Children who have received radiation therapy for the treatment of brain tumors have been shown to experience neurocognitive deficits which appear to increase over time. The purpose of this study was to examine the memory functioning of 22 children irradiated for brain tumor and 22 healthy children of the same age who had not received irradiation. Subjects were administered a brief form of the WISC-III, to obtain an IQ, and the Wide Range Assessment of Memory and Learning (WRAML), to evaluate visual and verbal memory. Results indicated that, although there were no significant differences between the IQ scores of healthy children and children who had been irradiated, children who have received radiation therapy for brain tumor evidence memory deficits which effect visual and verbal memory abilities. Among the children who had been irradiated, as time since treatment increased, visual memory and overall memory functioning appeared to decline. Findings also suggested that children who received total tumor resection may evidence greater memory deficits than those who received only a partial resection. Visual memory was more closely related to IQ in the children irradiated for brain tumor than in the healthy children. The overall importance of research with this population lies in refining the understanding of memory deficits and strengths in order to formulate more effective remediation compensation, strategies.

To study potential hyperactivity and hyperconnectivity based on the latent resource hypothesis, this study assessed functional connectivity in survivors of childhood brain tumors compared to their healthy peers during an attention task using psychophysiological interaction (PPI) analyses and evaluated for a relationship with performance. Twenty-three survivors and 23 healthy controls completed a letter n-back task in the scanner. An empirically-based seed was placed in the parietal lobe, a theoretical seed was placed in the hippocampus, and a control seed was placed in the occipital lobe. Differences in both performance and functional connectivity networks from each seed emerged between groups, with some findings supporting the latent resource hypothesis and other networks showing compensatory function in survivors. Attention networks, phonological networks, and executive function networks were all found to differ between controls and survivors.

Gliomas are brain tumors that present with neurological symptoms including seizures and cognitive deficits. Starting at early stages of tumor development glioma cells diffusely infiltrate brain tissue where they interact with non-neoplastic cells including neurons and can perturb normal brain function. While the clinical consequences of glioma induced cortical dysfunction are well established, the neuronal alterations that underlie cortical dysfunction in glioma are unknown. We hypothesize that glioma cells infiltrate surrounding brain tissue and induce alterations in neurons that may contribute to the neurological symptoms associated with gliomas. Due to intermingling of glioma cells and neurons it has been challenging to isolate and characterize neurons from glioma brain tissue while preserving complex neuronal morphology. To address this issue we developed a new mouse glioma model that allowed us to obtain a neuron specific gene expression profile, otherwise obscured by the predominantly large population of glioma cells within the tumor. In this thesis I use this model to test the hypothesis that infiltrating glioma cells induce phenotypic alterations in neurons that contribute to the neurological symptoms associated with glioma. The Camk2a-Ribotag mouse glioma model enabled us to isolate neuron specific transcripts from glioma brain tissue. The Ribotag mouse has a conditional HA-tagged ribosomal protein (Rpl22) that can be expressed upon Cre-recombination. Camk2a is specifically expressed in excitatory neurons, the Camk2a-Cre mouse induces Cre-recombination in the Ribotag mouse so that Camk2a+ neurons selectively express HA-tagged Rpl22. We used the Camk2a-Ribotag glioma model to isolate neuron specific ribosome bound transcripts to characterize neuronal alterations in glioma. In chapter 2 of this thesis I describe how we developed and characterized the Camk2a-Ribotag mouse glioma model. We first obtained mouse glioma cells that have p53 deletion and overexpress PDGFRa, then we injected these cells in the Camk2a-Ribotag mouse and use this as our glioma model to extract neuron specific ribosome bound transcripts. This method is referred to as translating ribosome affinity purification (TRAP) which is used to obtain cell type specific translational profiles. Using this approach we identified alterations in neuronal gene expression, specifically we show that there is an upregulation of actin binding genes associated with dendritic spine morphology and a downregulation of synaptic genes associated synaptic regulation. We demonstrate that drebrin, an actin binding protein in dendritic spines, is upregulated in tumor brain synaptosomes, we also show a downregulation of dendritic spine density in HA-tagged neurons which suggests that these neuronal alterations contribute to synaptic dysfunction in our glioma model. Dendritic spines are dynamic structures that regulate synaptic function in response to diverse stimuli. mTOR signaling can regulate brain specific functions such as synaptic plasticity. Alterations in mTOR signaling can result in cognitive deficits, epilepsy and brain abnormalities that are associated with neurological disease. We hypothesized that mTOR regulates the neuronal alterations we identified in our glioma model. In chapter 3 of this thesis I describe how we tested this hypothesis by acutely inhibiting mTOR signaling with the ATP competitive inhibitor AZD8055 in the Camk2a-Ribotag mouse glioma model. Using TRAP we show that acute mTOR inhibition reverses many neuron specific alterations that occurs in the glioma infiltrated cortex, actin binding genes that were upregulated in tumor brains were downregulated after mTOR inhibition and synaptic genes that were downregulated in tumor brains were upregulated after mTOR inhibition. These results suggest that key neuron specific alterations are regulated by mTOR signaling in our glioma model. In chapter 4 of this thesis I describe how we used ribosome profiling to identify translational alterations in our Camk2a-Ribotag mouse glioma model. Ribosome profiling in an RNA sequencing based method that is used to measure translation efficiency by calculating the number of ribosomes per transcript. Using this approach we identified an upregulation in the translation of DNA methylation and demethylation gene ontologies. These results suggest that alterations in specific DNA methylation and demethylation gene ontologies are regulated at the level of translation and warrant further analysis of cell type specific translational alterations using ribosome profiling. The work described in this thesis demonstrates 1) use of the Camk2a-Ribotag mouse glioma model for the identification of neuron specific alterations, 2) neuron specific alterations include the upregulation of dendritic spine genes, downregulation of synaptic genes and downregulation of dendritic spine density, 3) acute mTOR inhibition reverses many of these neuronal alterations, 4) ribosome profiling revealed the translational upregulation of epigenetic genes in our mouse glioma model. The findings described in this thesis provide the first characterization of neuron specific transcriptional and translational alterations in glioma infiltrated cortex that and provide new insights into the mechanisms that underlie the devastating neurological symptoms in glioma patients.

Chan Ching Yin. / Thesis submitted in: October 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 197-221). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.iii / Abstract in Chinese --- p.vi / List of abbreviations --- p.ix / List of tables --- p.xv / List of figures --- p.xvi / Contents --- p.xviii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1. --- What are astrocytic tumors? --- p.1 / Chapter 1.1.1. --- Histological characteristics and classification --- p.2 / Chapter 1.1.2. --- Epidemiology --- p.2 / Chapter 1.1.3. --- Treatment and patient survival --- p.4 / Chapter 1.2. --- "Cytogenetics, molecular genetics and epigenetics of astrocytic tumors" --- p.6 / Chapter 1.2.1. --- Cytogenetics --- p.6 / Chapter 1.2.2. --- Genetic imbalances --- p.7 / Chapter 1.2.3. --- Tumor suppressor genes --- p.13 / Chapter 1.2.4. --- Oncogenes --- p.22 / Chapter 1.2.5. --- Primary and secondary GBMs --- p.26 / Chapter 1.3. --- Major pathways involved in astrocytic tumorigenesis --- p.30 / Chapter 1.3.1. --- Cell cycle dysregulation and suppression of apoptosis --- p.30 / Chapter 1.3.2. --- Promotion of proliferation and survival --- p.33 / Chapter 1.4. --- BRAF mutation in human cancers --- p.38 / Chapter 1.5. --- Other CNS tumors included in the current study --- p.52 / Chapter 2. --- Aims of study --- p.61 / Chapter 3. --- Materials and methods --- p.64 / Chapter 3.1. --- Clinical materials --- p.64 / Chapter 3.2. --- Cell lines --- p.75 / Chapter 3.3. --- Cell culture --- p.77 / Chapter 3.4. --- DNA extraction --- p.78 / Chapter 3.4.1. --- Pre-treatment of samples --- p.78 / Chapter 3.4.2. --- Cell lysis and protein removal --- p.80 / Chapter 3.4.3. --- Precipitation of DNA --- p.81 / Chapter 3.4.4. --- Determination of DNA concentration --- p.81 / Chapter 3.5. --- Mutation analysis of BRAF by cycle sequencing --- p.83 / Chapter 3.5.1. --- Amplification of BRAF exons --- p.83 / Chapter 3.5.2. --- Cycle sequencing and automated gel electrophoresis --- p.84 / Chapter 3.6. --- Immunohistochemistry of B-Raf and GFAP --- p.87 / Chapter 3.6.1. --- Pre-treatment of samples --- p.87 / Chapter 3.6.2. --- Detection of B-Raf and GFAP antigens by ABC method --- p.88 / Chapter 3.6.3. --- Controls --- p.90 / Chapter 3.7. --- Quantification of EGFR gene dosage by TaqMan based real-time PCR --- p.91 / Chapter 3.7.1. --- Preparation of gene constructs --- p.92 / Chapter 3.7.2. --- Primers and TaqMan probes --- p.93 / Chapter 3.7.3. --- Experimental condition and PCR program --- p.95 / Chapter 3.7.4. --- DNA standards --- p.95 / Chapter 3.7.5. --- Controls --- p.96 / Chapter 3.7.6. --- Experimental layout --- p.96 / Chapter 3.8. --- Microsatellite analysis of chromosome 14q in astrocytic tumors --- p.97 / Chapter 4. --- Results --- p.101 / Chapter 4.1. --- Mutation analysis of BRAF --- p.101 / Chapter 4.2. --- Immunohistochemistry of B-Raf protein --- p.107 / Chapter 4.3. --- Quantification of EGFR gene dosage --- p.117 / Chapter 4.4. --- Correlation between EGFR dosage and BRAF mutation --- p.128 / Chapter 4.5. --- Correlation between EGFR dosage and B-Raf expression --- p.129 / Chapter 4.6. --- Microsatellite analysis of chromosome 14q in astrocytic tumors --- p.131 / Chapter 5. --- Discussions --- p.149 / Chapter 5.1. --- BRAF mutations as common events in human cancers --- p.149 / Chapter 5.2. --- BRAF mutation in CNS tumor specimens --- p.150 / Chapter 5.2.1. --- Tumorigenic effect of the V599E substitution --- p.153 / Chapter 5.2.2. --- V599E B-Raf mutant activation independent of Ras activation --- p.155 / Chapter 5.2.3. --- Autocrine stimulation of Ras signaling in V599E B-Raf mutant --- p.156 / Chapter 5.3. --- BRAF expression in astrocytic tumors --- p.159 / Chapter 5.4. --- Mutually exclusive pattern between EGFR amplification and BRAF expression --- p.161 / Chapter 5.4.1. --- Similar effect of EGFR activation and B-Raf activation --- p.163 / Chapter 5.4.2. --- Mutual effects between Ras/Raf/Mek/Erk and Akt signaling --- p.164 / Chapter 5.5. --- Microsatellite analysis of chromosome 14q in human cancers --- p.167 / Chapter 5.6. --- Microsatellite analysis of chromosome 14q in astrocytic tumors --- p.170 / Chapter 5.6.1. --- Finer mapping of common regions of deletion --- p.170 / Chapter 5.6.2. --- Genes within the common regions of deletion --- p.173 / Chapter 5.6.3. --- Overlapping deletion regions in astrocytic and non-CNS tumors --- p.186 / Chapter 6. --- Further studies --- p.190 / Chapter 6.1. --- Role of BRAF alterations in astrocytic tumors --- p.190 / Chapter 6.2. --- B-Raf expression in astrocytic tumors and correlation with EGFR overexpression --- p.193 / Chapter 6.3. --- Microsatellite analysis of 14q in astrocytic tumors --- p.194 / Chapter 7. --- Conclusions --- p.195 / Chapter 8. --- References --- p.198

The third ventricle region houses several neuroanatomical structures that are primary components of the human memory system, and provides pathways through which these brain regions communicate with critical regions of the frontal and medial temporal lobes. Archival data was obtained for 42 children with cerebellar or third ventricle tumors, and was examined for tumor and treatment related confounds. Children with third ventricle tumors were hypothesized to exhibit; 1) better performance on a measure of auditory attention, 2) greater impairment in learning across trials, 3) greater memory loss over a 20-minute delay, and 4) greater impairment across delayed memory tests than the cerebellar group. Children with third ventricle tumors demonstrated significantly better auditory attention, but greater impairments in verbal learning, and greater verbal memory loss following a 20-minute delay. In contrast, children with third ventricle tumors did not demonstrate significantly greater memory impairments across long delay memory tests.

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Os gliomas representam mais de 70% dos tumores cerebrais primários. Os glioblastomas multiformes são gliomas malignos caracterizados por baixa incidência, mas altas taxas de mortalidade. Apesar da responsividade inicial ao tratamento padrão realizado com o quimioterápico alquilante temozolomida (TMZ), poucos foram os avanços para o prognóstico dos pacientes nos últimos 10 anos. Isso deve-se ao fato desses tumores serem raramente passíveis de ressecção cirúrgica e apresentarem alta taxa de recorrência. Além disso, a eficácia de seu tratamento encontra barreiras como efeitos colaterais indesejáveis e resistência quimioterápica. Nesse cenário, a descoberta de novas substâncias que possam atuar com efeito aditivo ou sinérgico e aumentem a sensibilização de células tumorais ao tratamento, torna-se uma estratégia terapêutica no campo da oncologia. O ditelureto de difenila (DTDF) é um composto orgânico contendo telúrio que apresenta interessantes efeitos biológicos in vitro, como antioxidante, quimioprotetivo, citotóxico e antitumoral. Sendo assim, o objetivo deste estudo foi avaliar os efeitos do DTDF e do quimioterápico TMZ, em regimes isolados e em associação. Para tal foram investigados seus efeitos citotóxicos, após exposição aguda e crônica, em culturas celulares de glioma não-resistente (M059J) e resistente à TMZ (GBM). No ensaio de viabilidade celular a TMZ apresentou citotoxicidade para as linhagens celulares testadas, com valor de IC50 maior na linhagem resistente do que quando comparado à linhagem não-resistente. Este dado foi confirmado pelo teste de duplicação cumulativa de população e, também, pela coloração com laranja de acridina, após o tratamento de 120 h, por observar-se um aumento na frequência de células positivas para a formação de organelas vesiculares ácidas em ambas as linhagens, sendo predominantemente em células GBM. Além disso, foi observado que o tratamento associando DTDF e a TMZ apresentou uma maior citotoxicidade quando comparado aos tratamentos isolados, após 120 h de tratamento. Portanto, o DTDF sensibilizou as células ao tratamento com TMZ. Essa sensibilização ocorreu em níveis aproximados para ambas as linhagens, sendo os efeitos do DTDF independentes do perfil de resistência à TMZ. Em conjunto, os dados desse trabalho sugerem o uso do DTDF em associação à TMZ como uma estratégia para reduzir as doses de TMZ empregadas na clínica e diminuir efeitos colaterais aos pacientes em tratamento de glioma / Gliomas represent more than 70% of primary brain tumors. Malignant gliomas are characterized by low incidence, but high mortality rates. Despite the initial responsiveness to the standard treatment with the chemotherapeutic alkylating temozolomide (TMZ), few advances have been made in the prognosis of patients in the last 10 years. This is due to the fact that these tumors are rarely amenable to surgical resection and have a high rate of recurrence. Moreover, the effectiveness of this treatment encounters barriers such as undesirable side effects and chemotherapeutic resistance. In this scenario, the discovery of new substances that may act with additive or synergistic effect and increase the sensitization of tumor cells to the treatment becomes a therapeutic strategy in the field of oncology. Diphenyl ditelluride (DPDT) is a derivative of tellurium used in various reactions of organic synthesis and has interesting in vitro biological effects, as antioxidant, chemoprotective, cytotoxic and antitumor agent. Therefore this work aimed to evaluate the cytotoxic effects of this organotellurium compound and the chemotherapeutic, TMZ, in isolated and in association regimens, after acute and chronic exposure, of non-resistant (M059J) and TMZ- resistant (GBM) glioma cells. Through the cell viability assay, it was shown that TMZ is cytotoxic for both cell lines tested, showing a higher IC50 value in the resistant line when compared to the other line. This data was confirmed by the cumulative population doubling test. In addition, by the acridine orange staining, it was verified that autophagy might favor the chemoresistance, although not being the main resistance mechanism in the lines tested. It was observed that DPDT clearly has a dose-dependent cytotoxic effect on the M059J and GBM cell lines, in a lower concentration range than that used with TMZ. DPDT sensitized the cells to TMZ treatment as evidenced by the decline in cell viability. It is important to point out that this sensitization occurred in low and approximate IC50 values after both 24 h and 120 h of treatment, being the effects of the DPDT independent of the resistance profile to TMZ. Taken together, data from this work suggest the use of DPDT in association with TMZ as an interesting strategy to reduce the doses of TMZ used in the clinic and to reduce side effects to patients under treatment of glioma.

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