tumor growth. Glioblastoma multiforme (GBM) is the most frequent glioma. Its aggressive and infiltrative growth renders it extremely difficult to treat. Median survival after diagnosis is currently only 14 months. </p><p>The present thesis describes the use of retroviral tagging to identify candidate cancer-causing genes that cooperate with PDGF in brain tumor formation. Newborn mice were injected intracerebrally with a Moloney murine leukemia retrovirus carrying the <i>sis</i>/PDGF-B oncogene and a replication competent helper virus. Brain tumors with many characteristics of human glioblastomas developed after 13-42 weeks. </p><p>Analysis of proviral integrations in the brain tumors identified almost 70 common insertion sites (CISs). These CISs were named brain tumor loci and harbored known but also putative novel cancer-causing genes.</p><p>An array with over 15000 unique cDNAs was used to screen for differentially expressed genes in the mouse brain tumors compared to normal brain. Known tumor genes and markers of immature cells were upregulated in the tumors. Short latency tumors were further distinguished as fast growing and GBM-like. Long latency tumors resembled slow-growing oligodendrogliomas and contained significantly less integrations as compared to short latency tumors.</p><p>The gene <i>Prkg2</i>, encoding the cGMP-dependent protein kinase II, was targeted by insertions in two brain tumors. Overexpression of <i>Prkg2</i> in human glioma cell lines led to a reduction in colony formation, cell proliferation and migration. A glioma cell line expressing markers of immature stem cells showed loss of cell adhesion, G1 cell cycle arrest and decreased activation of the survival signaling protein Akt upon stimulation with a cGMP analog that activates the <i>Prkg2</i> protein. The present thesis shows that proviral tagging may be a useful tool in the search for candidate glioma genes.</p>

Malignant primary brain tumors, gliomas, often overexpress both platelet-derived growth factor (PDGF) ligands and receptors providing an autocrine and/or paracrine boost to tumor growth. Glioblastoma multiforme (GBM) is the most frequent glioma. Its aggressive and infiltrative growth renders it extremely difficult to treat. Median survival after diagnosis is currently only 14 months. The present thesis describes the use of retroviral tagging to identify candidate cancer-causing genes that cooperate with PDGF in brain tumor formation. Newborn mice were injected intracerebrally with a Moloney murine leukemia retrovirus carrying the sis/PDGF-B oncogene and a replication competent helper virus. Brain tumors with many characteristics of human glioblastomas developed after 13-42 weeks. Analysis of proviral integrations in the brain tumors identified almost 70 common insertion sites (CISs). These CISs were named brain tumor loci and harbored known but also putative novel cancer-causing genes. An array with over 15000 unique cDNAs was used to screen for differentially expressed genes in the mouse brain tumors compared to normal brain. Known tumor genes and markers of immature cells were upregulated in the tumors. Short latency tumors were further distinguished as fast growing and GBM-like. Long latency tumors resembled slow-growing oligodendrogliomas and contained significantly less integrations as compared to short latency tumors. The gene Prkg2, encoding the cGMP-dependent protein kinase II, was targeted by insertions in two brain tumors. Overexpression of Prkg2 in human glioma cell lines led to a reduction in colony formation, cell proliferation and migration. A glioma cell line expressing markers of immature stem cells showed loss of cell adhesion, G1 cell cycle arrest and decreased activation of the survival signaling protein Akt upon stimulation with a cGMP analog that activates the Prkg2 protein. The present thesis shows that proviral tagging may be a useful tool in the search for candidate glioma genes.

The invasive nature of glioblastoma (GBM) represents a significant challenge to the standard of care and contributes to poor clinical outcomes. Invasion of tumors into healthy brain restricts chemotherapeutic access and complicates surgical resection. The central hypothesis of the thesis is that an effective anti-invasive agent can enhance the standard chemotherapeutic response in invasive brain tumors. Through a screen of novel compounds, a new anti-invasive small molecule, Imipramine Blue (IB), was identified. This triphenylmethane compound inhibits invasion of highly invasive glioma in vitro and in vivo. To elicit a response in vivo, Imipramine Blue was liposomally encapsulated to yield better delivery to tumor. Using this formulation, it is shown that IB attenuates invasion of glioma in vivo leading to a more compact tumor in an aggressively invasive rodent glioma model. Further, it is shown that this novel compound binds NADPH oxidases and alters expression of actin regulatory elements to elicit this anti-invasive activity. To test our hypothesis that anti-invasive therapy coupled with chemotherapy will enhance efficacy, nano-IB therapy was followed by liposomally encapsulated doxorubicin (DXR) chemotherapy. Additionally, a co-encapsulated formulation of IB and DXR was developed and tested in vivo. This combination therapy significantly enhanced survival compared to IB or DXR alone, resulting in long-term survival in the syngeneic invasive rat astrocytoma model RT2. It was seen that sequential treatment was more effective than the co-encapsulated treatment indicating a benefit of pre-treating the tumor with the anti-invasive. This thesis demonstrates that novel anti-invasive IB mediated 'containment' of diffuse glioma significantly enhances the efficacy of DXR chemotherapy compared to chemotherapy or anti-invasive therapy alone.

Brain and central nervous cancer presents a significant clinical burden, accounting for 2.4% of all cancer deaths. High grade glioma is particularly deadly, with 5 year survival times of 35% or less. Traditional treatment includes tumor resection followed by radiation therapy or chemotherapy. Aggressive resection is essential in order to prolong patient life. In fact, several studies have shown that life expectancy increases with increased extent of resection. Extent of resection is burdened by the fact that surgeons must be careful not to remove functional brain tissue. Resection is incomplete more often than not due to lack of visual cues for the surgeon. He must rely on tactile sensation to distinguish tumor from healthy tissue. Methods such as intraoperative MRI and CT exist, but these require expensive equipment and special training that is not available in all surgical environments. Some laboratories have proposed small molecule dyes to solve this problem, but these are insufficient when used in an invasive tumor model. It was the goal of this research to provide an objective cue in the form of a nanoencapsulated visible dye without the need for additional equipment of changes to the surgery process itself other than injection of the dye. We hypothesized that the nanocarrier would allow staining of the tumor through passive targeting by taking advantage of the enhanced permeability and retention effect. Once the nanocarriers have reached the desired target, they would not diffuse out into healthy tissue due to their large size compared to small molecule dyes, which readily diffuse out and stain healthy tissue. To test this hypothesis, we prepared and characterized a liposomal nanocarrier encapsulating Evans blue dye. The nanocarrier was tested for safety in vitro and in vivo, then used to delineate tumor margins in an invasive rat glioma model in vivo. Microscopic analysis was then conducted to ensure only tumor tissue was stained by the nanocarrier. This thesis presents a successful method of tumor border delineation to provide surgeons with positive visual cues without the need for changes in surgical environment or techniques.

No description available.