Aberrations in Epidermal Growth Factor Receptor (EGFR/ErbB1) signalling are the most common oncogenic stimuli in human glioblastoma multiforme (GBM). Interactions between mutant and wildtype ErbB family members in GBMs are of biological and potential therapeutic importance. In this thesis, we describe our work developing and optimizing a novel in situ proximity ligation assay (PLA) for dimerization and activation analysis of EGFR mutants prevalent in GBMs. Utilizing this novel in situ platform for EGFR dimerization analysis, we seek to systematically interrogate the dimerization capacity and activation status amongst EGFR and EGFR mutants. Our in vitro analysis using this platform demonstrates the aberrant homo-/hetero-dimeric properties of EGFRvIII and EGFRc958 mutants, the two most common mutants associated with EGFR amplification in GBMs. In addition, dimer phospho-activation status can be detected using in situ PLA with ≥ 16-fold sensitivity and ≥ 17-fold signal-to-noise than phospho-EGFR measurements currently undertaken with IHC or IF. These aberrant features are not overexpression dependent but appear independent of cellular expression levels, suggesting inherent properties of the mutant receptors. This EGFR dimerization/activation detection platform may also be useful for evaluating novel anti-EGFR therapeutics. Our data suggests that various EGFR monoclonal antibody therapies have unique dimerization blocking abilities and that certain mutant EGFR dimer configurations can evade blockage by anti-EGFR treatments. Furthermore, we report for the first time the detection of wt- and EGFRvIII dimerization in GBM specimens, in keeping with our prior cell line data, and a potential feature of prognostic or diagnostic value in GBMs harbouring them. Additionally, we demonstrate the utility of this platform for measuring pEGFR and total EGFR expression on tissue samples, which has not been efficacious to date with conventional antibody-mediated techniques. Results from this thesis may therefore provide novel insights into the interaction and activation characteristics of EGFR mutants prevalent in GBMs, as well as the efficacy of current anti-EGFR therapies to target these mutants. In summary, these findings demonstrate the successful application of a novel in situ EGFR molecular detection platform which may have clinical utility in diagnostic evaluation or stratification of GBM patient subgroups for prognosis and treatment. Furthermore, since PLA allows specimen assessment of not only expression and activation, but also dimerization, which is not evaluated by current IHC techniques, it will likely serve as a way to evaluate promising anti-EGFR strategies directed at preventing EGFR dimerization and activation.

El glioblastoma multiforme és una de les neoplàsies més agressives de l’adult, malgrat els esforços realitzats en millorar el seu tractament, la supervivència dels pacients continua sent dolenta. L’objectiu global d’aquesta tesi ha estat l’estudi dels mecanismes moleculars implicats en la inducció de mort, parada de cicle pels inhibidors de MDM2 i els inhibidors de survivina en el glioblastoma multiforme humà, a més ha permès l’establiment de cultius primaris de glioblastoma humà. L’efecte dels compostos s’ha estudiat ex vivo en els cultius primaris i línies cel•lulars de glioblastoma multiforme humà. Les Nutlines són antagonistes de MDM2 que actuen, amb una gran especificitat, desplaçant la unió de la proteïna p53 del seu inhibidor MDM2 a un rang de dosi nanomolar, el qual comporta l’estabilització de la molècula de p53 i la posterior activació de la seva via de senyalització. Els resultats del tractament de cultius primaris i línies cel•lulars de glioblastoma demostren que la Nultina-3a és capaç de induir parada de cicle cel•lular (p21), apoptosi (Noxa i Puma), a més de induir senescència (marcatge amb SA-βGal) en aquelles cèl•lules amb un p53 funcional. Per el contrari, les línies cel•lulars i cultius primaris amb un p53 no funcional, són resistents al tractament degut a l’incapacitat de aquestes cèl•lules per activar la via de p53. El pretractament amb Nutlina-3a sensibilitza la línia cel•lular U87-MG enfront la radioteràpia. Això suggereix que la utilització de inhibidors de MDM2 podria ser una nova estratègia terapèutica, ja sigui de forma individual o combinada amb radioteràpia en el tractament de pacients amb glioblastoma multiforme amb una proteïna p53 funcional. YM155 (1-(2-metoxietil)-2-metil-4,9-dioxo-3-(piracina-2 ymetil)-4,9-dihidro-1H-nafto[2,3-d ]bromuro de imidazolium), és una petita molècula inhibidora de la proteïna antiapoptòtica Survivina. YM155 va ser seleccionat mitjançant un cribratge d’alt rendiment com supressor específic de l'expressió de survivina per inhibició del seu promotor. La survivina es troba sobreexpressada en la majoria dels tumors. La survivina es troba altament expressada en gliomes a més està associada amb una menor supervivència i amb un augment de la recurrència tumoral. En aquest apartat de la tesi es va estudiar l’efecte de YM155 en línies cel•lulars i un cultiu primari de glioblastoma humà. Els resultats obtinguts mostren que el tractament amb YM155 provoca una disminució de la viabilitat cel•lular, així com inducció d’apoptosi, que s’acompanya d’una disminució dels nivells proteics de survivina en totes les línies cel•lulars de glioma avaluades. La incubació de les línies cel•lulars i del cultius primari mostra canvis en el patró de cicle cel•lular, provocant aturada tant en fase G0/G1 o en fase S depenent de la línia cel•lular avaluada. El pretractament amb YM155 abans de la radioteràpia indueix un augment en el nombre de focus de γ-H2AX. Els resultats de aquest apartat de la tesis suggereixen que la inhibició de la Survivina podria ser una nova eina terapèutica que permetria augmentar la sensibilitat a la radioteràpia en pacients amb glioblastoma multiforme. / Glioblastoma multiforme is the most common and aggressive primary brain tumor in adults, despite efforts to improve their treatment, patients survival remains poor. The purpose of this thesis is the study of molecular mechanism involved in the induction of apoptosis, cells cycle arrest as a consequence of the treatment with MDM2 and Survivin inhibitors in human glioblatoma multiforme. This effect has been studied ex vivo in glioma cell lines and primary cultured glioblastoma. Nutlins small-molecule antagonist of MDM2, have been developed to inhibit p53-MDM2 interaction and activate p53 signaling in cancer cells. Nutlin-3a can induce cell cycle arrest (p21), apoptosi (PUMA and Noxa) in those cells with a functional p53 protein. In addiction Nutlin can induce cellular senescence (SA-βGal). In primary cultures and cells lines with a mutated p53 protein, Nutlin-3a fails to induce apoptosis and cell cycle arrest. Moreover Nutlin-3a pre-treatment in U87 cells sensitizes to radiotherapy. YM155 is a small molecule inhibitor of antiapoptotic protein survivin. Survivin is overexpressed in glioma and is associated with decreased survival and increases tumor recurrence. YM155 treatment induces a decrease in cell viability, as well as an apoptosis induction. This is accompanied by a decreased in survivin protein levels in all glioma cell lines tested. Glioma cell line and primary culture incubation show changes in the cell cycle pattern, causing arrest either in G0/G1 or S depending of the cell line tested.

Thesis (M.S.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains vii, 78 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 70-78).

Glioblastoma multiforme (GBMs) are the most common and severe form of malignant brain tumors. Despite recent advancement in the fields of surgical resection, radiotherapy and chemotherapy, the prognosis for patients with GBMs remains poor with the median survival rate of approximately a year. Recently, our laboratory has demonstrated the oncogenic role of cell cycle-related kinase (CCRK), a 42-kD protein kinase responsible for regulating cell growth in GBM carcinogenesis, suggesting that CCRK is a candidate oncogene in GBMs. Nevertheless, the regulation of CCRK expression and the cellular mechanism for its overexpression in GBMs remain elusive. Understanding the regulation of human CCRK expression in GBMs should therefore shed light on the development of better prognostic and therapeutic methods for this deadly disease. This study aims to characterize the human CCRK gene and the regulation of its expression in GBMs. We first characterized the 5’ upstream sequence of CCRK by in silico analysis, which revealed the absence of TATA box but the presence of three potential transcription factor binding sites for Sp1, c-Myc and CREB, and identified the transcription start site by 5’-RACE at 240 bp upstream of the start codon. In vitro analysis of the CCRK promoters revealed the presence of nucleotide polymorphisms in three high-grade glioblastoma cell lines U-87 MG, U-138 MG and U-373 MG, and the control fibroblast cell line CCD19Lu. Furthermore, three CpG islands within the CCRK promoter were identified and the CCRK promoter was hypomethylated in these cell lines. Sp1, c-Myc and CREB binding sites as well as the nucleotide polymorphisms on the CCRK promoter were further investigated. The results from electrophoretic mobility shift assay showed that these transcription factors interacted with the corresponding cis-regulatory elements on the CCRK promoter, removal of the potential Sp1 and c-Myc binding sites lowered the CCRK promoter activity by 46 – 66 % in vitro. In addition, mutations introduced to the nucleotide polymorphisms reduced the CCRK promoter activity by 62 – 81 % in U-87 MG cells and enhanced the CCRK promoter activity by 1.35-fold in U-138 MG cells, suggesting their importance in regulating CCRK expression. c-Myc is a proto-oncogene with its overexpression associated with a large variety of tumors. As c-Myc binding site was identified in the CCRK promoter, therefore, the effect of c-Myc on CCRK expression was examined. c-Myc overexpression resulted in significant enhancements in the CCRK promoter activity by 30.74-fold in U-87 MG cells, 26.5-fold in U-373 MG cells and 6.09-fold in U-138 MG cells. On the contrary, c-Myc knockdown reduced the CCRK promoter activity by 49 % in U-87 MG cells, 35 % in U-373 MG cells and 17% in U-138 MG cells. In summary, this study represents the first molecular characterization of the human CCRK gene and findings of this study would prime others for future research on the molecular pathogenesis of CCRK-mediated GBMs and for developing CCRK as a potential therapeutic and diagnostic target for GBMs and possibly other cancers. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Proliferating tissues, including embryonic and tumour tissues, preferentially employ aerobic glycolysis to support cell growth. This reliance on glycolysis even in the presence of oxygen, referred to as the “Warburg Effect”, may confer a proliferative, survival and invasive advantage and be exploited therapeutically. In this thesis, we demonstrate that the glycolytic enzyme Hexokinase 2 (HK2) is crucial for the “Warburg Effect” in human Glioblastoma Multiforme (GBM), the most common and therapeutically resistant malignant brain tumour. In contrast to normal brain and low-grade gliomas, GBMs exhibited a marked increase in HK2 expression, but not HK1, particularly in perinecrotic, hypoxic regions and its expression predicted poor overall survival of GBM patients. Stable loss of HK2 in GBM cells restored oxidative phosphorylation (OXPHOS)-mediated glucose metabolism, with increased oxygen consumption and decreased lactic acid production, an effect not seen with loss of glycolytic enzymes HK1 or PKM2. Furthermore, HK2 depletion resulted in decreased proliferation in vitro and in vivo and increased sensitivity to apoptotic inducers such as radiation and chemotherapy, both common adjuvant therapies of GBMs. Intracranial xenografts of GBM cells with reduced HK2 demonstrated significantly increased survival with decreased proliferation and angiogenesis yet enhanced invasiveness. In contrast, exogenous HK2 expression in GBM cells promoted proliferation, therapeutic resistance and intracranial growth. This was dependent partly on the PI3K/AKT dependent translocation of HK2 to the mitochondrial membrane. Stable loss of glycolytic enzymes HK2, HK1 and PKM2 reduced GBM proliferation but differentially altered the PI3K/AKT/mTOR and AMPK signaling pathways, the extent to which may influence whether a cell preferentially undergoes autophagy or apoptosis as the primary mode of cell death. Collectively, targeting enzymes employed by the tumour to modulate its energy metabolism, such as HK2 in GBMs, may favourably alter its therapeutic sensitivity to radiation and both classical and novel chemotherapeutic agents.

No description available.

Proliferating tissues, including embryonic and tumour tissues, preferentially employ aerobic glycolysis to support cell growth. This reliance on glycolysis even in the presence of oxygen, referred to as the “Warburg Effect”, may confer a proliferative, survival and invasive advantage and be exploited therapeutically. In this thesis, we demonstrate that the glycolytic enzyme Hexokinase 2 (HK2) is crucial for the “Warburg Effect” in human Glioblastoma Multiforme (GBM), the most common and therapeutically resistant malignant brain tumour. In contrast to normal brain and low-grade gliomas, GBMs exhibited a marked increase in HK2 expression, but not HK1, particularly in perinecrotic, hypoxic regions and its expression predicted poor overall survival of GBM patients. Stable loss of HK2 in GBM cells restored oxidative phosphorylation (OXPHOS)-mediated glucose metabolism, with increased oxygen consumption and decreased lactic acid production, an effect not seen with loss of glycolytic enzymes HK1 or PKM2. Furthermore, HK2 depletion resulted in decreased proliferation in vitro and in vivo and increased sensitivity to apoptotic inducers such as radiation and chemotherapy, both common adjuvant therapies of GBMs. Intracranial xenografts of GBM cells with reduced HK2 demonstrated significantly increased survival with decreased proliferation and angiogenesis yet enhanced invasiveness. In contrast, exogenous HK2 expression in GBM cells promoted proliferation, therapeutic resistance and intracranial growth. This was dependent partly on the PI3K/AKT dependent translocation of HK2 to the mitochondrial membrane. Stable loss of glycolytic enzymes HK2, HK1 and PKM2 reduced GBM proliferation but differentially altered the PI3K/AKT/mTOR and AMPK signaling pathways, the extent to which may influence whether a cell preferentially undergoes autophagy or apoptosis as the primary mode of cell death. Collectively, targeting enzymes employed by the tumour to modulate its energy metabolism, such as HK2 in GBMs, may favourably alter its therapeutic sensitivity to radiation and both classical and novel chemotherapeutic agents.

Made available in DSpace on 2015-12-10T14:23:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-02-17. Added 1 bitstream(s) on 2015-12-10T14:29:14Z : No. of bitstreams: 1 000851655.pdf: 1706170 bytes, checksum: 990d3d441221c8d5284639c96fcf57c8 (MD5) / Os tumores cerebrais primários mais comuns são denominados gliomas. Eles são definidos patologicamente pela presença de características histológicas e imuno-histoquímicas que evidenciam diferenciação glial. De acordo com a suposta linhagem de origem, eles são classificados como astrocitomas, oligodendrogliomas ou ependimomas. Dentre eles, os astrocitomas são os mais comuns e agressivos. O tratamento atualmente utilizado inclui remoção cirúrgica seguida de quimioterapia com temozolamida (TMZ) e radioterapia, porém sua eficácia é muito baixa devido à alta resistência das células tumorais. Buscando encontrar genes associados com a elevada resistência dos astrocitomas, realizamos um estudo anterior de expressão gênica diferencial utilizando uma coleção de genes de reparo de DNA. Nesta análise foram identificados sete genes significantemente superexpressos em glioblastoma multiforme (GBM), o tipo mais agressivo de astrocitoma. Estes genes são: APEX1, BRCA2, BRIP1, EXO1, NEIL3, RAD54L e XRCC2. Através de RT-PCR quantitativo, avaliamos os níveis de expressão destes genes em um painel expandido de 54 casos clínicos de astrocitomas de diferentes graus de malignidade e em 5 linhagens celulares de GBM. Todos os genes analisados mostraram-se mais expressos nos astrocitomas, com exceção de RAD54L em amostras de astrocitoma de grau II. Além disso, a superexpressão dos 7 genes avaliada isoladamente não exerce influência direta na sobrevida dos pacientes. Evidenciou-se ainda a superexpressão mais acentuada de EXO1 e NEIL3, que foram selecionados para realização de ensaios funcionais de silenciamento, e avaliação do ciclo celular e taxas de apoptose/morte efetiva das células. Estes ensaios foram realizados com as linhagens celulares T98G e U138MG, que apresentaram maiores níveis de expressão destes genes. Nos ensaios funcionais, observamos que o silenciamento... / Gliomas are the most common type of primary brain cancers. They are pathologically defined by the presence of histological and immunehistochemical characteristics that evidence glial differentiation. According to the hypothetical cell of origin they are classified in: astrocytomas, oligodendrogliomas and ependimomas. Among them, astrocytomas are the more common and aggressive type. The treatment currently used for GBM includes surgical resection of tumor followed by chemotherapy with temozolamide (TMZ) and radiotherapy, but this protocol is still insufficient due to the high resistance of cancer cells. Searching for repair genes associated with the high resistance of astrocytomas, we developed a previous study of differential gene expression using a collection of DNA repair genes. In this analysis, we identified seven genes significantly overexpressed in glioblastoma multiforme (GBM), namely: APEX1, BRCA2, BRIP1, EXO1, NEIL3, RAD54L and XRCC2. Using quantitative RT-PCR, we evaluated the expression of these genes in an expanded panel of samples with 54 clinical cases of different grade astrocytomas and five GBM cell lines. All genes showed expression significantly higher in astrocytomas, except RAD54L in grade II astrocytomas. Moreover, the overexpression of this 7 genes evaluated individually doesn't exert direct influence upon patient's survival rate. Remarkably, EXO1 and NEIL3 showed the higher fold changes and were chosen for functional silencing assays. This experiments were performed with T98G and U138MG cell lines that showed the higher expression levels among the GBM cell lines analyzed. In the functional assays, we observed that the silencing of EXO1 or NEIL3 doesn't induce changes in the apoptosis and cell death rates and doesn't change the distribution of cells in cycle. Beyond this, the silencing of this two genes doesn't sentisizes cells to ionizing radiation.

Made available in DSpace on 2014-06-11T19:33:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-03-01Bitstream added on 2014-06-13T19:04:25Z : No. of bitstreams: 1 salgado_ds_dr_arafo_parcial.pdf: 429004 bytes, checksum: f6208d49236c547d4d549611720de1cb (MD5) Bitstreams deleted on 2015-03-16T11:30:11Z: salgado_ds_dr_arafo_parcial.pdf,Bitstream added on 2015-03-16T11:30:59Z : No. of bitstreams: 1 000647903.pdf: 1029961 bytes, checksum: e2b31866e98357928fb646c544fe8be5 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O presente projeto de pesquisa tem como objetivo avaliar a prevalência, os aspectos clínicos, a severidade e os fatores de risco das manifestações gengivais de doenças de origem imunológica, além de avaliar a efetividade de medidas de controle da placa bacteriana na melhora dos sinais e sintomas de pacientes que apresentam líquen plano bucal com envolvimento gengival. Para isso, foram elaborados três estudos. No primeiro deles, com o objetivo de avaliar a epidemiologia da gengivite descamativa em um Serviço de Medicina Bucal, foram selecionados casos de líquen plano, penfigóide das membranas mucosas, pênfigo vulgar, lupus eritematoso e eritema multiforme com manifestações gengivais, atendidos no Serviço de Medicina Bucal da Faculdade de Odontologia de Araraquara - Unesp, no período de janeiro de 1995 a dezembro de 2004. Dos 4.776 prontuários avaliados, 48 apresentaram lesões de manifestações gengivais, sendo 68.8% mulheres, com idade média de 43.3 anos. As doenças mais comuns encontradas na amostra foram o líquen plano, o penfigóide das membranas mucosas e o pênfigo vulgar. A maioria dos pacientes avaliados apresentava sintomatologia dolorosa (83.3%) e apenas 35.4% dos casos apresentaram lesões restritas à gengiva. Nossos resultados contribuíram para mostrar a importância do diagnóstico precoce e consequentemente do adequado tratamento das manifestações gengivais associadas a doenças de origem imunológica. O Segundo estudo, também retrospectivo, objetivou avaliar a prevalência de lesões gengivais em portadores de líquen plano bucal, bem como os aspectos e fatores de risco associados, a partir de dados de prontuários de pacientes atendidos no mesmo Serviço, no período compreendido entre janeiro de 1995 e dezembro de 2009. Dos 9.475 prontuários analisados, 84 apresentaram... / This research project aims to assess the prevalence, clinical aspects, severity and risk factors of gingival disease of immune origin, and to evaluate the effectiveness of control plaque in the improvement of signs and symptoms of patients lichen planus presenting with gingival involvement. For this, we prepared three studies. In the first, in order to assess the epidemiology of desquamative gingivitis in a Department of Oral Medicine, were selected cases of lichen planus, oral pemphigoid, pemphigus vulgaris, lupus erythematosus and erythema multiforme in the gingiva, the Service's Oral Medicine Faculty of Dentistry of Araraquara - UNESP, from January 1995 to December 2004. Of 4,776 records evaluated, 48 had lesions associated with desquamative gingivitis, and 68.8% women, mean age of 43.3 years. The most common disease found in the sample were lichen planus, oral pemphigoid and pemphigus vulgaris. Much of the patients had painful symptoms (83.3%) and only 35.4% of cases had lesions limited to the gingiva. Despite the small sample size, the results helped show the importance of early diagnosis and consequently the appropriate treatment of diseases associated with desquamative gingivitis. The second study, also retrospective study aimed to assess the prevalence of gingival lesions in patients with lichen planus, as well as the aspects and associated risk factors in medical records of patients attending the same service in the period between January 1995 and December 2009. Of 9,475 charts analyzed, 84 had lichen... (Complete abstract click electronic access below)

Glioblastoma multiforme (GBM) is among the most aggressive and mortal cancers of the central nervous system. Maximal safe surgical resection, followed by radiotherapy accompanied with chemotherapy is the standard of care for GBM patients. Despite this intensive treatment with conventional approaches, the management of GBM remains poor. The infiltrative nature of cancer cells makes the complete tumour removal by surgery virtually impossible. In addition, the blood-brain barrier’s (BBB) lack of permeability limits the number of effective chemotherapy drugs for GBM. Temozolomide (TMZ) is the most widely used chemotherapeutic agent for GBM because of its ability to pass the BBB. However, high systemic doses required to achieve brain therapeutic level, resulting in numerous side effects. The recurrence of GBM is almost inevitable due to the aforementioned shortcomings of conventional methods of treatment. Therefore, a great deal of effort has been focused on the development of new treatment methods capable of providing a high concentration of chemotherapy drug at the tumour site. Microspheres made from biodegradable polymers hold great potential to keep the chemotherapeutic agent intact within the carrier and locally deliver the drug over an extended period. However, the encapsulation of amphiphilic drug molecules such as TMZ within poly (d, l-lactide-co-glycolide) (PLGA) microspheres with conventional emulsion methods, oil-in-water (o/w), water-in-oil-in-water (w/o/w), is a major challenge. The extremely low encapsulation efficiencies obtained for TMZ-loaded PLGA microspheres using the aforementioned techniques (<7%) hampers the ability to scale up this process. Additionally, the injected microspheres to the tumour site tend to dislocate due to the cerebral flow which reduces the effectiveness of this localized drug delivery strategy. This study has focused on the development of a 3D bioprinted hydrogel-based mesh containing TMZ-loaded PLGA microspheres with high encapsulation efficiency (GlioMesh). To accomplish this, oil-in-oil (o/o) emulsion solvent evaporation technique was used to prepare PLGA microspheres loaded with TMZ. The poor solubility of TMZ in the external oil phase, liquid paraffin, resulted in obtaining encapsulation efficiencies as high as 61%. We then used the 3D bioprinting technology to embed TMZ-loaded PLGA microspheres into an alginate mesh. This provides the advantage of immobilizing the microspheres at the tumour site. Additionally, the flexibility and porosity of 3D bioprinted mesh allow for easy implantation and nutrients transportation to the brain tissue. The incorporation of polymeric microspheres within alginate fibres led to achieving an extended release of TMZ over 50 days. The functionality of GlioMesh in inducing cell cytotoxicity was evaluated by performing in vitro cell viability tests on U87 human glioblastoma cells. Higher cytotoxic effects were observed in the case of treatment with GlioMesh compared to the free drug because of the sustained release properties of our mesh. These data suggest that GlioMesh holds great promise to be used as an implant in the treatment of GBM. / Graduate / 2019-04-19