Thesis (MMed (Biomedical Sciences. Anatomy and Histology))--University of Stellenbosch, 1993. / Glial fibrillary acidic protein (GFAP) is found in varying amounts in the cytoplasm of most normal and neoplastic cells of astroglial origin. Though not glial specific, immunoelectron microscopy has shown that vimentin and GFAP are coexpressed as monomers of glial intermediate filaments. These structures display irreversible assembly and a slow metabolic turnover. Although currently applied as astroglial markers, these intermediate filament proteins may reflect the functional and developmental differentiation status of the cells in which they are expressed. Some authors have tried to apply these aspects as diagnostic parameters for grades of malignancy and anaplasia whilst other workers have indicated variable concentrations of GFAP in different astroglial cell types and entities. Different processing protocols, including the use of epoxy and acrylic resins, omission of osmium tetroxide and variations in concentration and incubation time of primary fixatives, were evaluated to find a compromise between antigen availability and acceptable ultrastructure. Thin sections were labelled on grid for GFAP (Dako A561) and vimentin (Dako M725) by means of the indirect immunogold method. For semi- quantification of relative antigen concentrations, a novel method was devised to calculate the labelling density, percentage heterogeneity of the particle distribution and the surface area investigated. This allowed expression of labelling results as a three figure unit. Standardized post-embedding immunoelectron microscopy was performed on 11 normal and neoplastic human tissue specimens. The tissue was exposed to conventional immersion fixation in glutaraldehyde and osmium tetroxide prior to modified embedding in LR White resin. The validity of these results was verified by correlation with conventional histopathological, immunohistochemical and clinical data obtained for each specimen. The presence of epoxy resin in thin sections was shown to reduce antigen availability to such an extent that very low to negative labelling was encountered. Acrylic LR White resin allowed more acceptable immunodetection, but at the cost of inferior ultrastructure and greater instability of thin sections in the electron beam. This masked the effects of glutaraldehyde fixation on the density of the tissuefixative matrix which included destruction of the vimentin and some GFAP associated epitopes. Although osmium tetroxide was required for acceptable ultrastructure, it reduced the labelling sensitivity by 20% and was responsible for premature curing of acrylic resin during impregnation of tissue. Despite superior resolution gained by electron microscopy and the advantage of semi-quantification of labeling results, the labelling sensitivity of this technique is lesser than that of light microscopical immunohistochemistry. Immunoelectron microscopy confirmed the association between GFAP and glial intermediate filaments in almost all the glial tumours studied, correlating well with GFAP expression in matching specimens demonstrated at light microscopical level. In the absence of intermediate filaments, no positivity for GFAP or vimentin was found in oligodendroglial components of mixed tumours. GFAP positivity in astrocytomas was demonstrated by between 17 and 126 particles / µm2, whilst lower figures were obtained for the glioblastoma (PD = 8) and some of the mixed gliomas (Pd = 6). Rosenthal fibres showed both peripheral and central positive labelling for GFAP, thus providing more evidence for their hypothetical degenerative, astroglial nature. The meningioma studied, was GFAP negative, but produced low density positivity for vimentin. Coexpression of GFAP and vimentin was demonstrated in an astroblastoma and degenerative infant brain tissue, thus supporting the presence of both these proteins development of glial structures. Although sites of likely glial intermediate filament synthesis were found, the antigen availability for vimentin was too low to allow a reliable assessment of specific vimentin localization and determination of the GFAP : vimentin ratio in individual intermediate filaments and/or astroglial fibres. Variations in particle densities (PD) which demonstrated GFAP in the various astroglial entities studied, were considered to be a result of variable technical and tissue processing factors rather than truly significant differences in expression of GFAP in individual intermediate filaments. This lead to the conclusion that the GFAP concentration / glial intermediate filament area is likely to be constant for mature glial intermediate filaments and therefore cannot be used to distinguish between different astroglial cells or entities. Whether each cell has a different number of glial intermediate filaments, has not been established satisfactorily. Following complementary conventional immunohistochemistry and careful orientation of biopsy material, the procedure can be applied to suitable specimens for the electron microscopical localization of high concentrations of aldehyde resistant, cytoplasmic antigens.

Yang, Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 222-244). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Awards and Presentations --- p.ii / Abstract in English --- p.iii / Abstract in Chinese --- p.vii / Table of Contents --- p.x / List of Tables --- p.xv / List of Figures --- p.xvii / List of Abbreviations --- p.xx / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Gliomas --- p.1 / Chapter 1.1.1 --- Oligodendroglial tumors (OTs) --- p.3 / Chapter 1.1.2 --- Glioblastoma multiforme (GBM) --- p.3 / Chapter 1.1.3 --- Molecular pathology of gliomas --- p.4 / Chapter 1.1.3.1 --- Genetic alterations in OTs --- p.4 / Chapter 1.1.3.2 --- Prognostic and predictive factors in OTs --- p.7 / Chapter 1.1.3.3 --- Genetic alterations in GBM --- p.8 / Chapter 1.1.3.4 --- Prognostic and predictive factors in GBM --- p.10 / Chapter 1.2 --- microRNA(miRNA) --- p.13 / Chapter 1.2.1 --- miRNA biogenesis and function --- p.13 / Chapter 1.2.2 --- miRNA involvement in cancer --- p.17 / Chapter 1.2.2.1 --- Dysregulation of miRNAs in human malignancies --- p.17 / Chapter 1.2.2.2 --- Function and potential application of miRNAs --- p.17 / Chapter 1.2.3 --- Role of miRNAs in glioma --- p.19 / Chapter 1.2.3.1 --- miRNAs in OTs --- p.19 / Chapter 1.2.3.2 --- miRNAs in GBM --- p.20 / Chapter 1.3 --- miR-137 --- p.30 / Chapter 1.3.1 --- Biology of miR-137 --- p.30 / Chapter 1.3.2 --- Role of miR-137 in carcinogenesis --- p.33 / Chapter 1.3.2.1 --- Deregulation of miR-137 in cancer --- p.33 / Chapter 1.3.2.2 --- Regulation of miR-137 expression in cancer --- p.33 / Chapter 1.3.2.3 --- Biological functions of miR-137 in cancer --- p.37 / Chapter 1.3.3 --- Role of miR-137 in differentiation and neurogenesis --- p.39 / Chapter CHAPTER 2 --- AIMS OF STUDY --- p.43 / Chapter CHARPTER 3 --- MATERIALS AND METHODS --- p.45 / Chapter 3.1 --- Tumor samples --- p.45 / Chapter 3.2 --- Cell lines and culture conditions --- p.48 / Chapter 3.3 --- Fluorescence in situ hybridization (FISH) --- p.49 / Chapter 3.4 --- Cell transfection --- p.52 / Chapter 3.4.1 --- Transfection of oligonucleotides --- p.52 / Chapter 3.4.1.1 --- Oligonucleotide preparation --- p.52 / Chapter 3.4.1.2 --- Optimization of transfection condition --- p.52 / Chapter 3.4.2 --- Cotransfection of plasmids and miRNA mimic --- p.53 / Chapter 3.4.2.1 --- Optimization of transfection condition --- p.53 / Chapter 3.4.2.2 --- Procedure of transfection --- p.54 / Chapter 3.5 --- Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) --- p.55 / Chapter 3.5.1 --- RNA extraction from frozen tissues and cell lines --- p.55 / Chapter 3.5.2 --- qRT-PCR for miR-137 --- p.56 / Chapter 3.5.3 --- qRT-PCR for CSE1L and ERBB4 transcripts --- p.57 / Chapter 3.6 --- 5-aza-2'-deoxycytidine (5-aza-dC) and Trichostatin A (TSA) treatment --- p.61 / Chapter 3.7 --- Western blotting --- p.62 / Chapter 3.7.1 --- Preparation of cell lysate --- p.62 / Chapter 3.7.2 --- Measurement of protein concentration --- p.62 / Chapter 3.7.3 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.63 / Chapter 3.7.4 --- Electroblotting of proteins --- p.67 / Chapter 3.7.5 --- Immunoblotting --- p.67 / Chapter 3.8 --- Dual-luciferase reporter assay --- p.70 / Chapter 3.8.1 --- Construction of reporter plasmids --- p.70 / Chapter 3.8.1.1 --- Experimental outline --- p.70 / Chapter 3.8.1.2 --- PCR Amplification of MREs --- p.70 / Chapter 3.8.1.3 --- TA cloning --- p.71 / Chapter 3.8.1.4 --- Transformation --- p.72 / Chapter 3.8.1.5 --- Blue/white screening and validation of recombinants --- p.72 / Chapter 3.8.1.6 --- Subcloning of 3'UTR fragments into pMIR-reproter vector --- p.73 / Chapter 3.8.2 --- Site-directed mutagenesis --- p.74 / Chapter 3.8.3 --- Plasmid and miRNA mimic cotransfection --- p.76 / Chapter 3.8.4 --- Determination of luciferase activity --- p.76 / Chapter 3.9 --- Functional assays : --- p.79 / Chapter 3.9.1 --- Cell growth and proliferation assay --- p.79 / Chapter 3.9.1.1 --- "3-(4，5-Dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay" --- p.79 / Chapter 3.9.1.2 --- Cell counting --- p.80 / Chapter 3.9.1.3 --- 5-Bromo-2'-deoxyuridine (BrdU) incorporation assay --- p.80 / Chapter 3.9.2 --- Apoptosis assay --- p.82 / Chapter 3.9.3 --- Anchorage-independent growth assay --- p.82 / Chapter 3.9.4 --- Wound healing assay --- p.83 / Chapter 3.9.5 --- Matrigel invasion assay --- p.84 / Chapter 3.9.6 --- Cell differentiation assay --- p.85 / Chapter 3.10 --- Immunohistochemical analysis --- p.86 / Chapter 3.10.1 --- H&E staining --- p.86 / Chapter 3.10.2 --- Detection of Ki-67 expression --- p.87 / Chapter 3.10.3 --- Detection of CSE1L expression --- p.87 / Chapter 3.10.4 --- Scoring methods --- p.88 / Chapter 3.11 --- Bioinformatic analysis --- p.90 / Chapter 3.12 --- Statistical analysis --- p.92 / Chapter CHAPTER 4 --- RESULTS --- p.93 / Chapter 4.1 --- Expression of miR-137 in glioma cells and clinical significance --- p.93 / Chapter 4.1.1 --- Description of 36 OT samples --- p.93 / Chapter 4.1.2 --- miR-137 level in oligodendroglial tumors and glioma cells --- p.102 / Chapter 4.1.3 --- "Association of miR-137 expression with clinicopathological features, lp/19q status and Ki-67 expression" --- p.104 / Chapter 4.2 --- miR-137 levels in glioma cells after demethylation treatment --- p.113 / Chapter 4.3 --- Biological effects of miR-137 overexpression in glioma cells --- p.118 / Chapter 4.3.1 --- Cell growth --- p.118 / Chapter 4.3.1.1 --- Cell viability --- p.118 / Chapter 4.3.1.2 --- Cell number --- p.123 / Chapter 4.3.1.3 --- Cell cycle analysis : --- p.127 / Chapter 4.3.2 --- Anchorage-independent cell growth --- p.130 / Chapter 4.3.3 --- Cell apoptosis --- p.134 / Chapter 4.3.4 --- Cell motility --- p.136 / Chapter 4.3.5 --- Cell differentiation : --- p.142 / Chapter 4.4 --- Identification of miR-137 targets --- p.144 / Chapter 4.4.1 --- In silico prediction of potential miR-137 targets --- p.144 / Chapter 4.4.2 --- Experimental validation of miR-137 targets by dual-luciferase reporter assay --- p.147 / Chapter 4.4.3 --- "Expression of miR-137 candidate targets, CSE1L and ERBB4 in glioma cells" --- p.152 / Chapter 4.4.4 --- Effects of miR-137 on CSE1L transcript and protein levels --- p.154 / Chapter 4.5 --- Expression of CSE1L in OTs --- p.156 / Chapter 4.5.1 --- CSE1L expression in OTs by qRT-PCR and IHC --- p.156 / Chapter 4.5.2 --- Correlation of CSE1L expression with clinicopathological features --- p.165 / Chapter 4.6 --- Effects of CSE1L knockdown in glioma cells --- p.168 / Chapter 4.6.1 --- Cell growth --- p.170 / Chapter 4.6.1.1 --- Cell viability --- p.170 / Chapter 4.6.1.2 --- Cell number --- p.173 / Chapter 4.6.1.3 --- Cell cycle analysis --- p.176 / Chapter 4.6.2 --- Anchorage-independent cell growth --- p.179 / Chapter 4.6.3 --- Cell apoptosis --- p.182 / Chapter 4.6.4 --- Cell motility --- p.184 / Chapter CHAPTER 5 --- DISCUSSION --- p.190 / Chapter 5.1 --- Expression of miR-137 transcript level in OTs and glioma cell lines --- p.190 / Chapter 5.2 --- Association of miR-137 expression with OT clinical and molecular parameters --- p.192 / Chapter 5.3 --- Prognostic significance of clinical features and miR-137 expression in OTs --- p.194 / Chapter 5.4 --- Inactivation mechanisms of miR-137 in glioma --- p.196 / Chapter 5.5 --- Biological effects of miR-137 overexpression in glioma cells --- p.198 / Chapter 5.6 --- CSE1L is a novel miR-137 target in glioma --- p.200 / Chapter 5.7 --- Expression of CSE1L in glioma --- p.203 / Chapter 5.8 --- Intracellular distribution of CSElL in OTs --- p.206 / Chapter 5.9 --- Correlation of CSE1L expression with clinicopathological and molecular features in OTs --- p.208 / Chapter 5.10 --- CSE1L mediates effects of miR-137 in glioma cells --- p.210 / Chapter 5.11 --- Biological roles of CSE1L in glioma cells 226}0Ø. --- p.212 / Chapter 5.11.1 --- CSE1L in glioma cell proliferation --- p.212 / Chapter 5.11.2 --- CSE1L in glioma cell apoptosis --- p.213 / Chapter 5.11.3 --- CSE1L in glioma cell invasion --- p.215 / Chapter CHAPTER 6 --- CONCLUSIONS --- p.216 / Chapter CHAPTER 7 --- FUTURE STUDIES --- p.219 / Chapter 7.1 --- Expression Molecular mechanisms for miR-137 inactivation in glioma --- p.219 / Chapter 7.2 --- Identification of more miR-137 targets in glioma --- p.219 / Chapter 7.3 --- Role of miR-137 and CSE1L in drug-induced apoptosis in glioma --- p.220 / Chapter 7.4 --- Deciphering dysregulated and clinical relevant miRNAs in glioma --- p.220 / Chapter 7.5 --- Effects of miR-137 in vivo and the therapeutic potential in glioma treatment --- p.221 / REFERENCES --- p.222

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.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Lectinas sÃo proteÃnas que pertencem a um grupo heterogÃneo de molÃculas com capacidade de ligaÃÃo especÃfica e reversÃvel a carboidratos. Desde sua descoberta, as lectinas se tornaram importantes ferramentas para a investigaÃÃo de fenÃmenos como a adesÃo, a migraÃÃo e a proliferaÃÃo celular, em condiÃÃes normais e patolÃgicas. Durante o processo de diferenciaÃÃo, cÃlulas tumorais apresentam vÃrios graus de modificaÃÃo na expressÃo de glicoproteÃnas de membrana. Neste aspecto, a investigaÃÃo da estrutura da membrana tumoral pode ser compreendida como um mÃtodo sensÃvel e especÃfico de diagnÃstico. Portanto, o reconhecimento de marcadores capazes de identificar e quantificar estas caracterÃsticas, pode ser usado como ferramenta de diagnÃstico. Neste trabalho propomos um modelo experimental para detectar marcadores de membrana de tumores primÃrios do Sistema Nervoso Central usando lectinas vegetais (Con A e Con Br) acopladas à cromÃforos. Amostras de tumores foram obtidas de pacientes que tinham diagnÃstico clÃnico e radiolÃgico de tumores do Sistema Nervoso Central, apÃs cirurgias realizadas no ServiÃo de Neurologia da Santa Casa de MisericÃrdia de Sobral. As amostras foram processadas e investigadas com tÃcnicas imunohistoquÃmicas, usando lectinas vegetais acopladas à fluoresceÃna, e microscopia de fluorescÃncia. Os resultados mostraram que meningiomas e gliomas apresentaram um padrÃo diferente de interaÃÃo com Con Br-FITC e Con A-FITC quando comparados ao controle (BSA/FITC). Estes dados sugerem que as lectinas vegetais estudadas podem ser ferramentas Ãteis na identificaÃÃo de marcadores de membrana em tumores primÃrios do Sistema Nervoso Central. / Lectins are proteins that belong to a heterogeneous group of molecules capable of binding specifically and reversibly to carbohydrates. Since its discovery, lectins have become important tools for investigating phenomena such as adhesion, migration and cell proliferation in normal and pathological conditions. In the process of differentiation, tumor cells display different degrees of modification in the expression of membrane glycoproteins. In this respect, the investigation of membrane structure tumor can be understood as a sensitive and specific method of diagnosis. Therefore, the recognition of markers capable of identifying and quantifying these characteristics can be used as diagnostic tool. In this paper we propose an experimental model to detect markers of the membrane of primary tumors of the central nervous system using plant lectins (Con A and Con Br) attached to the chromophores. Tumor samples were obtained from patients who had clinical and radiological diagnosis of tumors of the central nervous system after surgeries performed at the Department of Neurology, Santa Casa de Misericordia de Sobral. The samples were processed and investigated with immunohistochemical techniques, using plant lectins coupled to fluorescein, and fluorescence microscopy. The results showed that meningiomas and gliomas showed a different pattern for interaction with Con Br-FITC and Con A-FITC compared to control (BSA / FITC). These data suggest that the studied plant lectins can be useful tools in identifying membrane markers in primary tumors of the central nervous system.

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.

Measles virus (MV) causes an acute childhood disease, associated in certain cases with the infection of the central nervous system (CNS). MV induces a profound immunosuppression, resulting in high infant mortality. The major cellular receptor for MV is CD150, which binds MV hemagglutinin (MV-H). As dendritic cell (DC) dysfunction is considered to be essential for the MV immunopathogenesis, we analyzed consequences of MV-H interaction with DCs. We developed an experimental model allowing us to analyze the direct CD150-MV-H interaction in the absence of infectious context. This interaction caused the downregulation of surface expression of CD80, CD83, CD86 and HLA-DR molecules and inhibition of IL-12 production in DCs. DCs also failed to activate T cell proliferation. The CD150-MV-H interaction in DCs and B cells decreased the phosphorylation of JNK1/2, but not ERK1/2 kinases, after subsequent CD150 ligation with anti-CD150 antibodies. Moreover, MV-H by itself induced Akt phosphorylation via CD150 in DCs and B cells. Engagement of CD150 by MV-H in mice transgenic for human CD150 decreased the inflammatory reaction, contact hypersensitivity response, confirming the immunosuppressive effect of CD150-MV-H interaction in vivo. Furthermore, our studies revealed the CD150 expression in CNS tumors and identified the novel CD150 isoform, containing an additional 83bp exon expressed in lymphoid cells, DCs and CNS tumors. Although its isoforms remain intracellular in tumor cells, CD150 may represent a new marker for human brain tumors. Novel mechanism of CD150-induced immunosuppression and new CD150 isoform identified in these studies shed new light on its immunoregulatory role and CD150 isoform diversity and open perspectives for their clinical applications.

Le virus de la rougeole (MV) provoque une maladie sévère chez les enfants qui induit une forte immunosuppression et peut dans certains cas infecter le système nerveux central (SNC). La protéine CD150, principal récepteur cellulaire du virus, permet l’entrée du MV en se liant à l’hémagglutinine (MV-H). L’altération fonctionnelle des cellules dendritiques (DC) étant considérée comme essentielle dans l’immunopathogénèse du MV, nous avons analysé les conséquences de l’interaction de MV-H avec les DC. Nous avons développé un modèle expérimental qui nous permet d’étudier l’interaction directe entre CD150 et MV-H hors contexte infectieux. Nos résultats montrent que cette interaction provoque une diminution de l’expression des molécules de surface CD80, CD83, CD86, et HLA-DR, de la production d’IL-12 par les DC, et de la capacité des DC à stimuler la prolifération des lymphocytes T. L’interaction CD150-MV-H a inhibé la phosphorylation des protéines kinases JNK1/2 dans les DC et les lymphocytes B (LB) induite par la stimulation de CD150 par un anticorps spécifique, mais pas celle des kinases ERK1/2. Par ailleurs MV-H seule induit la phosphorylation d’Akt via CD150 dans les DC et les LB. La liaison de CD150 par MV-H a réduit la réponse inflammatoire chez les souris transgéniques exprimant CD150 humain, ce qui confirme l’effet de l’interaction de CD150 et MV-H in vivo. Nos études ont révélé l’expression de CD150 dans les tumeurs du SNC et l’existence d’une nouvelle isoforme de CD150. Cette isoforme contient un exon supplémentaire de 83 pb et est exprimée dans les cellules lymphoïdes et les DC en plus des tumeurs du SNC. Bien que l’expression de CD150 soit uniquement intracellulaire dans les cellules tumorales, elle peut représenter un nouveau marqueur pour les tumeurs cérébrales humaines. Cette étude apporte un éclairage nouveau sur le rôle immunorégulateur de CD150 et sur la diversité de ses isoformes, et ouvre ainsi de nouvelles perspectives pour leurs applications thérapeutiques. / Measles virus (MV) causes an acute childhood disease, associated in certain cases with the infection of the central nervous system (CNS). MV induces a profound immunosuppression, resulting in high infant mortality. The major cellular receptor for MV is CD150, which binds MV hemagglutinin (MV-H). As dendritic cell (DC) dysfunction is considered to be essential for the MV immunopathogenesis, we analyzed consequences of MV-H interaction with DCs. We developed an experimental model allowing us to analyze the direct CD150-MV-H interaction in the absence of infectious context. This interaction caused the downregulation of surface expression of CD80, CD83, CD86 and HLA-DR molecules and inhibition of IL-12 production in DCs. DCs also failed to activate T cell proliferation. The CD150-MV-H interaction in DCs and B cells decreased the phosphorylation of JNK1/2, but not ERK1/2 kinases, after subsequent CD150 ligation with anti-CD150 antibodies. Moreover, MV-H by itself induced Akt phosphorylation via CD150 in DCs and B cells. Engagement of CD150 by MV-H in mice transgenic for human CD150 decreased the inflammatory reaction, contact hypersensitivity response, confirming the immunosuppressive effect of CD150-MV-H interaction in vivo. Furthermore, our studies revealed the CD150 expression in CNS tumors and identified the novel CD150 isoform, containing an additional 83bp exon expressed in lymphoid cells, DCs and CNS tumors. Although its isoforms remain intracellular in tumor cells, CD150 may represent a new marker for human brain tumors. Novel mechanism of CD150-induced immunosuppression and new CD150 isoform identified in these studies shed new light on its immunoregulatory role and CD150 isoform diversity and open perspectives for their clinical applications.

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> / Doctorat en sciences médicales / info:eu-repo/semantics/nonPublished

Gliomas are the most common primary brain tumors and include astrocytomas, oligodendrogliomas and oligoastrocytomas. Astrocytomas have a high frequency of occurrence as compared to the other gliomas and several studies including ours have focused on understanding the etiology, biology and genetics of this disease. Based on the degree of malignancy, astrocytomas have been graded from I to IV. Grade I or pilocytic astrocytomas are benign tumors and have limited infiltration. On the contrary, Grade II-IV astrocytomas also referred to as diffusely infiltrating astrocytomas (DA, Grade II), anaplastic astrocytomas (AA, Grade III) and glioblastoma multiforme (GBM, Grade IV), have the tendency of diffusely infiltrating the normal brain parenchyma. GBM is characterized by uncontrolled proliferation and resistance to apoptosis, rampant invasion, recalcitrance to most established therapies etc which makes them the most aggressive of all gliomas with a median survival of about 12 months. This makes it imperative to initiate further studies to understand the molecular basis of this disease. Gene expression profiling studies have been central to this effort. In recent years, several Microarray studies have provided crucial insights into the biological role of novel genes not previously associated with astrocytomas. In a previous Microarray study, several differentially regulated genes in astrocytoma were identified in our laboratory. In addition to many up regulated genes, several down regulated genes were also identified in this study. Down regulated genes are interesting to study because of their relevance as possible tumor suppressor genes. Hence, we decided to characterize the regulation and functional significance of few down regulated genes. The specific objectives of the study are as follows 1)To validate novel down‐regulated genes in astrocytomas identified by a previous Microarray study. 2)To understand the mechanism of down-regulation of a few selected gene. 3)Functional characterization of DIRAS2, a novel astrocytoma down‐regulated gene with respect to its possible role in astrocytoma progression. Towards these objectives, we identified 21 genes as differentially down-regulated across all grades of astrocytoma based on a previous Microarray study from our lab and data from literature. Real time qRT-PCR analysis performed on these 21 genes confirmed their down-regulation in all grades of astrocytoma as compared to normal brain tissues. From these 21 genes, we short-listed 10 of the most consistently down-regulated genes for further analysis. These genes were DIRAS2, IGFBP9, MAL2, MBP, OLFM1, PACSIN1, RAB26, SYT1, SYT5 and VSNL1. We also confirmed the expression of two of the genes, OLFM1 and RAB26 at the protein level by performing immunohistochemical analysis on an independent set of 38 tissues that included 10 normal tissues and 28 tissues from different grades of astrocytoma. OLFM1 was found to be down-regulated in a grade specific manner. RAB26 expression was found to be strikingly high in all the low grade astrocytomas in comparison to high grade astrocytomas which made it an interesting gene to study functionally. On functional characterization, we found that RAB26 over‐expressing LN229 cells showed significantly reduced invasion compared to the vector transfected cells suggesting RAB26 could have a tumor-suppressing role in astrocytomas. In order to investigate whether transcriptional modulation could play a role in the down-regulation of these 10 genes, we searched for transcription factor binding sites in approximately 2kb 5’ flanking region of each gene. Intriguingly one or more PAX6 binding sites were present in all their promoters. In light of the fact that PAX6 has been proposed as a tumor‐suppressor in astrocytomas, we predicted that some of these genes could be targets of PAX6 transactivation and could possibly mediate some of the tumor‐suppressive actions of PAX6. PAX6 has been proposed as a down stream target of Notch signaling in the context of eye development. Similar to this observation, upon activation of Notch signaling with a virus expressing human intracellular domain of Notch (Ad-NIC-1), PAX6 expression was found to be induced in glioma cell lines suggesting PAX6 to be a novel NOTCH target in astrocytomas. In addition, Ad-NIC-1 infection could also induce the expression of OLFM1, RAB26, MAL2 and MBP in U343 cells. We could also demonstrate that Ad-NIC-1 co-operates with PAX6 in the regulation of these four genes in cell lines expressing endogenous PAX6, namely U343 and U251. Intriguingly, in a cell-line lacking PAX6 expression (LN229), Ad-NIC-1 could not induce OLFM1, RAB26 and MBP, although we could see induction of MAL2. Interestingly, PAX6 overexpression in LN229 cells in the absence of Ad-NIC-1 could induce OLFM1, RAB26 and MAL2. In contrast, infection of Ad-NIC-1 on the PAX6 over-expressing cells seemed to have an antagonistic effect on the expression of OLFM1, RAB26 and MBP, suggesting that Ad-NIC-1 antagonizes PAX6 actions in these cells. Ad-NIC-1 infection resulted in increased apoptosis in a PAX6 independent manner in U343 cells, which as previously mentioned has high levels of PAX6 endogenous expression. Conversely, Ad-NIC-1 could not induce apoptosis in LN229 cells, which has negligible expression of PAX6. We could also demonstrate that apoptosis induced in U343 cells could be in a p53 dependent manner. Activation of AMPK pathway and inhibition of the mTOR pathway as a consequence of p53 induction could also explain the Ad-NIC-1 mediated apoptosis that was seen in these cells. Thus, we have proposed that Notch signaling could possibly have a tumor-suppressing role in the presence of PAX6. We also suggest that down-regulation of OLFM1, RAB26, MAL2 and MBP via the NOTCH-PAX6 axis could be a possible molecular mechanism for the down-regulation of these genes. With respect to the third objective, we sought to characterize DIRAS2 with respect to its function in astrocytomas. DIRAS2 was identified as a down‐regulated gene in all grades of astrocytoma by our Microarray study. We were also able to validate the down‐regulation of DIRAS2 in all grades of astrocytomas. DIRAS2 also bears significant homology to RIG1 (also known as DIRAS1), which has been proposed as a tumor suppressor gene in astrocytomas. In the light of these data, we predicted that DIRAS2 could be a tumor suppressor gene in astrocytomas. Overexpression of DIRAS2 in two glioma cell lines U87 and C6 did not reveal any appreciable change in proliferation. Strikingly when the DIRAS2 over-expressing clones were grown in the absence of serum, there was marked increase in proliferation with respect to vector transfected clones along with a distinct change in morphology. Decorin expression in the DIRAS2 over-expressing clones was found to be up regulated and could be responsible for the altered morphology as well as enhanced viability in absence of serum. Interestingly along with Decorin expression, we also observed an increase in phosphor-SMAD2 levels indicative of activated TGF‐β signaling in the DIRAS2 over-expressing clones in the absence of serum. In the soft agar and migration/invasion assays, the results across the two cell lines, U87 and C6 were contrasting. DIRAS2 over-expressing clones of U87 cells formed visibly larger and increased number of colonies as compared to vector transfected clones and there was about a three fold increase in invasion with respect to that seen in vector transfected clones in the matrigel invasion assay. On the other hand, DIRAS2 over-expressing C6 clones formed colonies of smaller size compared to vector transfected clones and a marked decrease in migration was observed in the DIRAS2 over-expressing clones of C6. The discrepancies in the results in these two cell lines could be attributed to the presence of other regulators of DIRAS2 function unique to each of the two cell lines. Although in the present study, the results with respect to its predicted function as a tumor-suppressor gene has not been conclusive, the role of DIRAS2 in tumorigenesis may depend on the cellular context in which the protein is expressed. Overall in this study, we have identified a novel down regulated gene signature in astrocytomas consisting of OLFM1, RAB26, MAL2 and MBP. Furthermore, we have proposed that inhibition of NOTCH and PAX6 signaling pathways could be responsible for the down-regulated expression of OLFM1, RAB26, MAL2 and MBP in astrocytomas. Collectively, these results suggest that astrocytomas with activated Notch1 and/or Pax6 signaling could have good prognosis due to the tumor suppressive actions of OLFM1, RAB26, MAL2 and MBP

Indiana University-Purdue University Indianapolis (IUPUI)