Role of Chaperone-Mediated Autophagy and Histone Deacetylases in Glioblastoma and Brain Aging-A focus on Stem Cell Maintenance.

226 p.En un primer momento, los procesos de envejecimiento y cáncer podrían parecer opuestos. Sin embargo, el hecho de que compartan tanto la acumulación del daño celular como origen común, como un sello molecular similar, hace que se conviertan en dos manifestaciones distintas de los mismos procesos subyacentes. Entre estas alteraciones moleculares se encuentra la desregulación del conjunto de células madre. En esta tesis doctoral, demostramos que la autofagia mediada por chaperonas promueve la capacidad tumorigénica de las células madre del glioma, regulando vías proteómicas y transcriptómicas asociadas a la proliferación, auto-renovación, metabolismo, respuesta inmune e interacciones con la matriz extracelular. Por otro lado, revelamos el enriquecimiento de la expresión de HDAC6 en la subpoblación de células madre del glioma, e identificamos un nuevo inhibidor de HDAC6 con gran selectividad de diana y efecto citotóxico en células madre de glioma, incluso más potente que la de otros inhibidores de deacetilasas de histonas actualmente disponibles. Además, ampliamos el estudio de las deacetilasas de histonas al contexto del envejecimiento cerebral, mostrando evidencias preliminares que indican el aumento en la expresión de las deacetilasas de histonas en varias regiones del cerebro, como es el caso del hipocampo, con el envejecimiento, correlacionando a su vez con el envejecimiento microglial

The Impact of Radiation on Glioblastoma Evolution

Glioblastoma (GB) is the most common and malignant primary adult brain cancer with a median survival of 15 months despite treatment with surgical resection followed by chemo-radiotherapy. The clonal diversity and evolutionary dynamics inherent to GBs is considered a major obstacle to effective treatment response. While studies have focused on temozolomide, a role for radiotherapy as an independent driver of GB evolution has not been investigated. We addressed the impact of radiation on glioblastoma evolution and potential treatment implications by examining the influence of intratumoral heterogeneity (ITH) on intrinsic radiosensitivity, by determining the effects of radiation on glioma stem-like cell (GSC) initiated orthotopic xenografts, and by assessing radioresistance with a reirradiation protocol. To determine the impact of ITH on intrinsic radiosensitivity, we performed whole-exome sequencing (WES) of multiple tumour fragments and corresponding patient-derived cell lines that underwent γH2AX foci analysis and limiting dilution assay analysis. Cell lines from the same tumour seem to display similar levels of intrinsic radiosensitivity despite genomic differences, suggesting that radiotherapy regimens may be effective for the whole of the tumour. To test the ability of radiation to drive GB evolution, we utilised GSC-initiated orthotopic xenograft models treated with or without fractionated radiation (3x5Gy) to examine differences in survival, morphology/histology, Viral integration site analysis (VISA), and WES. Irradiated mice experienced a survival advantage and harboured less invasive tumours compared to control mice. VISA revealed that control tumours harbour fewer clones than in vitro lines and that irradiated tumours harbour the fewest clones of all suggesting that radiation, particularly in the context of the brain microenvironment, drives GBM evolution. WES results demonstrated that variants from irradiated tumours mapped to different COSMIC mutational signatures and displayed a considerable amount of subpopulation shifting compared to control tumours, consistent with radiation-induced evolution and subpopulation selection. By adding a reirradiation protocol to this GSC-initiated orthotopic xenograft model, we sought to better understand the functional impact of radiotherapy on recurrent GB evolution and to establish an in vivo model for studying reirradiation. After initial treatment, mice were rerandomised into control (3x5Gy-Control) and radiation therapy groups (3x5Gy-3x5Gy) and retreated once the average BLI ratio began to increase. A further survival advantage was found for mice undergoing reirradiation compared to mice receiving only one course of radiation. This survival advantage was supported by clonogenic survival and reimplantation studies of cell lines derived from control and irradiated NSC11 tumours that did not demonstrate a difference in survival after radiation regardless of the previous tumour’s treatment regimen. Whereas radiation-induced evolution may not influence radioresponse, it may lead to the identification of novel targets for sensitisation which may ultimately yield more effective treatment strategies. Our results demonstrate that radiation, a treatment component for almost all glioblastoma patients, can have wide-ranging effects on the evolution of this dynamic tumour. In particular, the pressures imposed by radiation treatment seem to lead to the selection of a reduced number of clones. This selection may have future implications for tumour evolution and the treatment of recurrent GB. In addition, we have demonstrated for the first time the utility of a GSC-initiated orthotopic xenograft model for studying retreatment protocols and recurrent GB biology. This reirradiation model may provide the opportunity to design and test more effective recurrent GB treatment strategies centered around recurrent biology.Financial support for Chapter 4-6 was provided by Division of Basic Sciences, Intramural Program, National Cancer Institute (Z1ABC011372, Z1ABC011373) to P.J. Tofilon. The project has also been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this thesis does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organisations imply endorsement by the U.S. Government. Tissue was accessed through the Human Research Tissue Bank supported by the NIHR Cambridge Biomedical Research Centre and Addenbrooke's Hospital

Network and Algebraic Topology of Influenza Evolution

Evolution is a force that has molded human existence since its divergence from chimpanzees about 5.4 million years ago. In that same amount of time, an influenza virus, which replicates every six hours, would have undergone an equivalent number of generations over only a hundred years. The fast replication times of influenza, coupled with its high mutation rate, make the virus a perfect model to study real-time evolution at a mega-Darwin scale, more than a million times faster than human evolution. While recent developments in high-throughput sequencing provide an optimal opportunity to dissect their genetic evolution, a concurrent growth in computational tools is necessary to analyze the large influx of complex genomic data. In my thesis, I present novel computational methods to examine different aspects of influenza evolution. I first focus on seasonal influenza, particularly the problems that hamper public health initiatives to combat the virus. I introduce two new approaches: 1. The q2-coefficient, a method of quantifying pathogen surveillance, and 2. FluGraph, a technique that employs network topology to track the spread of seasonal influenza around the world. The second chapter of my thesis examines how mutations and reassortment combine to alter the course of influenza evolution towards pandemic formation. I highlight inherent deficiencies in the current phylogenetic paradigm for analyzing evolution and offer a novel methodology based on algebraic topology that comprehensively reconstructs both vertical and horizontal evolutionary events. I apply this method to viruses, with emphasis on influenza, but foresee broader application to cancer cells, bacteria, eukaryotes, and other taxa

Heterodimeric glycolipid complexes as targets for neuropathy associated pathogenic autoantibodies and other lectins

There is plentiful evidence that the pathology of Guillain Barré syndrome (GBS) is driven by autoantibodies generated following infection. A number of inconsistencies with this theory remain, and in many clinical cases such antibodies are not detected. Recent descriptions of ganglioside complex (GSC) antibodies suggest a potential explanation for this. This study aimed to further investigate GSCs and associated antibodies with a particular focus on GBS. GSCs were found to modulate the binding of other lectins such as bacterial toxins, immunomodulatory receptors, and monoclonal antibodies. The development of a semi-automated array system allowed screening of a large cohort of GBS sera against multiple complexes, revealing a greater antibody detection rate (particularly in demyelinating forms) than had previously been achieved. Binding that was both enhanced and attenuated by complexes was seen, and this varied between disease and control sera. Immunisation experiments provided insights into the generation of the GSC immune response. A transgenic mouse model of GBS was also developed, demonstrating that local axonal expression of gangliosides does not induce systemic tolerance. The work described in this thesis has thus significantly advanced knowledge in the field of glycolipid complexes, particularly with respect to anti-glycolipid complex antibodies and their association with inflammatory neuropathies such as Guillain-Barré syndrome

Assembly and repeat annotation of the nothobranchius furzeri genome

In der Alternsforschung an Vertebraten sind die relativ langen Lebensspannen der derzeitigen Modellorganismen ein Hindernis bei der Durchführung von Experimenten. Der Türkise Prachtgrundkärpfling Nothobranchius furzeri weist eine für Wirbeltiere sehr kurze Lebensspanne (4-12 Monate) auf, was ihn zu einem geeigneten Modellorganismus für die Alternsforschung macht. In der vorliegenden Arbeit wird der mehrstufige Prozess zur Erzeugung der Genomsequenz von N.furzeri beschrieben. Basierend auf umfangreichen Sequenzierungsadaten und zusätzlichen Kartenressourcen wurde eine qualitativ hochwertige Genomassemblierung mit einer Größe von 1,24 Gb (N50 57,4 Mb; 19 Chromosomen) erstellt. Weiterhin wurde die Zusammensetzung repetitiver Sequenzen im N. furzeri Genom untersucht. Neben etablierten Methoden zur Repeatanalyse wurde zudem eine neue Softwarelösung (RepARK) entwickelt und angewendet. Es wurden 35,5% der Genomassemblierung als Repeats eingestuft. Basierend auf dieser Repeatannotation zeigte sich, dass die Genomassemblierung zwar circa 90% der unikalen Sequenzen enthält aber rund 60% der Repeats fehlen. Eine Analyse von unassemblierten Sequenzen zeigt zudem einen genomischen Repeatanteil von 56-70%. Diesen fehlenden Sequenzanteil gilt es in Zukunft insbesondere mit Sequenziertechnologien der dritten Generation möglichst vollständig zu erfassen, um auf dieser Basis die Rolle von repetitiven Elementen in biologischen Prozessen wie dem Altern erforschen zu können.In aging research, the long lifespan of the current vertebrate model organisms challenges the feasibility of research efforts. The turquoise killifish Nothobranchius furzeri has the shortest lifespan for vertebrates known so far (4-12 months) making it to a valuable new model in aging research. In this thesis, the multi-step process of building genome reference sequence of N. furzeri is described. Based on a broad range of sequencing data and additional map resources, a high-quality genome assembly with a size of 1.24 Gb (N50 57.4 Mb, 19 chromosomes) was achieved. Furthermore, the composition of repetitive sequences in the N. furzeri genome was analyzed. In addition to established repeat detection methods a new software solution (RepARK) was developed and applied. In this analysis, 35.5% of the genome assembly was annotated as repetitive. Using this repeat annotation, it was estimated that the genome assembly contains 90% of the unique sequence while about 60% of the repeats are absent. An analysis in not-assembled data suggests a repeat content in the N. furzeri genome ranging from 56% to 70%. This missing fraction needs to be resolved by further third generation sequencing efforts to study the role of repeats in biological processes such as aging

INTEGRATIVE ANALYSIS OF OMICS DATA IN ADULT GLIOMA AND OTHER TCGA CANCERS TO GUIDE PRECISION MEDICINE

Transcriptomic profiling and gene expression signatures have been widely applied as effective approaches for enhancing the molecular classification, diagnosis, prognosis or prediction of therapeutic response towards personalized therapy for cancer patients. Thanks to modern genome-wide profiling technology, scientists are able to build engines leveraging massive genomic variations and integrating with clinical data to identify “at risk” individuals for the sake of prevention, diagnosis and therapeutic interventions. In my graduate work for my Ph.D. thesis, I have investigated genomic sequencing data mining to comprehensively characterise molecular classifications and aberrant genomic events associated with clinical prognosis and treatment response, through applying high-dimensional omics genomic data to promote the understanding of gene signatures and somatic molecular alterations contributing to cancer progression and clinical outcomes. Following this motivation, my dissertation has been focused on the following three topics in translational genomics. 1) Characterization of transcriptomic plasticity and its association with the tumor microenvironment in glioblastoma (GBM). I have integrated transcriptomic, genomic, protein and clinical data to increase the accuracy of GBM classification, and identify the association between the GBM mesenchymal subtype and reduced tumorpurity, accompanied with increased presence of tumor-associated microglia. Then I have tackled the sole source of microglial as intrinsic tumor bulk but not their corresponding neurosphere cells through both transcriptional and protein level analysis using a panel of sphere-forming glioma cultures and their parent GBM samples.FurthermoreI have demonstrated my hypothesis through longitudinal analysis of paired primary and recurrent GBM samples that the phenotypic alterations of GBM subtypes are not due to intrinsic proneural-to-mesenchymal transition in tumor cells, rather it is intertwined with increased level of microglia upon disease recurrence. Collectively I have elucidated the critical role of tumor microenvironment (Microglia and macrophages from central nervous system) contributing to the intra-tumor heterogeneity and accurate classification of GBM patients based on transcriptomic profiling, which will not only significantly impact on clinical perspective but also pave the way for preclinical cancer research. 2) Identification of prognostic gene signatures that stratify adult diffuse glioma patientsharboring1p/19q co-deletions. I have compared multiple statistical methods and derived a gene signature significantly associated with survival by applying a machine learning algorithm. Then I have identified inflammatory response and acetylation activity that associated with malignant progression of 1p/19q co-deleted glioma. In addition, I showed this signature translates to other types of adult diffuse glioma, suggesting its universality in the pathobiology of other subset gliomas. My efforts on integrative data analysis of this highly curated data set usingoptimizedstatistical models will reflect the pending update to WHO classification system oftumorsin the central nervous system (CNS). 3) Comprehensive characterization of somatic fusion transcripts in Pan-Cancers. I have identified a panel of novel fusion transcripts across all of TCGA cancer types through transcriptomic profiling. Then I have predicted fusion proteins with kinase activity and hub function of pathway network based on the annotation of genetically mobile domains and functional domain architectures. I have evaluated a panel of in -frame gene fusions as potential driver mutations based on network fusion centrality hypothesis. I have also characterised the emerging complexity of genetic architecture in fusion transcripts through integrating genomic structure and somatic variants and delineating the distinct genomic patterns of fusion events across different cancer types. Overall my exploration of the pathogenetic impact and clinical relevance of candidate gene fusions have provided fundamental insights into the management of a subset of cancer patients by predicting the oncogenic signalling and specific drug targets encoded by these fusion genes. Taken together, the translational genomic research I have conducted during my Ph.D. study will shed new light on precision medicine and contribute to the cancer research community. The novel classification concept, gene signature and fusion transcripts I have identified will address several hotly debated issues in translational genomics, such as complex interactions between tumor bulks and their adjacent microenvironments, prognostic markers for clinical diagnostics and personalized therapy, distinct patterns of genomic structure alterations and oncogenic events in different cancer types, therefore facilitating our understanding of genomic alterations and moving us towards the development of precision medicine

Somatic regulation of germ cell development in the planarian Schmidtea mediterranea: the role of systemic and local factors

Germline development in animals is closely coordinated with those aspects of physiology that are influenced by environmental conditions. The soma acts as a vehicle for the germline and plays a critical role in regulating its development and maintenance by providing systemic and local instructive signals. Investigating the relationship between soma and germline is essential for understanding the mechanisms of germline development across animals and, in particular, in the context of human reproductive physiology. Planarians provide a unique system for studying somatic regulation of germline development: they display remarkable plasticity in maintenance of their germline, with the ability to develop or dismantle reproductive tissues in response to systemic and environmental cues. The planarian nervous system plays a key role in systemic regulation of germ cell development through signaling molecules such as neuropeptide Y-8 (NPY-8). In this study, we explored different aspects of NPY-8 biosynthesis and signaling in planarians and expanded our work to examine the role of G protein-coupled receptors (GPCRs) in systemic and local regulation of germ cell differentiation. First, we revealed the expression of NPY-8 in central and peripheral nervous systems of the planarian and used mass spectrometric methods to deduce the structure of endogenous NPY-8. We demonstrated that two neuropeptide processing enzymes, prohormone convertase 2 and peptidylglycine alpha-hydroxylating monooxygenase, are essential for planarian reproductive development, likely through post-translational modification of NPY-8. Next, we sought to identify the NPY-8 receptor by genome-wide analysis of planarian GPCRs. We identified 566 putative planarian GPCR genes and classified them into conserved and phylum-specific subfamilies. By functional analysis of a subset these genes, we found that neurally expressed neuropeptide y receptor-1 is the NPY-8 receptor and required for reproductive maturation of the planarian. Additionally, we screened the complement of GPCRs with enriched expression in sexual planarians and identified a chemoreceptor family member, ophis, that is required for germ cell differentiation. ophis is expressed in somatic cells of male and female gonads, as well as in accessory reproductive tissues. We have previously shown that somatic gonadal cells are required for male germline specification and maintenance in planarians. However, ophis is not essential for germline specification or maintenance, and therefore, defines a secondary role for planarian gonadal niche cells in promoting germ cell differentiation. In addition to characterization of NPY-8 expression and biosynthesis, our studies uncover the complement of planarian GPCRs and reveal previously unappreciated roles for these receptors in systemic and local control of germ cell development

Impact of extracellular vesicles’ cargo in the chemoresistance transfer in glioblastoma

Tese de Mestrado, Biologia Molecular e Genética, 2021, Universidade de Lisboa, Faculdade de CiênciasSegundo a Organização Mundial de Saúde (WHO), o glioblastoma (GBM) é uma neoplasia de classe IV com diferenciação astrocística, responsável por aproximadamente 45% de todos os tumores cerebrais primários malignos. Devido à sua natureza altamente invasiva e rápida taxa de proliferação, a ressecção cirúrgica completa do tumor não é exequível. Adicionalmente, a grande heterogeneidade genética típica deste tumor cerebral possibilita um rápido desenvolvimento de resistência à quimioterapia e radioterapia utilizadas, e uma consequente célere recidiva tumoral. Para além da sua grande variabilidade genética intra-paciente, o GBM é também notório pela sua heterogeneidade inter pacientes, o que se revela de forma clara no comportamento patológico diferencial deste tumor, como por exemplo, na resposta aos diferentes tratamentos. Por este motivo, o GBM foi agrupado em diferentes grupos, de acordo com a natureza das suas mutações e com a sua expressão fenotípica. Esta informação pode ser utilizada para personalizar terapias para pacientes com GBM, aumentando assim a sua eficácia. De acordo com o último relatório da WHO de 2016, que clarifica a classificação de tumores do sistema nervoso central através da integração tanto de critérios fenotípicos como genotípicos, o GBM pode ser dividido em dois grupos: Desidrogenase de isocitrato (IDH)-estirpe selvagem e IDH-mutante. Existe ainda um terceiro grupo que inclui todos os diagnósticos de GBM no qual a avaliação do gene IDH é inconclusiva. O perfil genotípico mais comum ocorre em cerca de 90% de todos os casos e está associado a pacientes que apresentam a estirpe selvagem do gene IDH. Esta variante está associada a GBM primários, isto é, tumores com as suas principais mutações a ocorrem de novo, em pacientes com mais de 55 anos, com uma idade mediana de 62 anos aquando do diagnóstico. Este grupo está também associado com uma maior incidência no sexo masculino, com um rácio de 1,42:1 em comparação com o sexo feminino. A taxa de sobrevivência geral de pacientes neste grupo é de apenas 15 meses após diagnóstico. Associado a aproximadamente 10% de todos os casos, a segunda variante mais comum deste tumor está associada com GBM secundários que se desenvolvem em pacientes mais novos, por volta dos 44 anos. Os pacientes neste grupo apresentam mutações no gene IDH, e normalmente possuem um histórico clínico de casos de gliomas difusos de baixo grau. A taxa de sobrevivência nesta variante ronda os 31 meses após o diagnóstico, um pouco mais do dobro quando comparada com a variante IDH estirpe selvagem. Até ao momento, não existe ainda um claro conjunto de sintomas associados com nenhuma variante específica. Os sintomas para ambas as variantes podem incluir défices neurológicos focais progressivos, danos cognitivos e motores, náusea, dor de cabeça e convulsões. Estes sintomas derivam normalmente de fatores como a localização do tumor, destruição direta de tecido cerebral por necrose, ou da pressão intracraniana elevada devido ao volume tumoral. Devido a este conjunto de sintomas ambíguos e a falta de grandes manifestações clínicas, o GBM é normalmente diagnosticado num estádio avançado, revelando assim uma falta de estratégias de diagnóstico eficientes. Os métodos de diagnóstico atualmente utilizados consistem em testes neurológicos, neuroimagem e sobretudo realização de biópsias, apesar do seu caracter invasivo e das limitações deste método em avaliar mudanças na dinâmica tumoral em tempo real. O último grande avanço no tratamento do GBM ocorreu em 2005, quando o pró-fármaco temozolomida (TMZ) foi introduzido em combinação com a radioterapia após ressecção cirúrgica máxima em segurança do tumor. A TMZ é um agente alquilante capaz de causar quebras na molécula de ácido desoxirribonucleico (DNA) através da formação de incompatibilidades no emparelhamento de bases que iniciam ciclos fúteis de reparação de DNA, resultando em morte celular. Este agente é um pró-fármaco que não necessita de ativação metabólica, uma vez que a TMZ é convertida espontaneamente ao seu princípio ativo, o catião eletrofílico altamente reativo metildiazónio. Ao contrário de outros fármacos comumente utilizados em quimioterapia, a TMZ tem a capacidade de atravessar a barreira hematoencefálica e atingir a localização tumoral em concentrações adequadas. Esta capacidade deve-se não só ao facto de que este pró-fármaco possui uma massa molecular de apenas 194 Da, mas principalmente devido ao seu padrão de degradação a diferentes valores de pH. O efeito citotóxico e citostático mediado por TMZ é geralmente causado pela metilação de uma guanina na posição O6, resultando no seu mal emparelhamento com uma timina durante a replicação que despoleta os vários ciclos fúteis de reparação de DNA mencionados anteriormente. No entanto, este aduto pode ser reparado pela enzima O6-metilguanina-DNA metiltransferase (MGMT). Metade dos pacientes diagnosticados com GBM possuem o promotor da enzima MGMT metilado, resultando numa menor expressão da mesma e num maior efeito citotóxico e citostático. Todavia, a outra metade dos pacientes é geralmente intrinsecamente resistente a TMZ devido a não possuírem a expressão de MGMT silenciada. Adicionalmente, após realização de quimioterapia com TMZ, existe uma pressão seletiva sobre as células para a perda do silenciamento do promotor do gene MGMT, resultando assim em quimiorresistência a TMZ. Uma vez que existe uma escassez de fármacos aprovados para o tratamento de primeira linha do GBM, é importante aprofundar o conhecimento das vias biológicas que levam ao desenvolvimento de resistência à TMZ, com o objetivo de potencialmente melhorar a eficácia do medicamento e retardar o desenvolvimento de quimiorresistência. As vesículas extracelulares (EVs) são um grupo heterogéneo de nanoestruturas de bicamada lipídica, que atuam como transportadoras biológicas de macromoléculas, como lipídios, proteínas e ácidos nucleicos. A biogénese destas estruturas é altamente dependente do tipo de célula, do seu estado fisiológico ou patológico e dos estímulos do microambiente celular. Devido às suas funções na comunicação celular, as EVs estão implicadas em vários processos fisiológicos e patológicos, incluindo funções importantes na biologia tumoral. Exemplos destas são: a promoção da proliferação e invasão celular, auxílio no aparecimento de metástases e angiogénese, e desenvolvimento de resistência às terapias utilizadas. Por este motivo, o estudo de EVs no contexto da biologia tumoral tem recebido grande atenção. Isto deve-se ao facto destas vesículas terem a potencialidade de servirem como biomarcadores do estado patológico das células cancerígenas em tempo real, mas também devido ao seu potencial valor terapêutico. O objetivo principal do presente estudo foi compreender melhor o impacto das EVs na transferência de quimiorresistência à TMZ entre linhas celulares com diferentes níveis de resistência. Deste modo, foram isoladas EVs através de um método baseado na ultracentrifugação diferencial em gradiente de densidade, sendo posteriormente validadas biologicamente. Estas vesículas foram isoladas de uma linha celular resistente a este pro-fármaco, T98G, e de uma linha celular sensível, U-251. De modo a serem identificadas possíveis diferenças na carga de vesículas extracelulares de células tratadas com TMZ, estas também foram isoladas de células cultivadas na presença deste pró-fármaco. Com a finalidade de estabelecer a concentração de TMZ utilizada nestas experiências, foram realizados dois ensaios, um referente à viabilidade celular com MTT e outro para avaliar a proliferação utilizando sistemas de análise Incucyte®. Para este trabalho, foi estipulado que a concentração de TMZ utilizada sobre as células T98G e U-251 seria 300 µM e 50 µM, respetivamente. Utilizando sistemas de análise Incucyte®, ao avaliar a proliferação de células U-251 tratadas com TMZ juntamente com EVs previamente isoladas, foi possível observar uma maior proliferação celular nestas condições quando comparadas às células que foram tratadas apenas com TMZ. Apesar da falta de significado biológico deste resultado, vários estudos publicados no decorrer desta dissertação corroboraram que as EVs têm a capacidade de transferir quimiorresistência entre células com diferentes graus de sensibilidade à TMZ. Por exemplo, foi demonstrado que EVs com níveis elevados de ácido ribonucleico longo não codificante (lncRNA) SBF2-AS1 derivadas de células com resistência a TMZ, foram capazes de transmitir quimiorresistência a células sensíveis em GBM. Outro estudo do mesmo laboratório, demonstrou que EVs derivadas de células de GBM resistentes a TMZ contendo níveis elevados de microRNA 1238 (miR-1238) foram também capazes de conferir resistência em células sensíveis. Embora se tenha tornado evidente que as vesículas extracelulares estão implicadas em vários processos biológicos, incluindo quimiorresistência no GBM, a tradução deste conhecimento em aplicações clínicas exigirá uma compreensão mais profunda das EVs. Além do já mencionado valor intrínseco das vesículas extracelulares como biomarcadores do estado patológico, há uma grande janela de oportunidade no uso destas mesmas vesículas como alvos ou vetores terapêuticos num futuro próximo.Glioblastoma (GBM) is a class IV neoplasm with astrocytic differentiation, which accounts for approximately 45% of all primary malignant brain tumours. Due to its highly invasive nature and rapid proliferation rates, complete tumour resection is not viable, while its great genetic heterogeneity enables a fast tumour recurrence and chemoresistance emergence. The last major advance in GBM treatment occurred in 2005, when temozolomide (TMZ) was introduced in combination to radiotherapy after maximal safe tumour resection. Opposed to many chemotherapeutic drugs, TMZ has the capacity to cross the blood-brain barrier and reach intracranial tumour sites in appropriate concentrations. TMZ is an alkylating agent which causes deoxyribonucleic acid (DNA) strand breaks through the formation of base mismatches that initiate futile DNA repair cycles, thus leading to cell death. However, the majority of the patients are either intrinsically chemoresistant or acquire resistance shortly after chemotherapy. Since there is a limited number of drugs available for GBM treatment, there is a need to better understand the biological pathways that leads to TMZ chemoresistance in order to potentially improve drug efficacy. Extracellular vesicles (EVs) are a heterogeneous group of lipid bilayer nanostructures, which acts as biological carriers of macromolecules, such as lipids, proteins and nucleic acids. The biogenesis of these structures is highly dependent on cell-type, microenvironmental stimuli and physiological status of the cells. Due to their role in the interplay between cells, EVs are implicated in several physiological and pathological processes, including important roles in tumour biology. The main goal of the present study was to further understand the impact of extracellular vesicles in the transfer of temozolomide chemoresistance between cell lines with different resistance levels. With the aim of extending the knowledge on the EVs impact on chemoresistance transfer of TMZ in GBM, these vesicles were isolated from a TMZ resistant GBM cell line, T98G, and from a sensitive cell line, U-251. In order to identify possible differences in the cargo of extracellular vesicles from cells treated with temozolomide, these vesicles were also isolated from cells which were cultured in the presence of TMZ. By assessing the cell proliferation of U-251 cells treated with TMZ and isolated EVs, it was possible to observe a less extensive impairment on the cell proliferation when compared to cells that were solely treated with temozolomide. Despite the lack of biological significance of these results, several studies published in the course of this dissertation corroborated that EVs have the capacity to transfer resistance between cells with different degree of TMZ sensitiveness. Although it has become evident that EVs are implicated in several biological processes, including chemoresistance in GBM, the translation of this knowledge into clinical applications will require a deeper understanding of the extracellular vesicles. Besides the intrinsic value of EVs as biomarkers of GBM status, there is a great window of opportunity in using these vesicles as targets or therapeutic delivery vehicles in the near future