Though most primary brain gliomas present as a single mass lesion in the brain, this potential diagnosis must be considered in the differential diagnosis when faced with a case of multifocal brain mass lesions. Among the most common brain tumors in humans, glioblastomas can be classified into four classes, one of which consists of anaplastic astrocytomas (AA). Due to its significant malignant potential, a prompt stereotactic brain biopsy should be considered to allow for early diagnosis. Karyotypic analysis of the specimen may allow for the discovery of 1p12q and IDH132 gene mutations. This knowledge can be used to best determine prognosis and guide therapy.

Glioblastoma (GBM) is the most common and malignant of the glial tumors. These tumors may develop from lower-grade astrocytomas (diffuse astrocytoma; grade II or anaplastic astrocytoma; grade III) through a progressive pathway, but, more frequently, they manifest de novo without any evidence of a pre-malignant lesion. The treatment of GBM includes surgery, radiotherapy, and chemotherapy with temozolomide. Despite improvements in treatment protocols, the median survival of GBM patients remains very low at 12-15 months. The cause of glioma (either development or progression) can be genetic and epigenetic modification driven changes. In contrast to genetic modifications, where DNA sequence is changed, epigenetic modifications are those gene expression regulatory mechanisms which do not involve the change in the DNA sequence. It includes DNA methylation, chromatin modifications and miRNA mediated changes in gene expression. Aberrant DNA methylation is one of the common molecular lesions occurring in the cancer cell. The 5th position of cytosine (CpG) is the most preferred site of DNA methylation in mammalian cells. The methylated cytosines are prone to undergo oxidative deamination, and get mutated to thymine in DNA. Consequently, this led to decrease in CpG abundance in the genome. In normal conditions, promoters of majority of the genes escape methylation, because of which CpG of these regions remain same. This phenomenon led to the restriction of CpGs in the promoter regions of most of the genes. These CpG rich regions of the promoters are known as CpG islands, and the methylation status of these islands have a major role in regulating gene expression. The cancer genome is shown to undergo genome-wide hypomethylation whereas CpG islands undergo hypermethylation compared to normal tissue, resulting in net loss of total methylation, as the CpGs from non-island areas far exceed in number compared to the CpGs from islands. The most studied change of DNA methylation in neoplasms is the silencing of the tumor suppressor genes by CpG island promoter hypermethylation. Apart from few studies, the role of DNA methylation in glioma development and progression is poorly known. With this background, we have focused our study on DNA methylation changes in GBM. To identify GBM specific DNA methylation alterations, we have performed the genome wide methylation profile of 44 GBM and 8 normal samples using Infinium methylation array. Beta value, which is a measure of methylation, was calculated for all the CpG probes. Beta value ranges between 0-1 (from no methylation to complete methylation). We sought to understand the clinical importance, with particular importance to patient survival, of the DNA methylation pattern observed. We also undertook steps to understand the contribution of the differential DNA methylation and the associated gene expression changes in GBM development. This work has been divided into three parts: Part I –Identification of GBM specific methylome and development of a DNA methylation prognostic signature for GBM To identify the differentially methylated genes in GBM, we compared the methylation levels of 27,578 CpGs between GBM and normal control samples using statistical methods. We then compared the list of differentially methylated genes with the expression data generated by The Caner Genome Atlas (TCGA) to find out genes whose expression oppositely correlates with the DNA methylation status. This resulted in the identification of 62 genes hypermethylated and down regulated, while 65 genes hypomethylated and up regulated. We believe that this set of differentially methylated genes may play important role in glioma development. Next, to identify GBM specific DNA methylation survival signature, we correlated the survival data of 44 GBM patients with beta values of all the 27,578 probes. Using Cox regression method, we identified a set of 9 genes, whose methylation predicted the survival in GBM patients. A risk score was then calculated using methylation values and regression co-efficient of each of the genes. The methylation risk score was found to be an independent predictor of survival in a multivariate analysis in TCGA data set and the Bent et al data set (independent validation sets). Using methylation risk score, we were able to divide the patients into low and high risk groups with significant difference in survival. To discover the biology behind the difference in the survival of low and high risk groups, we performed network analysis, using differentially expressed genes between low and high risk patients, which revealed an activated NFkB pathway association with poor prognosis. The inhibition of NFkB pathway sensitized the glioma cells for chemotherapeutic drugs only in NFkB activated cell lines, suggesting a pivotal role for NFkB pathway imparting chemoresistance in poor surviving group. Part II -NPTX2, a methylation silenced gene, inhibits NFkB through a p53-PTEN-PI3K-AKT signaling pathway To understand the mechanism behind the prediction of survival by methylation of 9 genes, we took NPTX2 as a candidate gene for further investigation. NPTX2, a risky methylated gene, is highly methylated in high risk group with poor survival, which suggests that it may have a growth inhibitory activity in GBM. Bisulphite sequencing confirmed the hypermethylation status of NPTX2 promoter in GBM samples and glioma cell lines compared to normal brain tissue. As expected, NPTX2 transcript level was significantly down regulated in GBMs and glioma cell lines compared to normal samples, and could be re-expressed upon methylation inhibitor treatment in glioma cells. Exogenous over expression of NPTX2 inhibited proliferation, colony formation and sensitized glioma cells to chemotherapeutic drugs. Moreover, NPTX2 also inhibited soft agar colony formation in vitro, which confirms its growth inhibitory function in GBM. As NPTX2 was methylated and silenced in the high risk group, which has high activation of NFkB pathway, we then checked if NPTX2 could inhibit NFkB activity. Indeed, we observed that NPTX2 overexpression inhibited expression from NFkB dependent luciferase reporter, sequence-specific DNA-binding of NFkB, nuclear translocation of NFkB sub unit (p65) and it also significantly repressed key NFkB target genes. We also show that NPTX2 mediated inhibition of NFkB could be abrogated by co-expression of constitutively active forms of PI3 kinase, AKT and IKKα, suggesting an involvement of PI3K-AKT-IKKα axis in NPTX2 mediated NFkB inhibition. Further, we found that NPTX2 repressed NFkB activity by inhibiting AKT through an ATM-p53-PTEN-PI3K dependent pathway. Thus, these results explain the need for hypermethylation and down regulation of NPTX2 in high risk GBM wherein the NFkB pathway is activated. Part III -Methylation silencing of ULK2, an autophagy gene, is important for astrocyte transformation and cell growth Among the differentially methylated genes (see part I), ULK2 was one of the most hypermethylated and down regulated genes. ULK2 is a known initiator protein in autophagy pathway, which is a type II cell death mechanism. There are many contradictory reports with respect to the role of autophagy in GBM development. For example, it has been shown that autophagy has a tumor suppressor activity and is essential for temozolomide mediated cell toxicity in GBM cells, whereas others studies implicate its involvement in tumor growth and progression. Hence, we carried out experiments to understand the role of ULK2 in GBM development. Using bisulphite sequencing, we validated ULK2 promoter hypermethylation status in GBM and glioma cell lines. In good correlation, ULK2 was found to be down regulated in GBMs and glioma cell lines, which was reexpressed by methylase inhibitor treatment in glioma cell lines. The over expression of ULK2 was found to inhibit colony formation, proliferation and soft agar colony formation of glioma cells. As expected, ULK2 overexpressing cells showed higher autophagy, compared to control cells. Interestingly, we also found increased apoptosis in ULK2 overexpressing cells. The cell death caused by ULK2 overexpression was compromised when cells were treated with 3-MA (an autophagy inhibitor) or Z-VAD-FMK (a pan caspase inhibitor). However, ULK2 failed to inhibit cell growth in autophagy deficient cells (ATG5-/-), thereby suggesting the importance of autophagy in ULK2 induced cell death. Further, ULK2 overexpression, increased catalase degradation and Reactive Oxygen Species (ROS) generation, which suggests that increase in ROS may play a role in ULK2 dependent cell death. In good correlation, N-Acetyl Cysteine, a ROS inhibitor, treatment rescued the cells from ULK2 mediated cell death, confirming the role of ROS in ULK2 induced cell death. Kinase deficient ULK2 overexpression failed to induce cell growth inhibition, autophagy and apoptosis, suggesting kinase activity of ULK2 is important for ULK2 function. Co-transfection of ULK2 inhibited Ras mediated transformation of immortalized normal human astrocytes. Taken together, we have identified and validated ULK2 as one of the DNA methylation silenced genes in GBM. ULK2 was found to be growth inhibitory in GBM cells by increasing autophagy dependent apoptosis. ULK2 inhibited Ras mediated transformation, suggesting essentiality of DNA methylation mediated ULK2 down regulation in GBM. In conclusion, the present work sheds light on the importance of methylation of genes in GBM progression. As observed, two of the genes, NPTX2 and ULK2 play as critical growth inhibitors in GBM. Also, we have identified a robust, independent and a highly sensitive 9 gene methylation signature, for GBM patient’s survival prediction.

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

Os astrocitomas são os tumores cerebrais primários mais frequentes, dentre os quais, o glioblastoma multiforme (GBM) é o tipo mais agressivo, sendo classificado como astrocitoma de grau IV. O tratamento envolve remoção cirúrgica seguida de quimio e radioterapias, porém essa abordagem não é eficaz devido à alta resistência das células tumorais. Em um trabalho anterior, buscando caracterizar os mecanismos associados à esta característica, investigamos a expressão de genes de reparo de DNA em astrocitomas de diferentes graus. Foram encontradas alterações em 19 genes. Através da combinação destas alterações em todos os arranjos possíveis, definimos um grande conjunto de assinaturas de expressão gênica que foi utilizado como filtro para a busca de correlação em bancos de dados públicos. No total, 421 assinaturas foram associadas à redução na sobrevida dos pacientes, sendo que cinco dos genes (EXO1, NEIL3, BRCA2, BRIP1 e DDB2) foram isoladamente relacionados ao pior prognóstico. Notavelmente, DDB2 foi o único gene subexpresso a apresentar esta correlação, levando a um risco de morte aproximadamente três vezes maior. No presente estudo in vitro, após radiação ionizante, observamos que células com baixos níveis de DDB2 reparam mais rapidamente as quebras induzidas no DNA, saem mais facilmente da parada de ciclo na fase G2/M e se tornam ainda mais resistentes a este tratamento. Além disso, o silenciamento de DDB2 induziu o aumento dos níveis de Zeb1, um importante promotor da transição epitélio-mesênquima, bem como dos índices de invasão e migração celular. Estes dados mostram que a redução nos níveis de DDB2 induz um fenótipo mais agressivo, corroborando a correlação com pior prognóstico dos pacientes observado anteriormente. Tomados em conjunto, esses resultados sugerem que a função de DDB2 não está limitada ao âmbito do reparo de DNA, apontam uma potencial relação com o controle da transição epitélio-mesênquima e mostram que este gene possui papel fundamental no estabelecimento da agressividade e resistência de GBM. / Astrocytomas are the most common primary brain tumors. Glioblastoma multiforme (GBM) is the most aggressive type and is classified as grade IV astrocytoma. The treatment involves surgical resection followed by chemo and radiotherapies, however this approach is not effective due to the high resistance of tumor cells. In a previous work, to characterize the cellular mechanisms associated to this characteristic, we investigated the expression of DNA repair genes in samples of different astrocytoma grades. We found alterations in 19 genes. By combining these expression changes in all possible arrangements, a large set of gene expression signatures was defined and used as a filter to seek correlations in public databases. As a result, 421 signatures were associated with reduced patient survival. Among them, five genes (EXO1, NEIL3, BRCA2, BRIP1 and DDB2) were individually related to worse prognosis. Notably, DDB2 was the only down regulated gene to exhibit this correlation, making the risk of death approximately three times higher. In the present in vitro study, after ionizing radiation, we observed that cells with low levels of DDB2 are capable of faster DNA breaks repair, easily exit the G2/M arrest and become even more resistant to this treatment. Furthermore, DDB2 silencing enhanced the levels of Zeb1, an important promoter of the epithelial-mesenchymal transition, as well as the rates of cell invasion and migration. These data indicate that DDB2 knockdown leads to a more aggressive cell behavior, corroborating the association with worse prognosis previously observed. Taken together, these results suggest that DDB2 function is not limited to DNA repair, they point out its potential participation in epithelial-mesenchymal transition control and show that this gene might play a key role in GBM\'s aggressiveness and resistance.

O estudo das alterações neoplásicas do sistema nervoso (SN) de cães apresenta enorme desafio devido suas particularidades, entre as espécies de animais domésticos o cão Canis familiares é o que apresenta a maior ocorrência destes neoplasmas. O desenvolvimento deste trabalho visa estabelecer o estudo anátomopatológico, molecular e epidemiológico em neurooncologia veterinária, avaliando pela técnica de imuno-histoquímica marcadores da proliferação celular PCNA e Ki-67, expressão da proteína p53 e marcadores de diferenciação celular. Foram utilizados neste estudo 18 animais, da espécie canina, que deram entrada ao Serviço de Patologia vinculado ao Hospital veterinário (HOVET) e ao Departamento de Patologia da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, e que tiveram diagnóstico de neoplasia intracraniana primária confirmado, entre eles (8) meningiomas e (3) astrocitomas. Não houve predileção sexual e a idade média do aparecimento tumoral foi de 9 anos. O valor médio do índice proliferativo (IP) de meningiomas benignos para PCNA é 4,8% e Ki-67 é 2,8%, no astrocitoma fibrilar o valor do IP é para PCNA 1% e Ki-67 1%, no astrocitoma anaplásico o valor do IP é para PCNA 10% e Ki-67 5% e no xantoastrocitoma pleomórfico o valor do IP é para PCNA 20% e Ki-67 4%.Não foi detectada alterações em p53 e o IP foi útil para definir o comportamento tumoral benigno ou maligno e dois tipos tumorais que não haviam sido descritos em cães: xantoastrocitoma pleomórfico e tumor de parênquima pineal de diferenciação intermediária puderam ser diagnosticados graças ao conjunto de informações coletadas, morfologia celular, marcadores de diferenciação celular e índice proliferativo. / The study of neoplastic alterations of dogs nervous system (NS) presents huge challenges due their particularities, between domestic animals species the dog Canis familiars presents the highest of these neoplasms. The development of this research aims to establish the anatomical-pathological, molecular and epidemiology study in veterinary neuro-oncology, evaluating by Immunohistochemistry technique markers of cell proliferation PCNA and Ki-67, expression of p53 protein and markers of cell differentiation. In this study were used 18 animals, dogs, which had entry at the Pathology Service of the Veterinary Hospital (HOVET) and at the Department of pathology from Faculdade de Medicina Veterinária e Zootecnia of University of São Paulo, which had confirmed diagnose of primary intracranial neoplasia, among them (8) meningiomas and (3) astrocytomas. There wasn\'t sexual preference and the mean age of tumor manifestation was 9 years old. The mean value of labelling index (LI) of benign meningiomas for PCNA is 4,8% and Ki-67 is 2,8%, in fibrillary astrocytoma the mean value of LI is 1% for PCNA and 1% for Ki-67, in anaplastic astrocytoma the mean value of LI is 10% for PCNA and 5% for Ki-67 and in pleomorphic xanthoastrocytoma the mean value of LI is 20% for PCNA and 4% for Ki-67. The p53 alterations wasn\'t detected and the LI was useful to set the benign or malign tumor behavior and two tumor types which had not been described in dogs: pleomorphic xanthoastrocytoma and pineal parenchymal tumour of intermediate differentiation could be diagnosed through the set of information collected, cell morphology, markers of cell differentiation and labelling index.

O estudo das alterações neoplásicas do sistema nervoso (SN) de cães apresenta enorme desafio devido suas particularidades, entre as espécies de animais domésticos o cão Canis familiares é o que apresenta a maior ocorrência destes neoplasmas. O desenvolvimento deste trabalho visa estabelecer o estudo anátomopatológico, molecular e epidemiológico em neurooncologia veterinária, avaliando pela técnica de imuno-histoquímica marcadores da proliferação celular PCNA e Ki-67, expressão da proteína p53 e marcadores de diferenciação celular. Foram utilizados neste estudo 18 animais, da espécie canina, que deram entrada ao Serviço de Patologia vinculado ao Hospital veterinário (HOVET) e ao Departamento de Patologia da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, e que tiveram diagnóstico de neoplasia intracraniana primária confirmado, entre eles (8) meningiomas e (3) astrocitomas. Não houve predileção sexual e a idade média do aparecimento tumoral foi de 9 anos. O valor médio do índice proliferativo (IP) de meningiomas benignos para PCNA é 4,8% e Ki-67 é 2,8%, no astrocitoma fibrilar o valor do IP é para PCNA 1% e Ki-67 1%, no astrocitoma anaplásico o valor do IP é para PCNA 10% e Ki-67 5% e no xantoastrocitoma pleomórfico o valor do IP é para PCNA 20% e Ki-67 4%.Não foi detectada alterações em p53 e o IP foi útil para definir o comportamento tumoral benigno ou maligno e dois tipos tumorais que não haviam sido descritos em cães: xantoastrocitoma pleomórfico e tumor de parênquima pineal de diferenciação intermediária puderam ser diagnosticados graças ao conjunto de informações coletadas, morfologia celular, marcadores de diferenciação celular e índice proliferativo. / The study of neoplastic alterations of dogs nervous system (NS) presents huge challenges due their particularities, between domestic animals species the dog Canis familiars presents the highest of these neoplasms. The development of this research aims to establish the anatomical-pathological, molecular and epidemiology study in veterinary neuro-oncology, evaluating by Immunohistochemistry technique markers of cell proliferation PCNA and Ki-67, expression of p53 protein and markers of cell differentiation. In this study were used 18 animals, dogs, which had entry at the Pathology Service of the Veterinary Hospital (HOVET) and at the Department of pathology from Faculdade de Medicina Veterinária e Zootecnia of University of São Paulo, which had confirmed diagnose of primary intracranial neoplasia, among them (8) meningiomas and (3) astrocytomas. There wasn\'t sexual preference and the mean age of tumor manifestation was 9 years old. The mean value of labelling index (LI) of benign meningiomas for PCNA is 4,8% and Ki-67 is 2,8%, in fibrillary astrocytoma the mean value of LI is 1% for PCNA and 1% for Ki-67, in anaplastic astrocytoma the mean value of LI is 10% for PCNA and 5% for Ki-67 and in pleomorphic xanthoastrocytoma the mean value of LI is 20% for PCNA and 4% for Ki-67. The p53 alterations wasn\'t detected and the LI was useful to set the benign or malign tumor behavior and two tumor types which had not been described in dogs: pleomorphic xanthoastrocytoma and pineal parenchymal tumour of intermediate differentiation could be diagnosed through the set of information collected, cell morphology, markers of cell differentiation and labelling index.

Yeung, Tak Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 181-197). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iv / Acknowledgements --- p.vi / List of Publications --- p.vii / Presentation --- p.vii / List of Abbreviations --- p.viii / Abbreviations in Figures --- p.xiii / Abbreviations in Symbols --- p.xiv / List of Cell Lines Used in this Study --- p.xv / Table of Contents --- p.xvi / List of Figures --- p.xxv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Cellular Redox State and Cancer Biology --- p.1 / Chapter 1.2 --- Reactive Oxygen Species (ROS) --- p.1 / Chapter 1.3 --- Regulation of Cellular Redox State by Intrinsic and Extrinsic Antioxidant Systems --- p.5 / Chapter 1.3.1 --- Intrinsic Antioxidant System --- p.6 / Chapter 1.3.2 --- Extrinsic Antioxidant System --- p.8 / Chapter 1.4 --- Glutathione --- p.9 / Chapter 1.4.1 --- General Information of Glutathione --- p.9 / Chapter 1.4.2 --- Functions of Glutathione --- p.12 / Chapter 1.4.2.1 --- As an Antioxidant and Free Radical Scavenger --- p.12 / Chapter 1.4.2.2 --- As a Detoxifier --- p.13 / Chapter 1.4.2.3 --- As a Regulator of Cell Signaling --- p.14 / Chapter 1.4.3 --- Synthesis of Glutathione --- p.15 / Chapter 1.4.4 --- Catabolism of Glutathione --- p.15 / Chapter 1.4.5 --- Transport and Uptake of Glutathione --- p.16 / Chapter 1.4.6 --- Glutathione in Cancer Biology --- p.18 / Chapter 1.4.6.1 --- "Role of Glutathione in the Regulation of Carcinogenesis, Growth and Apoptosis of Cancer Cells" --- p.18 / Chapter 1.4.6.1.1 --- Role of Glutathione in Carcinogenesis --- p.18 / Chapter 1.4.6.1.2 --- Role of Glutathione in the Growth of Cancer Cells --- p.20 / Chapter 1.4.6.1.3 --- Role of Glutathione in Apoptosis of Cancer Cells --- p.21 / Chapter 1.4.6.2 --- Role of Glutathione in the Regulation of Metastasis --- p.23 / Chapter 1.4.6.3 --- Role of Glutathione in Cancer Resistance and Therapy --- p.24 / Chapter 1.4.6.3.1 --- Role of Glutathione in Cancer Resistance --- p.24 / Chapter 1.4.6.3.2 --- Role of Glutathione in Cancer Therapy --- p.24 / Chapter 1.5 --- Aims of the Present Study --- p.25 / Chapter Chapter 2 --- In Vitro Study of Bαicαlein and Baicalin on Glutathione Depletion --- p.28 / Chapter 2.1 --- Introduction --- p.28 / Chapter 2.1.1 --- Scutellaria bαicαlensis Georgi --- p.28 / Chapter 2.1.1.1 --- General Clinical Applications to Treat or Prevent Diseases --- p.28 / Chapter 2.1.1.2 --- As an Antioxidant and Free Radical Scavenger --- p.29 / Chapter 2.1.1.3 --- Long History for Treatment of Cancers with the Obscure Mechanism --- p.30 / Chapter 2.1.1.4 --- Major Components --- p.31 / Chapter 2.1.2 --- Baicalein and Baicalin --- p.32 / Chapter 2.1.2.1 --- General Clinical Applications to Treat or Prevent Diseases --- p.32 / Chapter 2.1.2.2 --- As an Antioxidant and Free Radical Scavenger --- p.33 / Chapter 2.1.3 --- Hypothesis: Baicalein and Baicalin Induce Cancer Cell Death Via Glutathione Depletion --- p.35 / Chapter 2.2 --- Materials and Methods --- p.36 / Chapter 2.2.1 --- Chemicals --- p.36 / Chapter 2.2.2 --- Buffers and Solutions --- p.36 / Chapter 2.2.3 --- Animals --- p.37 / Chapter 2.2.4 --- Preparation of Rat Brain Microsomes --- p.37 / Chapter 2.2.5 --- Glutathione Depletion Assay In Vitro and Thiol Depletion Assay in Rat Brain Microsomes --- p.38 / Chapter 2.2.6 --- Statistical Analysis --- p.39 / Chapter 2.3 --- Results --- p.40 / Chapter 2.3.1 --- Effects of Baicalein and Baicalin on Sulfhydryl Contents of Glutathione --- p.42 / Chapter 2.3.2 --- Effects of Baicalein and Baicalin on Sulfhydryl Contents of Rat Brain Microsomes --- p.42 / Chapter 2.4 --- Discussion --- p.44 / Chapter Chapter 3 --- Effects of Baicalein and Baicalin on Proliferation of Different Human Cancer and Normal Cells --- p.45 / Chapter 3.1 --- Introduction-Importance of Developing A Novel Compound Inducing Cancer Cells to Cell Death with the Least Side Effects on Normal Cells --- p.45 / Chapter 3.2 --- Materials and Methods --- p.46 / Chapter 3.2.1 --- Instruments --- p.46 / Chapter 3.2.2 --- Chemicals and Cell Culture Reagents --- p.46 / Chapter 3.2.3 --- Buffers --- p.46 / Chapter 3.2.4 --- Cell Lines --- p.47 / Chapter 3.2.5 --- Cell Culture --- p.48 / Chapter 3.2.6 --- Determination of Cell Proliferation by MTT Assay --- p.49 / Chapter 3.3 --- Results --- p.51 / Chapter 3.3.1 --- Anti-Proliferative Effects of Baicalein and Baicalin on Different Cancer Cell Lines --- p.51 / Chapter 3.3.2 --- Effects of Baicalein on Different Normal Cell Lines --- p.56 / Chapter 3.4 --- Discussion --- p.58 / Chapter 3.4.1 --- Anti-Proliferative Effects of Baicalein and Baicalin on Different Cancer Cell Lines --- p.58 / Chapter 3.4.2 --- Effects of Baicalein on Cell Proliferation on Different Human Normal Cell Lines --- p.60 / Chapter Chapter 4 --- Glutathione-Depleting Effects of Baicalein on Cell Proliferation of Different Cell Lines --- p.61 / Chapter 4.1 --- Introduction-Brain Tumors --- p.61 / Chapter 4.1.1 --- Types and Classifications of Brain Tumors --- p.61 / Chapter 4.1.2 --- "Incidence Time, Patient Survival Time and Rate for" --- p.65 / Chapter 4.1.3 --- Symptoms and Diagnostic Methods for Brain Tumors --- p.66 / Chapter 4.1.4 --- "Treatments, Side Effects and Difficulties of Treatments for Brain Tumors" --- p.67 / Chapter 4.1.5 --- Glutathione Levels in Brain Normal and Cancer Cells --- p.69 / Chapter 4.2 --- Materials and Methods --- p.70 / Chapter 4.2.1 --- Instruments --- p.70 / Chapter 4.2.2 --- Chemicals --- p.70 / Chapter 4.2.3 --- Buffers --- p.70 / Chapter 4.2.4 --- Determination of Cell Proliferation by MTT Assay --- p.70 / Chapter 4.2.5 --- Determination of Intracellular Glutathione Depletion by Fluorescent Dye CMAC --- p.71 / Chapter 4.2.6 --- Determination of Cellular Reduced Glutathione Levels by DTNB-Coupled Glutathione Reductase Recycling Assay --- p.73 / Chapter 4.3 --- Results --- p.75 / Chapter 4.3.1 --- Effects of Baicalein on Intracellular GSH Levels and Cell Proliferation for Different Cell Lines --- p.75 / Chapter 4.3.2 --- Basal Intracellular Glutathione in Different Cell Lines --- p.81 / Chapter 4.4 --- Discussion --- p.84 / Chapter 4.4.1 --- Intracellular Glutathione Depletion and Cell Death Induction Effects of Baicalein on Different Cell Lines --- p.84 / Chapter 4.4.2 --- Relationship between Basal Glutathione Levels and Drug Susceptibilities --- p.85 / Chapter Chapter 5 --- Effects of Baicalein on Apoptosis and Caspase Pathways --- p.88 / Chapter 5.1 --- Introduction-Modes of Cell Death --- p.88 / Chapter 5.1.1 --- Necrosis --- p.88 / Chapter 5.1.2 --- Apoptosis --- p.89 / Chapter 5.2 --- Materials and Methods --- p.92 / Chapter 5.2.1 --- Chemicals --- p.92 / Chapter 5.2.2 --- Buffers --- p.92 / Chapter 5.2.3 --- Determination of Change of Mitochondrial Membrane Potential by JC-1 --- p.93 / Chapter 5.2.4 --- Determination of Apoptosis by Annexin V-Propidium Iodide Staining --- p.94 / Chapter 5.2.5 --- Determination of Cell Cycle Arrest by Propidium Iodide Staining --- p.95 / Chapter 5.2.6 --- "Determination of Caspase-3, -8 and -9 Activities by Fluorescent-Labeled Peptides" --- p.96 / Chapter 5.2.7 --- Determination of DNA Fragmentation --- p.97 / Chapter 5.2.8 --- Terminal Deoxynucleotidyl Transferase Mediated dUTP End Labeling (TUNEL) Assay --- p.99 / Chapter 5.2.9 --- Flow Cytometry --- p.101 / Chapter 5.3 --- Results --- p.102 / Chapter 5.3.1 --- Effects of Baicalein on Mitochondrial Membrane Potential by JC-1 Staining --- p.102 / Chapter 5.3.2 --- Effects of Baicalein on Apoptosis and Necrosis by Annexin V-Propidium Iodide Staining --- p.104 / Chapter 5.3.3 --- Effects of Baicalein on Cell Cycle Arrest by Propidium Iodide Staining --- p.108 / Chapter 5.3.4 --- "Effects of Baicalein on Caspase-3, -8 and -9 Activities" --- p.110 / Chapter 5.3.5 --- Effeets of Baiealein on DNA Fragmentation --- p.115 / Chapter 5.3.6 --- Effects of Baicalein on TUNEL Assay --- p.117 / Chapter 5.4 --- Discussion --- p.120 / Chapter Chapter 6 --- Pro-Oxidant Role of Baicalein on Reactive Oxygen Species Generation --- p.122 / Chapter 6.1 --- Introduction --- p.122 / Chapter 6.2 --- Materials and Methods --- p.122 / Chapter 6.2.1 --- Chemicals --- p.122 / Chapter 6.2.2 --- Determination of Cellular Reactive Oxygen Species Generation by Fluorescent Dye cDCFDA --- p.123 / Chapter 6.2.3 --- Determination of Mitochondrial Reactive Oxygen Species Generation by Fluorescent Dye Rhl23 --- p.124 / Chapter 6.3 --- Results --- p.125 / Chapter 6.3.1 --- Effects of Baicalein on Cellular ROS Generation by Fluorescent Dye cDCFDA --- p.125 / Chapter 6.3.2 --- Effects of Baicalein on Mitochondrial ROS Generation by Fluorescent Dye Rhl23 --- p.129 / Chapter 6.4 --- Discussion --- p.132 / Chapter Chapter 7 --- The Anticancer Mechanistic Study of Baicalein --- p.133 / Chapter 7.1 --- Introduction --- p.133 / Chapter 7.2 --- Materials and Methods --- p.134 / Chapter 7.2.1 --- Chemicals --- p.134 / Chapter 7.2.2 --- Reversibility of Baicalein-Induced GSH Depletion and Cell Death by Different Antioxidant Treatments --- p.134 / Chapter 7.2.3 --- Reversibility of Baicalein-Induced Cellular ROS Generation --- p.136 / Chapter 7.2.4 --- Reversibility of Baicalein-Induced Apoptosis by Co-Treatment of Different Antioxidants and Caspase Inhibitors --- p.137 / Chapter 7.2.5 --- "Reversibility of Baicalein-Induced Caspase-3, -8 and -9 Activation by Co-Treatment of Different Antioxidants" --- p.138 / Chapter 7.3 --- Results --- p.139 / Chapter 7.3.1 --- Reversibility of Baicalein-Induced GSH Depletion and Cell Death by Different Antioxidant Treatments --- p.139 / Chapter 7.3.1.1 --- Pre-treatments --- p.139 / Chapter 7.3.1.2 --- Co-treatments --- p.141 / Chapter 7.3.1.3 --- Post-treatments --- p.144 / Chapter 7.3.2 --- Reversibility of Baicalein-Induced Cellular ROS Generation by Co-Treatment of Different Antioxidants --- p.147 / Chapter 7.3.3 --- Reversibility of Baicalein-Induced Apoptosis by Co-Treatment of Different Antioxidants and Caspase Inhibitors --- p.152 / Chapter 7.3.4 --- Reversibility of Baicalein-Induced Caspase-3 Activation by Co-Treatment of Different Antioxidants --- p.156 / Chapter 7.3.5 --- Reversibility of Baicalein-Induced Caspase-8 and -9 Activation by Co-Treatment of Different Antioxidants --- p.160 / Chapter 7.4 --- Discussion --- p.164 / Chapter 7.4.1 --- Reversibility of Baicalein-Induced GSH Depletion and Cell Death --- p.164 / Chapter 7.4.2 --- "Reversibility of Baicalein-Induced ROS Generation," --- p.167 / Chapter 7.5 --- Concluding Remarks --- p.168 / Chapter Chapter 8 --- General Discussion --- p.169 / Chapter 8.1 --- Drug Delivery to Brain --- p.169 / Chapter 8.2 --- Protective Roles of Baicalein on Brain Cells --- p.170 / Chapter 8.2.1 --- Actions Against Oxidative Stress --- p.170 / Chapter 8.2.2 --- Actions Against Other Neurotoxic Damages --- p.171 / Chapter 8.2.3 --- Actions Against Neuronal Diseases --- p.172 / Chapter 8.3 --- Anticancer Roles of Baicalein on Astrocytoma --- p.173 / Chapter 8.4 --- Implications on the Dual Roles of Baicalein: Antioxidant and Pro-oxidant --- p.175 / Chapter 8.5 --- Future Perspectives --- p.175 / Chapter 8.5.1 --- Effects of Baicalein on Antioxidant System --- p.175 / Chapter 8.5.2 --- Effects of Baicalein on GSH Synthesis --- p.176 / Chapter 8.5.3 --- In Vivo Studies on Cytotoxic Effects of Baicalein --- p.177 / Chapter 8.5.4 --- In Vivo Studies on Anti-Tumor Effects and In Vitro Studies on Anti-Metastasis Effects of Baicalein --- p.178 / Reference List --- p.181

Gliomas are the most common primary brain tumours, and their capacity to invade surrounding normal brain prevents complete removal of the tumour. Malignant glioma has still a poor prognosis. However, with the rapid development of molecular biology our understanding about glioma has increased dramatically. Among known growth factors, EGF and its receptor are frequently amplified and over expressed in malignant glioma. Therefore, it is of interest to find approaches to hamper the activity of EGF/EGFR. The aim of this thesis was to identify and characterize human analogues to a recently identified gene in Drosophilia, kekkon-1, which negatively regulates the activity of Drosophilia EGF receptor. In the first part, we set up a quantitative real-time RT-PCR assay, which showed good linearity, reproducibility and uniformity. We analyzed the expression of the most commonly used reference genes, and showed that 18S was the most reliable endogenous reference gene in this study. In the second part, we cloned, identified, and sequenced a gene family, which we named leucine-rich repeats and immunoglobulin–like domains family (LRIG). The LRIG gene family had three vertebrate paralogs and one homolog in ascidiacea. The proteins encoded by human LRIG genes shared an overall structure with a signal peptide, 15 tandems leucine-rich repeats with N- and C- terminal flanking regions followed by 3 immunoglobulin-like domains, a transmembrane domain, and a cytoplasmic tail. Northern blot showed the mRNA sizes to be 5.5 kb for LRIG1, 4.8 kb for LRIG2, and 5.1 kb for LRIG3. LRIG1-3 mRNAs were detected in all human and mouse tissues analyzed, however, at various levels. FISH and BLAST analysis showed that LRIG1 was located at 3p14, LRIG2 at 1q13, and LRIG3 at 12q13. LRIG1 was shown to be down-regulated in several cancer cell lines and proposed to be a tumour suppressor gene. In the third part, we analysed the expression of LRIG gene family in human astrocytic tumours. LRIG1-3 mRNAs were detected in all human glioma cell lines, in primary tumour tissues and control-matched normal brain tissues, at various levels. Subcellular localizations of LRIG1-GFP fusion proteins were visualized in nuclear, perinuclear, and cytoplasmic compartment. According to the predicted protein sequences, short peptides were synthesized and used to raise antibodies in rabbits. The antibodies were used for immunohistochemical analysis of LRIG1-3 in 404 human astrocytic tumours in a tissue micro array. The pattern of immunoreactivity of LRIG1-3 was heterogeneous with staining in nuclear, perinuclear and cytoplasmic compartment of positive tumour cells. Perinuclear staining of LRIG1-3 displayed a significant inverse correlation with WHO grade and especially positive LRIG3 perinuclear and cytoplasmic staining correlated with a low proliferation index. The LRIGs correlated with survival, and LRIG3 perinuclear staining was in addition to tumour grade an independent prognostic factor. The results suggest that LRIGs may play a role in normal tissue, and may be of importance in the pathogenesis and prognosis of tumours. The exact function of LRIG1-3 remains to be established.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 78-93).

Gliomas are a common type of brain tumour and for the treatment of a patient it is important to determine the tumour’s grade of malignancy. This is done today by a biopsy, a histopathological analysis of the tumourous tissue, that is classified by the World Health Organization on a malignancy scale from I to IV. Recent studies have shown that the local image variance (LIV) and the intratumoural susceptibility signal (ITSS) in susceptibility-weighted MR images correlate to the tumour grade. This thesis project aims to develop a software program as aid for the radiologists when grading a glioma. The software should by image analysis be able to separate the gliomas into low grade (I-II) and high grade (III-IV). The result is a graphical user interface written in Python 3.4.3. The user chooses an image, draws a region of interest and starts the analysis. The analyses implemented in the program are LIV and ITSS mentioned above, and the code can be extended to contain other types of analyses as research progresses. To validate the image analysis, 16 patients with glioma grades confirmed by biopsy are included in the study. Their susceptibility-weighted MR images were analysed with respect to LIV and ITSS, and the outcome of those image analyses was tested versus the known grades of the patients. No statistically significant difference could be seen between the high and the low grade group, in the case of LIV. This was probably due to hemorrhage and calcification, characteristic for some tumours and interpreted as blood vessels. Concerning ITSS a statistically significant difference could be seen between the high and the low grade group (p < 0.02). The sensitivity and specificity was 80% and 100% respec- tively. Among these 16 gliomas, 11 were astrocytic tumours and between low and high grade astrocytomas a statistically significant difference was shown. The degree of LIV was significantly different between the two groups (p < 0.03) and the sensitivity and specificity were 86% and 100% respectively. The degree of ITSS was significantly different between the two groups (p < 0.04) and the sensitivity and specificity were 86% and 100% respectively. Spearman correlation showed a correlation between LIV and tumour grade (for all gliomas r = 0.53 and p < 0.04, for astrocytomas r = 0.84 and p < 0.01). A correlation was also found between ITSS and tumour grade (for all gliomas r = 0.69 and p < 0.01, for astrocytomas r = 0.63 and p < 0.04). The results indicate that SWI is useful for distinguishing between high and low grade astrocytoma with 1.5T imaging within this cohort. It also seems possible to distinguish between high and low grade glioma with ITSS.