Sobrevida após radioterapia em pacientes com glioma de alto grau Introdução: Gliomas de alto grau são os principais tumores primários do sistema nervoso central em adultos, cujo prognóstico permanece invariavelmente ruim, mesmo com a terapia atual: cirurgia e quimioirradiação. Na radioterapia utiliza-se de técnicas conformacionais (3DRT) e de intensidade modulada do feixe (IMRT). Objetivos: Avaliar a sobrevida dos pacientes tratados na instituição e os fatores que influenciam os resultados. Métodos: Análise retrospectiva dos tratamentos terapêuticos de pacientes diagnosticados com gliomas de alto grau entre 2009 e 2014 e tratados com radioterapia no Serviço de Radioterapia do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (HCRP-USP). Resultados: A sobrevida mediana obtida foi 16,63 meses, 29 pacientes (61,7%) foram à óbito e os demais encontravam-se em seguimento até o momento da análise. IMRT correspondeu à modalidade de tratamento em 68,1% dos casos, a duração média da radioterapia foi de 56 dias, com intervalo médio (demora) entre cirurgia e o início da radioterapia de 61,7 dias (27 a 123 dias). Observamos que idade, ressecção macroscópica total, tipo histológico e utilização de temozolomida adjuvante influenciaram a taxa de sobrevida (p<0,05). A sobrevida global estimada é de 18 meses (estimativa Kaplan Meyer). Nossa série avaliou os pacientes tratados e os dados corroboram os já relatados na literatura, respaldando o protocolo de tratamento institucional. / Introduction: High-grade gliomas are the main primary tumors of the central nervous system in adults, it`s prognosis remains invariably bad, even with current therapy: chemoradiation after surgery. Radiotherapy is currently employed as conformational techniques (3DRT) or intensity modulated beam (IMRT) with or without temozolomide. Objectives: To evaluate survival of patients treated with radiation and factors influencing results. Methods: Retrospective analysis of patients diagnosed with high-grade gliomas between 2009 and 2014 and treated with radiotherapy at the University Hospital of the Faculty of Medicine of Ribeirão Preto, University of São Paulo (HCRP-USP). Results: Median survival was 16.63 months, 29 patients (61.7%) died and the others were in follow-up at the time of analysis. IMRT was used in 68.1% of cases, the mean duration of radiotherapy obtained of 56 days, with a mean interval (delay) between the surgery/diagnosis and radiotherapy was 61.7 days (27-123 days). We found that age, total gross resection, histological type and use of adjuvant temozolomide influenced the survival rate (p <0.05). The estimated overall survival was 18 months (Kaplan Meyer estimator). Our data was similar to those reported in the literature, supporting the institutional treatment protocol.

Sobrevida após radioterapia em pacientes com glioma de alto grau Introdução: Gliomas de alto grau são os principais tumores primários do sistema nervoso central em adultos, cujo prognóstico permanece invariavelmente ruim, mesmo com a terapia atual: cirurgia e quimioirradiação. Na radioterapia utiliza-se de técnicas conformacionais (3DRT) e de intensidade modulada do feixe (IMRT). Objetivos: Avaliar a sobrevida dos pacientes tratados na instituição e os fatores que influenciam os resultados. Métodos: Análise retrospectiva dos tratamentos terapêuticos de pacientes diagnosticados com gliomas de alto grau entre 2009 e 2014 e tratados com radioterapia no Serviço de Radioterapia do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (HCRP-USP). Resultados: A sobrevida mediana obtida foi 16,63 meses, 29 pacientes (61,7%) foram à óbito e os demais encontravam-se em seguimento até o momento da análise. IMRT correspondeu à modalidade de tratamento em 68,1% dos casos, a duração média da radioterapia foi de 56 dias, com intervalo médio (demora) entre cirurgia e o início da radioterapia de 61,7 dias (27 a 123 dias). Observamos que idade, ressecção macroscópica total, tipo histológico e utilização de temozolomida adjuvante influenciaram a taxa de sobrevida (p<0,05). A sobrevida global estimada é de 18 meses (estimativa Kaplan Meyer). Nossa série avaliou os pacientes tratados e os dados corroboram os já relatados na literatura, respaldando o protocolo de tratamento institucional. / Introduction: High-grade gliomas are the main primary tumors of the central nervous system in adults, it`s prognosis remains invariably bad, even with current therapy: chemoradiation after surgery. Radiotherapy is currently employed as conformational techniques (3DRT) or intensity modulated beam (IMRT) with or without temozolomide. Objectives: To evaluate survival of patients treated with radiation and factors influencing results. Methods: Retrospective analysis of patients diagnosed with high-grade gliomas between 2009 and 2014 and treated with radiotherapy at the University Hospital of the Faculty of Medicine of Ribeirão Preto, University of São Paulo (HCRP-USP). Results: Median survival was 16.63 months, 29 patients (61.7%) died and the others were in follow-up at the time of analysis. IMRT was used in 68.1% of cases, the mean duration of radiotherapy obtained of 56 days, with a mean interval (delay) between the surgery/diagnosis and radiotherapy was 61.7 days (27-123 days). We found that age, total gross resection, histological type and use of adjuvant temozolomide influenced the survival rate (p <0.05). The estimated overall survival was 18 months (Kaplan Meyer estimator). Our data was similar to those reported in the literature, supporting the institutional treatment protocol.

Introdução: A capacidade funcional refere-se fundamentalmente à potencialidade humana para o desempenho ocupacional, imprescindível para uma melhor qualidade de vida. O objetivo desta pesquisa, aprovada pelo Comitê de Ética em Pesquisa do HCFMRP-USP (processo no 648/2008), foi avaliar a capacidade funcional de pacientes submetidos a neurocirurgia oncológica e sua relação com a qualidade de vida. Método: Durante o período de maio/08 a abril/09 (12 meses) foram avaliados 52 sujeitos adultos de ambos os sexos; o grupo experimental teve 26 pacientes, sendo 14 com diagnóstico de meningioma (grupo1) e 12 com diagnóstico de glioma de alto grau (grupo 2); e o grupo controle teve 26 sujeitos, subdivididos em grupos: 1A (pareado com o grupo 1) e 2A (pareado com grupo 2). Foram realizados dois tipos de dinamometria - avaliação de força de preensão (Grip TrackTM Testing) e de pinça (Pinch TrackTM Testing) com equipamento computadorizado Tracker SystemTM; aplicação de protocolos internacionais validados no Brasil - Hospital Anxiety and Depression Scale HAD, Item Short-Form Health Survey - SF-36 e Health Assessment Questionnaire HAQ, no período pré-operatório e no terceiro mês de pós-operatório. Na análise estatística foram aplicados os testes Mann-Whitney, Wilcoxon e Coeficiente de Correlação de Spearman Resultados: Constatou-se que nos três primeiros meses de pós operatório houve aumento da capacidade funcional dos sujeitos dos grupos experimentais (1 e 2) e diminuição dos sintomas de ansiedade e depressão; não houve diferença significativa nos testes de força no pré e no pós operatório, mas ambos os grupos apresentam diminuição de força no membro dominante em comparação com os grupos controle. Houve correlações de forte magnitude entre os dados coletados através dos testes Grip TrackTM Testing, Pinch TrackTM Testing; protocolos: HAD, HAQ e SF-36, com rho 0,600 a 0,969. Os domínios aspecto social e emocional do instrumento de qualidade de vida mostraram uma piora no pósoperatório imediato. Esses resultados corroboram a compreensão ampliada do conceito de funcionalidade, como proposto na Classificação Internacional de Funcionalidade e Incapacidade e Saúde\" (CIF). Conclusão: A funcionalidade está diretamente relacionada com a qualidade de vida, principalmente nos aspectos psicossociais, sendo necessária compreendê-la de forma mais ampla do que funções físicas específicas, para implementar planos de tratamentos mais adequados para os sujeitos com tumor cerebral, tanto no pré como no pós operatório, com acompanhamento de uma equipe multidisciplinar. / Introduction. Functional capacity refers mainly to the potential for human occupational performance, essential for a better quality of life. This research, approved by the Committee of Ethics in Research- HCFMRP USP (in case 648/2008), was to assess the functional capacity of patients undergoing neurosurgery oncology and its relationship to quality of life. Methods. During the period of the abril/09 May/08 (12 months) were assessed 52 subjects adults of both sexes, the experimental group had 26 patients, 14 with a diagnosis of meningioma (group1) and 12 diagnosed with high grade glioma (group 2); and the control group has 26 subjects, divided into groups: 1A (paired with group 1) and 2A (paired with group 2). Were performed two types of dinamometry - evaluation of grip strength (grip TrackTM Testing) and pinch (Pinch TrackTM Testing) SystemTM Tracker with computerized equipment, application of international protocols validated in Brazil - Hospital Anxiety and Depression Scale - HAD, Item Short- Form Health Survey - SF-36 and Health Assessment Questionnaire - HAQ in the preoperative period and in the third month postoperatively. Statistical analysis using the statistical Mann-Whitney, Wilcoxon and Spearman Correlation Coefficient. Results . It was found that the first three months postoperatively increased functional capacity of the subjects of the experimental groups (1 and 2) and decrease symptoms of anxiety and depression, no significant difference in the strength tests before and after surgery but both groups have reduced strength in the dominant limb compared with control groups. There were strong correlations between the magnitude of data collected through tests Grip TrackTM Testing, Testing Pinch TrackTM; protocols: HAD, HAQ and SF-36 with rho 0.600 to 0.969. The aspect of social and emotional domains of quality of life instrument showed a worsening in the immediate postoperative period. These results support the expanded understanding of the concept of functionality, as proposed in the \"International Classification of Functioning and Disability and Health (ICF). Conclusion. The functionality is directly related to the quality of life, especially on psychosocial aspects and needed to understand it more broadly than specific physical functions, to implement plans of treatment best suited to individuals with brain tumor, both pre and postoperatively, with the accompaniment of a multidisciplinary team.

Glioma is the most frequent primary tumor of the central nervous system. By using the RCAS/tv-a mouse glioma model, we have studied mechanisms controlling glioma development and the effect of cell of origin on these processes. SOX5 was identified as a brain tumor locus in a retroviral insertional mutagenesis screen of PDGF-B induced mouse gliomas. Here we found that SOX5 could suppress PDGFB-induced glioma development particularly in Ink4a-/- mice. Analysis of putative PDGF-B signaling pathways revealed that the underlying mechanism could involve the activation of AKT and p27, which caused an acute cellular senescence. When cultured in a highly selective serum free medium, glioma-initiating cells could be isolated from mouse GBMs and their self-renewal and proliferation was independent on exogenous EGF and FGF2. Addition of serum into the medium induced aberrant differentiation that was reversible. Specific depletion of viral PDGF-B demonstrated that PDGF-B was necessary for stemness and tumorigenicity of GICs by preventing them to differentiate. Subsequently, by applying the same culture conditions, GICs of APC, NSC and OPC origins were isolated from mouse GBMs. GICs derived from NSCs exhibited higher self-renewal, faster proliferation and more potent tumorigenicity than those of APC or OPC origin. Furthermore, addition of 5% serum significantly inhibited the proliferation of APC- and OPC-derived GICs, but did not in NSC-derived GICs. Transcriptome analysis revealed that GICs of the same cell of origin displayed distinct expression profiles. In the last study, we showed that OPCs could serve as the origin for astrocytic glioma. Results from immunostainings revealed that these tumors might belong to a different molecular subtype than the oligodendroglial tumors induced in OPCs. We also found differences in tumorigenic potential between OPCs in neonatal and adult mice, which suggest that developmental age of the cell of origin is important for its susceptibility to oncogenic transformation.

BACKGROUND Few high-quality observational studies have been conducted to examine clinically relevant features of emotional distress and Major Depressive Disorder (MDD) in adults with primary cerebral glioma. Our knowledge of these important complications of glioma is currently poor. AIMS This thesis aims to answer a series of relevant clinical questions. I have studied: [1] the frequency, independent clinical associations and course of general emotional distress measured using the NCCN Distress Thermometer (DT); [2] the utility of three depression screening tools for identifying MDD; [3] the frequency, independent clinical associations and course of MDD in glioma; [4] current patterns of practice, and the apparent tolerability of antidepressant treatment of depression in glioma; and [5] barriers to the effective management of MDD in glioma. METHODS I conducted a prospective, twin-centre, observational cohort study. Adults with a new histological diagnosis of primary supratentorial glioma were enrolled and interviewed three times: shortly after starting radiotherapy (T1), three months later (T2) and six months later (T3). At each time point participants completed the DT, the Hospital Anxiety and Depression Scale (Depression subscale, HAD-D), the Patient Health Questionnaire-9 (PHQ-9) and the Structured Clinical Interview for DSMIV MDD (SCID). Barriers to depression management were studied using questionnaires completed by the patient and their named GP. RESULTS During a two-year recruitment period, 223 patients were eligible and 155 provided useable data (57.4% male, mean age = 54.2 years, 85.8% high-grade glioma, 78.1% radical radiotherapy, 55.5% chemotherapy). [1] High distress (DT score ≥ 4/10) was consistently a frequent complication, occurring in between 36.4% ± 7.6% of patients at T1 to 33.7% ± 10.2% at T3. In a logistic regression analysis, high distress at T1 was independently associated with MDD, functional impairment and younger age (χ2 for model = 39.882, p < 0.001, R Square = 0.312). Patients who reported high distress at T1 (median DT score = 8; IQR 7 - 9) remained highly distressed on follow-up (T2 median score = 8, IQR 6 - 8; T3 median score = 7, IQR 5 - 8). [2] As screening tools, the HAD-D and PHQ-9 showed good internal consistency (α = 0.769 - 0.862 at any time point). The HAD-D displayed the best operating characteristics on ROC curve analysis. At a threshold of 7+, sensitivity = 0.933, specificity = 0.907 and Positive Predictive Value (PPV) = 0.56. A threshold of 8+ displayed similar PPV, however. [3] The cross-sectional prevalence of MDD was 13.5% ± 5.4% at T1, 14.8% ± 6.7% at T2 and 6.8% ± 5.8% at T3. Inter-rater diagnostic agreement was good (κ = 0.81, 95% CI 0.60 – 1.00). MDD was independently associated with a past history of depression (OR = 3.8, 95%CI 1.5 - 9.8), and with current functional impairment (OR = 3.6, 95%CI 1.4 - 9.4). MDD persisted for at least three months in 9/17 patients who could be followed up. [4] The frequency of antidepressant prescription was 8.4% ± 4.4% at T1, 7.4% ± 4.9% at T2 and 12.6% ± 6.9% at T3. Citalopram was the most frequent antidepressant choice. Antidepressant tolerability appeared to be good among patients who could be followed up. [5] Barriers to the management of depression included 78.4% of GPs regarding major depression as a normal reaction to having glioma, and 39.2% expressing a belief that major depression did not always require treatment. In addition, most patients expressed a degree of resistance to any kind of future depression treatment. DISCUSSION This is the largest cohort study of depression in consecutively presenting adults with glioma, and the first to utilise criterion standard structured interview diagnoses in a longitudinal design. There is a degree of theoretical uncertainty about the nosological validity of MDD in glioma, although the clinical relevance of this uncertainty can be debated. Methodological limitations to the presented study include an absence of alternative potential psychiatric diagnoses to MDD, the likelihood of selection bias in recruitment, and considerable attrition. Due to these and other limitations, findings from this study are tentative and should ideally be replicated. Clinicians should have a high index of suspicion for identifying low mood in glioma patients, particularly those with functional impairment or previous depressive episodes. The HAD-D (suggested threshold 8+) can reasonably be used to screen for depression, if desired. Caution is required when prescribing antidepressants. Clinicians should be educated about the frequency and consequences of MDD in glioma. Researchers interested in psychological neuro-oncology could convene a meeting to guide future projects, particularly since multi-centre studies may be necessary to recruit sufficient sample sizes in future.

<p>Gliomagenesis is driven by a complex network of genetic alterations and while the glioma genome has been a focus of investigation for many years; critical gaps in our knowledge of this disease remain. The identification of novel molecular biomarkers remains a focus of the greater cancer community as a method to improve the consistency and accuracy of pathological diagnosis. In addition, novel molecular biomarkers are drastically needed for the identification of targets that may ultimately result in novel therapeutics aimed at improving glioma treatment. Through the identification of new biomarkers, laboratories will focus future studies on the molecular mechanisms that underlie glioma development. Here, we report a series of genomic analyses identifying novel molecular biomarkers in multiple histopathological subtypes of glioma and refine the classification of malignant gliomas. We have completed a large scale analysis of the WHO grade II-III astrocytoma exome and report frequent mutations in the chromatin modifier, alpha thalassemia mental retardation x-linked (<italic>ATRX<italic>), isocitrate dehydrogenase 1 and 2 (<italic>IDH1<italic> and <italic>IDH2<italic>), and mutations in tumor protein 53 (<italic>TP53<italic>) as the most frequent genetic mutations in low grade astrocytomas. Furthermore, by analyzing the status of recurrently mutated genes in 363 brain tumors, we establish that highly recurrent gene mutational signatures are an effective tool in stratifying homogeneous patient populations into distinct groups with varying outcomes, thereby capable of predicting prognosis. Next, we have established mutations in the promoter of telomerase reverse transcriptase (<italic>TERT<italic>) as a frequent genetic event in gliomas and in tissues with low rates of self renewal. We identify <italic>TERT<italic> promoter mutations as the most frequently mutated gene in primary glioblastoma. Additionally, we show that <italic>TERT<italic> promoter mutations in combination with <italic>IDH1<italic> and <italic>IDH2<italic> mutations are able to delineate distinct clinical tumor cohorts and are capable of predicting median overall survival more effectively than standard histopathological diagnosis alone. Taken together, these data advance our understanding of the genetic alterations that underlie the transformation of glial cells into neoplasms and we provide novel genetic biomarkers and multi &ndash; gene mutational signatures that can be utilized to refine the classification of malignant gliomas and provide opportunity for improved diagnosis.</p> / Dissertation

INTRODUÇÃO: Os gliomas são tumores neuroepiteliais e correspondem a aproximadamente 24,6% de todos os tumores primários cerebrais. Na última década, grande esforço tem sido feito em meio acadêmico para caracterização molecular dos gliomas na tentativa de descrever comportamento clínico, definir prognóstico e predizer resposta terapêutica. Ferramentas de bioinformática estimam que os microRNAs possam regular cerca de 60% dos genes humanos, incluindo um número significativo de oncogenes, genes supressores tumorais e genes relacionados a quimio e radioressistência. Uma problemática atual na prática clínica é a ausência de ferramentas moleculares para predizer a evolução dos gliomas classificados como baixo grau, uma vez que, durante o seguimento clínico-radiológico pós-cirúrgico, esses pacientes podem ter recidiva tumoral e confirmação histopatológica de glioma de alto grau. A transformação tumoral em glioma de alto grau implica em pior prognóstico e redução das possibilidades terapêuticas. OBJETIVOS: O objetivo deste trabalho é analisar a expressão dos microRNAs miR-124a, miR-138, miR-155, miR-1275 em amostras de tumor primário humano e sangue periférico de pacientes com diagnóstico de gliomas de baixo e alto graus (astrocitoma grau I,astrocitoma grau II, astrocitoma grau III, glioblastoma, oligodendroglioma grau II e oligodendroglioma grau III). PACIENTES E MÉTODOS: As análises da expressão dos microRNAs foram realizadas utilizando-se a técnica de PCR em tempo real. Foram analisados 65 pacientes adultos (entre 18 e 65 anos de idade) com diagnóstico histopatológico confirmado de glioma com material biológico tumoral criopreservado armazenado junto ao Banco de Tumores do Sistema Nervoso Central do HCFMRP. Foram coletadas informações contidas no prontuário médico referentes às características clínicas, epidemiológicas, de evolução clínica e radiológica e tempo para recidiva e óbito. Considerando-se um nível de significância de 5%, a associação das variáveis qualitativas categóricas e expressão de microRNAs foi realizada pelo teste de Mann-Whitney. A análise de sobrevida foi realizada pelo método não-paramétrico de Kaplan-Meier. RESULTADOS: Os microRNAs em estudo não apresentaram hiperexpressão em tecido tumoral de pacientes diagnosticados com Glioblastoma. Apenas o miR-1275 apresentou expressão aumentada em pacientes com diagnóstico de astrocitoma pilocítico e oligodendroglioma grau II. Além disso, tumores de linhagem oligodendroglial, de baixo e alto graus, demonstraram hiperexpressão do miR-1275. Em sangue periférico, observou-se significativa hipoexpressão dos miR-1275, miR-124 e miR-138 em amostra de glioblastoma. Observou-se, ainda, acentuada hipoexpressão do miR-138 em amostras de oligodendroglioma grau III. CONCLUSÃO: Os microRNAs miR-124a, miR-138, miR-155 e miR-1275 não apresentaram hiperexpressão em tecido tumoral de pacientes diagnosticados com GBM. O miR-1275 apresentou expressão aumentada em pacientes com diagnóstico de astrocitoma pilocítico e o miR-155 foi hiperexpresso apenas em amostras de oligodendroglioma grau II. Além disso, tumores de linhagem oligodendroglial, de baixo e alto graus, demonstraram hiperexpressão do miR1275 e miR-124a. Em sangue periférico, observou-se significativa hipoexpressão dos miR-1275, miR-124 e miR-138 em amostra de glioblastoma assim como acentuada hipoexpressão do miR-138 em amostras de oligodendroglioma grau III / INTRODUCTION: Gliomas are neuroepithelial tumors and correspond to approximately 24.6% of all primary brain tumors. In the last decade, great effort has been made in academic circles to characterize gliomas in an attempt to describe clinical behavior, define prognosis and predict therapeutic response. Bioinformatics tools estimate that microRNAs can regulate about 60% of human genes, including a significant number of oncogenes, tumor suppressor genes, and chemo and radioresistance genes. A current problem in clinical practice is the absence of molecular tools to predict the evolution of gliomas classified as low grade, since during postoperative radiological follow-up these patients may have tumor recurrence with histopathological confirmation of high glioma degree. Tumor transformation in high-grade glioma has a worsening of prognosis and reduction of therapeutic possibilities OBJECTIVES: The objective of this project is to analyze the expression of miR124a, miR-138, miR-155, miR-1275 in human primary and peripheral blood samples from patients diagnosed with low and high grade gliomas (astrocytoma grade I, astrocytoma grade II, astrocytoma grade III, glioblastoma, oligodendroglioma grade II and oligodendroglioma grade III). PATIENTS AND METHODS: MicroRNA expression analyzes were performed using the real-time PCR technique. Sixty-five adult patients (18-65 years of age) with confirmed histopathological diagnosis of glioma with cryopreserved tumor biological material stored at the Bank of Tumors of the Central Nervous System of HCFMRP were analyzed. Information collected in the medical records regarding clinical, epidemiological, clinical and radiological characteristics and time for recurrence and death were collected. Considering a level of significance of 5%, the association of categorical qualitative variables and microRNA expression were performed by the Mann-Whitney test. Survival analysis was performed using the Kaplan-Meier non-parametric method. RESULTS: The microRNAs under study did not present hyperexpression in tumor tissue of patients diagnosed with glioblastoma. Only miR-1275 showed increased expression in patients diagnosed with pilocytic astrocytoma and grade II oligodendroglioma. In addition, tumors of low and high grade oligodendroglial lineage demonstrated overexpression of miR-1275. In peripheral blood, significant hypoexpression of miR-1275, miR-124 and miR-138 were observed in a glioblastoma sample. There was also a marked hypoexpression of miR-138 in samples of grade III oligodendroglioma. CONCLUSION: The microRNAs miR-124a, miR-138, miR155 and miR-1275 did not show hyperexpression in tumor tissue of patients diagnosed with GBM. MiR-1275 showed increased expression in patients diagnosed with pilocytic astrocytoma and miR-155 was hyperexpressed only in samples of grade II oligodendroglioma. In addition, tumors of oligodendroglial lineage, of low and high grades, demonstrated hyperexpression of miR-1275 and miR-124a. In peripheral blood, significant hypoexpression of miR-1275, miR-124 and miR-138 were observed in the GBM sample as well as marked hypoexpression of miR-138 in samples of grade III oligodendroglioma

Glioblastoma multiforme é o tipo mais frequente de tumor cerebral primário, sendo caracterizado por uma alta agressividade e um prognóstico bastante limitado. O ácido hidroxâmico suberoilanilida (SAHA) é um inibidor específico de histonas desacetilases aprovado para o tratamento de linfoma cutâneo de células T, e em fase de crescente investigação clínica e pré-clínica em tumores sólidos. Neste estudo, avaliamos a eficácia do ácido hidroxâmico suberoilanilida em tratamento combinado com temozolomida, o agente alquilante já utilizado em glioblastomas. Através de testes de viabilidade e analises por citometria de fluxo em células tumorais das linhagens U251MG e C6, observamos que não houve sinergismo de potenciação entre temozolomida e ácido hidroxâmico suberoilanilida, apenas efeito sinergismo de adição O tratamento combinado inicialmente promoveu parada do ciclo celular em fase G2/M (≥48 h) ao passo que a apoptose foi detectada apenas em exposição prolongada (≥96 h) aos fármacos em estudo. Ainda, as células tratadas com TMZ/SAHA apresentaram fenótipo autofágico, como determinado por citometria de fluxo e imunodetecção de proteínas marcadoras de autofagia como LC3 e o p62/SQSTM1. A autofagia temporalmente precedeu a apoptose e exerceu função citoprotetora, uma vez que o bloqueio da terminação autofágica com cloroquina promoveu uma significante redução na viabilidade celular, a qual foi associada a um aumento de apoptose em células de glioma tratadas com TMZ/SAHA. Portanto, os dados apresentados neste trabalho demonstram que a autofagia é um processo que diminui a eficácia antiglioma do temozolomida e do ácido hidroxâmico suberoilanilida, e a inibição deste fenômeno pode ser uma estratégia para aperfeiçoar a terapia com esses fármacos. / Glioblastoma multiforme (GBM) is the most frequent and aggressive type of primary brain tumor which has been associated with a dismal prognosis. In this study, we tested the efficacy of combining temozolomide (TMZ) with suberoylanilide hydroxamic acid (SAHA) - an inhibitor of HDACs 1, 2, 3, and 6 approved for the treatment of cutaneous T-cell lymphoma - in the viability of tumor cells. The data showed that potentiation synergism between TMZ e SAHA was not achieved due to activation of protective autophagy in vitro. The SAHA/TMZ treatment promoted arrest in the G2/M phase of the cell cycle as soon as 48 h after drug exposure whereas apoptosis was only detected after long-lasting exposure (≥96 h). In addition, SAHA and TMZ induced autophagy as detected by flow cytometry of acridine orange stained cells and immunodetection of the lipidated form of LC3 as well as decreases in p62/SQSTM1. Autophagy preceded apoptosis, and by blocking the termination step of autophagy with chloroquine promoted a significant reduction in the viability of glioma cells which was accompanied by increased apoptosis in SAHA/TMZ treatment. Overall, the herein presented data demonstrate that autophagy impairs the efficacy of combined TMZ/SAHA, and inhibiting this phenomenon could provide novel opportunities to improve the therapeutic potential of these compounds.

Dentre os tumores cerebrais primários, o glioblastoma (GBM) é o mais comum, apresentando alta taxa de mortalidade e morbidade. O tratamento convencional desses tumores tem surtido pouco efeito, com alta taxa de recorrência e progressão da doença. Neste trabalho, buscou-se aprimorar o conhecimento sobre a biologia destes tumores e, utilizando polifenóis amplamente distribuídos na natureza, procurar novas formas de terapia. Nesta tese demonstrou-se que o tratamento com resveratrol e quercetina diminuíram o número de células de maneira tempo e dose dependente em linhagens de GBM humano (U87 e U138), de camundongo (GL261) e de rato (C6), sendo a última a mais sensível e a utilizada neste trabalho. Esse efeito foi observado apenas em linhagens tumorais, não ocorrendo em cultura primária de astrócitos. Resveratrol em altas doses (50 M) induziu apoptose, necrose e parada no ciclo celular. Agudamente, o tratamento com doses baixas de resvetraol (10 M) mais quercetina (25 M) – 24 a 72 h - induziu apoptose, enquanto cronicamente – 12 dias – induziu senescência celular. Esses efeitos não ocorreram quando esses compostos foram utilizados isoladamente. Até então, a indução de senescência pela combinação destes compostos não tinha sido demonstrada. Como provável mecanismo de ação demonstrou-se que o tratamento combinado destes polifenóis induziu senescência através da indução de quebra dupla de DNA e ativação da via da p53. O tratamento com as drogas isoladas não induziu dano ao DNA. A partir de então, investigou-se o efeito destes compostos em um modelo in vivo de implante de gliomas em ratos. Inesperadamente, a quercetina (50 mg/kg/dia intraperitoneal (i.p.)) induziu aumento do volume tumoral, o que não foi observado com o resveratrol (30 mg/kg/dia i.p.) e com a combinação de resveratrol mais quercetina. Para explicar este efeito prevalente do resveratrol nós elaboramos as seguintes hipóteses: 1. necrose/angiogênese: por diminuir a necrose o resveratrol pode ter diminuido a angiogênese, que pode ter sido aumentada pela quercetina; 2. Modulação do sistema imunológico: a quercetina foi capaz de imunussuprimir os animais (por diminuir a proliferação das células T-periféricas após estimulação por fitohemaglutinina e concanavalina A) e a combinação de resveratrol mais quercetina aumentou a estimulação das células T periféricas. Hipoteticamente, atribuiu-se a divergência entre o efeito antitumoral in vitro e in vivo à ausência de senescência neste último, o que pode ser indicado pela ausência da diminuição do índice mitótico. Além disso, o estresse da cultura, um fator indutor de senescência celular, é perdido no modelo in vivo. Com base nos resultados aqui apresentados concluí-se que o resveratrol e a quercetina possuem um potencial antitumoral em gliomas in vitro que precisa de estudos mais aprofundados in vivo para melhorar o conhecimento dos efeitos exercidos por estes afim de se estabelecer uma dose segura para o tratamento dessa e de outras doenças para as quais essas moléculas possam vir a ser utilizadas. / Glioblastoma (GBM) is the most common primary brain tumor, with high mortality and morbidity. The conventional treatment of these tumors has little effect, with high recurrence and progression of the disease. In this work, we set out to better understand the biology of these tumors, looking for new treatment approaches, and, by using polyphenols widely spread on nature, seek for new forms of therapy. In this thesis we demonstrated that the resveratrol and quercetin treatment decreased the cell number in a time and dose dependent manner in GBM cell lines from human (U87 and U138), mouse (GL261) and rat (C6) being the last the most sensitive and therefore the cell line used in this work. This effect was not observed in primary astrocyte cell culture. Higher doses of resveratrol (50 M) induced apoptosis, necrosis and cell cycle arrest. Acutely, the treatment of lower doses of resveratrol (10 M) plus quercetin (25 M) – 24 to 72 h - induced apoptosis while chronically – 12 days – this treatment induced cell senescence. These effects did not occur when the compounds were utilized alone. Until now, the induction of senescence by the combination of these compounds had not been demonstrated. As a probable mechanism of action it was showed that the cotreatment of these polyphenols induced senescence through inducing DNA damage by generating DNA double strand breaks and activating the p53 pathway. The treatment with the drugs alone did not induce DNA damage. These findings lead us to investigate the effect of these compounds in an in vivo rat glioma experimental model. Unexpectedly, quercetin (50 mg/kg/day) induced an increase in the tumor volume, which was not observed for resveratrol (30 mg/kg/dia) and the combination of the resveratrol plus quercetin. To explain this prevalent effect of resveratrol it was elaborated the following hypotheses: 1. necrosis/angiogenesis: by decreasing the necrosis, resveratrol can diminish the angiogenesis, that can be increased by quercetin; 2. modulation of immunological system: quercetin was able to imunossupress the animals (by diminishing the peripherical T-cells proliferation upon phytohemmaglutinin and concanavalin A stimulation) and the combination of resveratrol plus quercetin increased the peripherical T-cell proliferation. Hypothetically, the divergence between the in vitro and in vivo antitumoral effect was attributed to the absence of senescence induction in the latter that can be indicated by the lack of mitotic index decrease. Besides, the culture stress, a senescence inductor factor, is lost in the in vivo model. Based in the results presented here, we concluded that resveratrol and quercetin have a potential antitumoral effect in GBM in vitro and this potential needs more in vivo studies to better harvest this potential and to establish a safe concentration to treat this and other diseases in which these compounds could be used.

Nucleotídeos extracelulares são envolvidos em diversos processos patofisiológicos no sistema nervoso central. Astrócitos são a maior fonte de nucleotídeos extracelulares da adenina no cérebro e também importantes alvos para as ações desses nucleotídeos via receptores purinérgicos P2. As ações induzidas pela sinalização purinérgica são reguladas pelas ecto-nucleotidases, que incluem membros da família das ecto-nucleosídeo trifosfato difosfoidrolase (E-NTPDase), ecto-5’-nucleotidase (ecto- 5’N) e ecto-adenosina deaminase (ADA). Culturas de astrócitos preparadas de hipocampo, córtex e cerebelo de ratos foram capazes de rapidamente converter ATP extracelular a ADP, que foi então hidrolizado a AMP. Os nucleosídeos tri-fosfatados foram hidrolisados preferencialmente aos difosfatados em todas as estruturas cerebrais. A análise cinética sugere que varias ecto-nucleotidases estão envolvidas nessa cascata enzimática. Análises preliminares de mRNA por PCR indicaram que astrócitos expressam múltiplos membros da família das NTPDases (NTPDase1 a NTPDase3 e NTPDase5/6). Por RT-PCR quantitativo (Real-time PCR), nós identificamos a NTPDase2 (CD39L1) como a NTPDase predominante expressa por astrócitos de hipocampo, córtex e cerebelo de ratos. Astrócitos do cerebelo apresentaram um padrão diferente para a hidrólise do AMP, com uma atividade específica 7 vezes maior, quando comparada com astrócitos de hipocampo e córtex. Uma maior expressão da ecto-5’N por RT-PCR foi identificada nessa estrutura. Não houve acúmulo de adenosina extracelular em todas as estruturas estudadas, indicando a presença de uma alta atividade ecto-adenosina deaminase em astrócitos. Dipiridamol aumentou significativamente os níveis de inosina no meio extracelular de astrócitos de hipocampo e córtex, mas não em astrócitos de cerebelo. Essas diferenças observadas podem indicar heterogeneidade funcional dos nucleotídeos no cérebro. Com o objetivo de investigar as enzimas envolvidas no catabolismo dos nucleotídeos como indicadoras da invasividade e agressividade dos gliomas malignos, nós avaliamos a degradação dos nucleotídeos extracelulares em cinco linhagens de gliomas diferentes e comparamos com astrócitos. Todas as linhagens de gliomas examinadas apresentaram baixas razões de hidrólise quando comparadas com astrócitos. Resultados preliminares sugerem que a falta de expressão da NTPDase1 e 2 possam ser responsáveis pela baixa hidrólise de ATP nas linhagens de gliomas. Considerando que o ATP é reconhecido como um fator mitogênico que induz a proliferação em células de gliomas, a substancial diminuição na hidrólise de ATP e ADP observadas em gliomas, sugere que alterações na via das ecto-nucleotidases pode representar um importante mecanismo associado com a transformação maligna desse tipo de tumor.