SETMAR est un gène chimérique constitué d’un domaine SET (codant des fonctions d’histone méthylase) et du domaine MAR (ayant conservé certaines fonctions de la transposase HsMAR1). Des études ont montré que les deux domaines sont biologiquement actifs et sont impliqués dans la stabilité et/ou dans la régulation de l’expression du génome humain. La littérature suggère que l’expression de SETMAR est plus forte dans les cellules cancéreuses que dans les cellules saines. Notre hypothèse de travail est que la protéine SETMAR est surexprimée en conditions pathologiques, permettant aux cellules de franchir les points de contrôle du cycle cellulaire, contribuant ainsi à augmenter l’instabilité génétique. Notre objectif est d’étudier la régulation de l’expression de SETMAR et son implication dans l’oncogenèse, gliale en particulier. / SETMAR is a chimeric gene consisting of a SET domain (encoding methylase histone functions) and a MAR domain (having retained some of the of the HsMAR1 transposase functions). Studies have shown that the two domains are biologically active and are involved in the stability and / or in the regulation of the human genome expression. The literature suggests that SETMAR expression is higher in cancer cells than in normal cells. Our working hypothesis is that SETMAR protein is overexpressed in pathological conditions, allowing cells to overcome the cellular cycle checkpoints, helping to increase the genetic instability. Our goal is to study the regulation of the SETMAR expression and its involvement in oncogenesis, glial in particular.

Das Glioblastom ist mit 70 % aller Gliome der häufigste humane Hirntumor mit sehr ungünstiger Prognose. Es konnte gezeigt werden, dass das natürlich vorkommende Dipeptid Carnosin (β-Alanyl-L-histidin) die Proliferation von Glioblastomzellen inhibiert. Diese Wirkung des Carnosins konnte ebenfalls in vivo nachgewiesen werden. Da Carnosin auch einen Einfluss auf den ATP-Haushalt der Glioblastomzellen besitzt, war das Ziel dieser Arbeit einen Wirkungsort von Carnosin zu identifizieren, womit die ATP mindernden und proliferationshemmenden Eigenschaften erklärt werden können. Es wurde untersucht, ob Carnosin den Energiemetabolismus der Glioblastome beeinflusst. Dabei konnte mithilfe zellbiochemischer Methoden gezeigt werden, dass die untersuchten Zelllinien nicht von der Energieversorgung durch die mitochondriale oxidative Phosphorylierung abhängen, da sich weder Hemmung (KCN) noch Entkopplung (DNP) der Elektronentransportkette auf den zellulären ATP-Gehalt auswirkten. Carnosin hingegen verringerte den ATP-Spiegel dieser Zellen. Die Hemmung der Glykolyse durch Oxamat (LDH-Hemmung), bewirkte einen starken Abfall des intrazellulären ATP-Spiegels, worauf Carnosin keinen zusätzlichen Effekt mehr besaß. Carnosin konnte eine Wirkung auf die glykolytische ATP-Synthese zugesprochen werden. Da ein direkter, molekularer Wirkungsort auf diesem Weg nicht identifiziert werden konnte, wurde parallel untersucht, ob sich über Proteomanalysen der Glioblastomzelllinie T98G ein Wirkungsort, bzw. -mechanismus bestimmen lässt. Anhand der Methode der zweidimensionalen Gelelektrophorese (2D-GE) konnten 31 signifikant differenziell exprimierte Proteine detektiert werden, von denen 6 Proteine (VBP-1, OLA-1, TALDO 1, UROD, BAG-2, GRPEL1) über MALDI-TOF-Analysen identifiziert wurden. In Western-Blot-Analysen konnte ein Protein (VBP-1), neben T98G, auch in primären Glioblastomzelllinien als differenziell exprimiert nachgewiesen werden. Anhand der zellbiologischen und proteinbiochemischen Untersuchungen konnte einerseits eine Verbindung des Carnosins zum HIF1α-Signalweg und andererseits zur generellen posttranslationalen Peptidprozessierung hergestellt werden. Der direkte Nachweis eines Einflusses von Carnosin auf HIF1α wurde aber bisher nicht erbracht.

Glioblastoma multiforme (GBM) is the most common and lethal form of primary brain tumor that mainly affects adults. GBM displays remarkable intra- and inter-tumoral heterogeneity and contains a subpopulation of cells named glioma stem cells that is believed to be responsible for tumor maintenance, progression and recurrence. We have established and characterized a biobank of 48 cell lines derived from GBM patients. The cells were explanted and maintained as adherent cultures in serum-free, defined neural stem cell medium. These GBM cells (GCs) displayed NSC marker expression in vitro, had orthotopic tumor initiating capability in vivo, harboured genomic alterations characteristic of GBM and represented all four TCGA molecular subtypes. Our newly established biobank is also connected with a database (www.hgcc.se) that provides all molecular and clinical data. This resource provides a valuable platform of valid in vitro and in vivo models for basic GBM research and drug discovery. By using RCAS/tv-a mouse models for glioma, we found that GBMs originating from a putative NSC origin caused more tumorigenic GCs that had higher self-renewal abilities than those originating from putative glial precursor cell origin. By transcriptome analysis a mouse cell origin (MCO) gene signature was generated to cluster human GCs and GBM tissue samples and a functional relationship between the differentiation state of the initially transformed cell and the phenotype of GCs was discovered, which provides the basis for a new predictive MCO-based patient classification. LGR5 was found to be highly expressed in the most malignant mouse GC lines of putative NSC origin and also enriched in proneural GBMs characterized by PDGFRA alterations and OLIG2 up-regulation. By overexpressing or depleting LGR5 we discovered that high LGR5 expression in proneural GC lines increased the tumorigenicity, self-renewal and invasive capacities of the cells and could potentiate WNT signalling through its ligand RSPO1. Through transcriptome analysis we identified the candidate genes CCND2, PDGFRA, OLIG2, DKK1 that were found to be regulated by LGR5. In the last study, we found that mouse OPCs could initiate both astrocytic and oligdendroglial gliomas, which indicated that oncogenic signalling is dominant to cell of origin in affecting the histology of gliomas.

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumour in adults. The employment of current standard of care management strategy, that is combining maximum but safe surgical resection, and concomitant chemoradiotherapy, only achieves very modest survival benefits. Antiangiogenesis is a widely studied therapeutic strategy, which restricts the tumour growth by cutting off blood supplement. Although several antiangiogenic agents are now under clinicaland preclinical trials, bevacizumab is still the only one that has been proven to be effective in the treatment of recurrent GBM. However, the clinical use of bevacizumab has encountered the emergence of drug resistance. Its therapeutic benefit is considered limited because of its single pathway targeting. Many researchers believe that the use of broad spectrum angiogenesis inhibitors may leadto better clinical outcomes by overcoming the shortcomings of bevacizumab. / Vastatin, the globular non-collagenous 1 (NC1) domain of collagen VIIIα1, was initially proved to inhibit the proliferation and migration of bovine aortic endothelial cells. Although vastatin is similar in origin to other collagen-derived antiangiogenic factors (CDAFs), its antiangiogenic capability in treatment of cancers has not been studied systematically. Our team members previously found that vastatin wasa safe and effective antiangiogenic therapeutic and a potential biomarker for liver cancer. In this thesis, I tried to explore the therapeutic potential of vastatin in treatment of GBM. / Using a recombinant adeno-associated virus mediated gene therapy, the antiangiogenic potential of vastatin was first confirmed in vitro that it inhibited proliferation, migration and tube formation of murine microvascular endothelial cells (MECs). These effects were further confirmed using another gene vector (H1) which was subsequently employed for the in vivo studies. H1 is a nanopolymer gene vector has high affinity with the folate receptors on tumour cells. Transfection ofH1/vastatin reduced MEC proliferation in a U87/MEC co-culture system, suggesting a paracrine inhibition. Mechanism studies showed that vastatin caused a wide range of changes in the global gene transcription level in MECs, indicating a broad spectrum of action. / Following the establishment of an orthotopic murine GBM model, the H1/DNA polyplexes were injected directly to the tumour area. Treatment induced a significant increase in intracranial mRNA level of the therapeutic gene. Both vastatin and endostatin, a positive control, prolonged the survivals of GBM bearing mice. Immunostaning showed that vastatin decreased microvessel density in the outer layer of the tumour, while decreased cell density and caused abnormal vessel structures inthe centre. No synergistic effect was observed when GBM was treated with the combination of H1/vastatin and temozolomide (TMZ) in this model. / Finally, the therapeutic effect of vastatin on a TMZ resistant model was studied. GBM cells with acquired TMZ resistance (ATR) were established by chronic exposure of U87 cells to TMZ. Animals grafted with the U87-ATR cells were proved to be tolerant of TMZ treatment. H1/vastatin injection significantly prolonged the survival in this model. More interestingly, H1/vastatin also resensitized these animals to TMZ treatment. Stem cell related drug resistance was supposed to be disturbed in this process. / In conclusion, the present study has demonstrated for the first time that vastatin, a broad spectrum endogenous angiogenesis inhibitor, is of therapeutic benefit in a murine orthotopic GBM model. Vastatin’s capability to reverse TMZ resistance highlights an important area for further research. / 胶质母细胞瘤(GBM)是成人最常见的恶性原发性脑肿瘤。目前的治疗手段包括了手术切除和放化疗，但是效果仍不能让人满意。与传统的化疗药不同，抗血管生成药物能通过抑制肿瘤内新血管的形成，切断血流供给，达到限制肿瘤生长的目标。贝伐单抗（Bevacizumab)是目前唯一获得批准用于临床GBM治疗的抗血管生成药物。然而Bevacizumab在临床应用中必须面对耐药性产生的问题， 而且因为Bevacizumab只单一性地阻断血管内皮生长因子相关的通路，所以它的治疗效果也受到了一定程度的限制，让肿瘤可以选择替代性的通路来获得新生血管。因此一些研究人员认为，改用多靶点或者广谱的抗血管生成药物，治疗效果应该会更好。 / Vastatin是VIII型胶原蛋白α1链上的球状非胶原裂解片断。人体内这一类的片段多被证明了具有抗血管生成的功能，它们统称为“源自胶原蛋白的抗血管生成因子”。Vastatin具有抑制牛主动脉内皮细胞增殖和迁移的作用，然而它在抗肿瘤血管生成方面的作用却没有被系统地研究过。我们之前的实验曾经发现Vastatin对肝癌模型中的血管生成具有明显的抑制效果，而本论文将对Vastatin是否同样具有治疗GBM的作用展开研究。 / 在体外，我们首先证明了重组腺相关病毒（rAAV）介导的Vastatin基因治疗能有效抑制MEC的增殖和迁移，并阻止其形成管状结构。我们同时也测试了另一种基因载体H1，以方便后续动物实验的开展。H1是一种纳米聚合物，对肿瘤细胞表面高表达的叶酸受体有高亲和力。H1 介导的Vastatin 基因治疗对肿瘤细胞和MEC都没有直接的作用，但在两种细胞的共培养体系中，Vastatin可以通过旁分泌的方式来抑制MEC的增殖。对机制的研究发现，Vastatin使MEC内基因转录的水平发生了大范围多通路的改变，说明了它的作用具有一定的广谱性。 / 实验进一步研究了Vastatin在小鼠原位GBM 模型中的作用。将H1/DNA 复合物直接注入瘤区可以明显提高颅内相应基因的转录水平。Vastatin和作为阳性对照的Endostatin都能有效地延长GBM小鼠的生存期。免疫组织化学的结果显示Vastatin 能降低肿瘤内部的微血管密度，并诱导组织坏死。这与之前报道过的Endostatin的作用相似。在同一模型上，我们还测试了Vastatin和Temozolomide(TMZ)结合给药的效果，但并没有了现明显的协同作用。 / 实验最后研究了Vastatin在TMZ耐药模型中的治疗效果。通过将U87细胞长期浸泡中含有TMZ的培养基中，我们成功地筛选出了具有TMZ耐药性的GBM细胞。用这些细胞建立的小鼠GBM模型对TMZ的作用不敏感。实验表明，H1/Vastatin基因疗法不仅能够明显延长模型小鼠的生存期，还可以逆转耐药性，使TMZ重新发挥作用。我们推测干细胞相关的耐药性的产生和维持可能在这个过程中受到了影响。 / 上述研究第一次阐明了Vastatin对GBM的治疗效果。Vastatin具有广谱的抗血管特性，能够通过作用于MEC抑制肿瘤内部新血管的生成。Vastatin不仅本身具有治疗作用，还能逆转动物模型对化疗药物的耐受性，因些具有很高的研究价值。相信对Vastatin更一步的探索不但可以拓宽我们对抗血管生成药物的理解，也可能意味着一个新的研究领域的出现。 / Li, Yi. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 102-110). / Abstracts also in Chinese. / Title from PDF title page (viewed on 05, January, 2017). / Li, Yi. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Glioblastoma multiforme (GBM) is an aggressive Grade IV astrocytoma with a poor survival rate. This is largely due to the GBM tumor cells migrating away from the primary tumor site along white matter tracts and blood vessels leading to secondary tumor sites. It is unknown whether the microenvironment nanotopography influences the biomechanical properties of the tumor cells. Although these tumor cells have an innate propensity to migrate, we believe that the nanotopography changes the biomechanical properties to enhance the migratory phenotype. To study this, we used an in vitro polycaprolactone aligned nanofiber film that mimics the nanotopography of the white matter tracts and blood vessels to investigate the mechanical properties of the GBM tumor cells. Our data demonstrate that the cytoskeletal stiffness, traction force, and focal adhesion area are inherently lower in invasive GBM tumor cells compared to healthy astrocytes. Moreover, the tumor cytoskeletal stiffness was significantly reduced when cultured on the aligned nanofiber films compared to smooth and randomly aligned nanofibers films. Analysis of gene expression also showed that tumor cells cultured on the aligned nanotopography upregulated key migratory genes and downregulated key proliferative genes. In addition, cell cycle analysis exhibited a reduced proliferative state on aligned nanofibers, highlighting the dichotomy between proliferation and migration observed in GBM. Finally, focal adhesions of tumor cells were larger and more elliptical when grown on the aligned fibers, suggesting a more migratory state. Therefore, our data demonstrate that the invasive potential is elevated when the tumor cells are cultured on an aligned nanotopography. This in vitro model can further be used to identify the GBM tumor cells’ response in a mimetic in vivo tumor microenvironment and elucidate how the aligned nanotopography transduces into altered gene and protein expression, thus providing a mechanism to target to inhibit the enhanced migratory behavior observed in these cells.

Orientador: Valeria Valente / Banca: Ana Lucia Fachin Saltoratto / Banca: Cleverton Roberto de Andrade / Resumo: O câncer é um conjunto de alterações celulares que favorecem a proliferação descontrolada e a aquisição de propriedades metastáticas. A ativação de oncogenes e/ou a perda de genes supressores tumorais leva a desbalanços nos mecanismos de controle do ciclo celular e/ou na inativação das vias apoptóticas, contribuindo para a instabilidade genômica presente em todos os tipos tumorais. Relatos na literatura têm sugerido que o estresse replicativo oriundo desse aumento proliferativo exacerbado é um fator importante na formação e progressão de muitos tipos de câncer. Dentro dessa perspectiva, é plausível pensar que as células tumorais tenham desenvolvido certas competências que as permitam lidar com o estresse replicativo para continuar se propagando. Resultados prévios de nosso laboratório sugerem que a proteína centromérica HJURP (Holliday Junction Recognizing Protein) esteja envolvida nesse ganho de competência. Dessa forma, esse trabalho teve como objetivo a análise do papel dessa proteína frente ao estresse replicativo em linhagens de glioblastoma. Nossos resultados demonstraram que a superexpressão de HJURP confere um aumento na capacidade proliferativa celular da linhagem U87MG. Além disso, vimos que em baixas concentrações de camptotecina as células superexpressoras de HJURP possuíam um comportamento proliferativo muito semelhante à condição não tratada. Os ensaios de citotoxicidade revelaram uma maior capacidade de recuperação frente ao estresse replicativo exógeno nas sit... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Cancer is characterized by a subset of features that supports uncontrolled cell proliferation and acquisition of metastatic properties. Oncogene activation and/ or the deactivation of tumor suppressor genes lead to disbalances in cell cycle control progression and/or inactivation of apoptotic pathways, followed by genomic instability present in all types of tumors. According to the literature, replicative DNA stress arising from uncontrolled cell growth is the major driver in the development and progression of many cancers. In this perspective, it is believed that cancer cells have developed some skills in order to deal with high levels of endogenous replicative stress for their continued proliferation. Previous results from our group suggest that the centromeric protein HJURP (Holliday Junction Recognizing Protein) is involved in this mechanism. Thus, in this work, we aimed to analyze the role of this protein in the cellular response to the replicative stress in glioblastoma U87MG cell line. Our data showed that cells who levels of HJURP were high, had an increase in cell growth. Besides that, we also observed that at lower camptothecin concentrations the curve proliferation of overexpressed HJURP cells had a similar behavior as the control cells. The cytotoxicity results demonstrated that cells overexpressing HJURP had a better replicative induced stress when compared to the control cells. Together, this data suggests... (Complete abstract click electronic access below) / Mestre

Dissertação (mestrado)—Fundação Universidade de Brasília, Programa de Pós-Graduação em Biologia Animal, 2018. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). / O câncer é o nome dado a um conjunto de doenças relacionadas responsáveis pelas maiores taxas de morte ao redor do mundo, tanto em países mais ou menos desenvolvidos. Dentre eles está o Glioblastoma Multiforme (GBM), originado nas células gliais e classificado como um astrocitoma de grau IV, caracteristicamente agressivo e invasivo. Sabendo da baixa expectativa de vida dos pacientes com esta condição, terapias alternativas têm sido incentivadas de forma a tentar combater o tumor com as mais diversas estratégias, visando atingir todas as células da massa tumoral. O uso de células mesenquimais (MSCs) como carreador de terapias à tumores já vem sendo investigado e tem apresentado bons resultados, entretanto, estabelecer um protocolo de administração efetivo é essencial. Uma das estratégias que se tem usado para entender esse potencial migratório é sua marcação com nanopartículas magnéticas. Por isso, este estudo teve como objetivo investigar e comparar a administração de MSCs por via intranasal e endovenosa em um modelo murino de glioma. Para tal, as MSCs foram isoladas de lipoaspirado humano e marcadas com nanopartículas magnéticas para visualização e quantificação in vivo. Foi feita a transdução lentiviral da linhagem tumoral U87MG com luciferase para facilitar o estabelecimento do modelo animal de glioma por meio de acompanhamento de sua bioluminescência. Posteriormente, as MSCs marcadas com nanopartículas foram administradas pelas vias intranasal e endovenosa em camundongos imunodeficientes previamente enxertados com células U87MG e, ao final, sua capacidade migratória foi avaliada por meio de analise histológica e determinação da biodistribuição de ferro por espectrometria de emissão óptica com plasma acoplado indutivamente. Os dados obtidos foram analisados conforme o teste estatístico apropriado e apresentados como média e erro padrão. Observamos por meio dos ensaios in vitro que a marcação das MSCs com nanopartículas foi eficiente, visto que foi possível evidenciar o ferro na célula e estas tiveram tendência migratória em direção ao campo magnético. O estabelecimento da linhagem tumoral modificada com luciferase foi efetivo, porém a transdução com GFP não foi efetiva, visto que poucas células se mostraram fluorescentes. Já nos ensaios in vivo, a administração das MSCs marcadas após o estabelecimento do modelo murino de glioblastoma demonstrou uma maior eficiência da via intranasal, pois teve um maior acúmulo de ferro no cérebro e menor concentração nos pulmões, em comparação com a via endovenosa. Dessa forma, a via intranasal se mostrou mais eficaz e deve ser priorizada quando o objetivo é atingir o cérebro e reduzir possíveis efeitos associados ao acúmulo do tratamento nos pulmões. / Cancer is the name given to a set of related diseases responsible for highest death rates around the world, both in underdeveloped or developed countries. Among them, is Glioblastoma Multiforme (GBM), which originates in glial cells and is classified as a grade IV astrocytoma, characteristically aggressive and invasive. Knowing low life expectancy of patients with this condition, alternative therapies have been encouraged in order to try to fight tumor with most diverse strategies, aiming to reach all cells of the tumor mass. The use of mesenchymal stromal cells (MSCs) as a carrier of tumor therapies has been investigated and has shown good results, however, establishing an effective administration protocol is essential. One of the strategies that has been used to understand this migratory potential is labbeling the cells with magnetic nanoparticles. Therefore, this study aimed to investigate and compare intranasal and intravenous administrations routes for MSCs in a murine model of glioma. For this, human MSCs were isolated from liposuction and labeled with magnetic nanoparticles for visualization and quantification in vivo. Transduction of the U87MG tumor cell line with luciferase lentiviral particles was performed to facilitate establishment of the animal model of glioma by monitoring bioluminescence. Subsequently, MSCs labeled with magnetic nanoparticles were injected intravenously and intranasally in immunodeficient glioblastoma mice model with U87MG cells and, ultimately, their migratory capacity was evaluated by both histological analysis and biodistribution of iron analised by optical emission spectrometry with inductively coupled plasma. Data were analyzed by using appropriate statistical tests and reported as mean and standard error. We observed through in vitro assays that labelling and further tracking of MSCs with nanoparticles was efficient, since it was possible to evidence the iron into the cell and these had a migratory tendency towards the magnetic field. Establishment of luciferase-expressing tumor line was effective, but the GFP transduction was not effective, since few cells were fluorescent. In the in vivo assays, after established of glioblastoma murine model, we observed a higher efficiency of intranasal administration route when comparing to endovenous route. Higher accumulation of iron in brain and a lower concentration in lungs were found in intranasal group, in comparison with intravenous route group. Thus, intranasal route has proven to be more effective and should be prioritized when the goal is to reach brain and reduce possible effects associated with accumulation of treatment in lungs.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Glioblastoma multiforme (GBM), a grade IV malignant astrocytic tumor according to WHO classification, is one of the most common and malignant brain tumor. Temozolomide (TMZ) is the current standard treatment for GBM. Nevertheless, resistance to chemotherapy in GBM is common and therefore a major obstacle to successful treatment. Adducin 3 (ADD3), a cytoskeletal protein, has been found to be associated with chemoresistance in osteosarcoma, but its potential role in glioblastoma is unclear. A TMZ-resistant model was established by chronically exposing the glioma cells (D54 cell line) to an increasing dose of TMZ. A resistant subclone (D54-R) was successfully generated. ADD3 expression level was found to be upregulated in the D54-R when compared to the parental D54 cells (D54-C). CD133 is a putative cancer stem cell marker. Its expression level was found also to be higher in D54-R when compared to D54-C cells. Among the D54-R cells, a subgroup of cells was found to express ADD3 intensely. The proportion of these spherical cells was higher in D54-R than D54-C. Moreover, these cells were spherical in morphology and expressed putative cancer stem cell markers: CD133, NANOG and OCT-3/-4. Therefore, ADD3 is associated with cancer stem cells in human glioma. The upregulation of ADD3 expression is associated with TMZ-resistance in GBM. / published_or_final_version / Surgery / Master / Master of Research in Medicine