The first experiment was conducted to determine the optimum in vitro culture system for one-cell bovine embryos. Subsequent experiments compared bisection and biopsy for acquisition of cellular material from bovine morulae for DNA amplification by the polymerase chain reaction technique (PCR), and evaluated the use of DNA microinjection, in vitro culture, morula bisection, and PCR for production and selection of transgenic bovine preimplantation embryos. In vivo fertilized one-cell bovine embryos were cultured in TCM-199 (n=46), co-cultured with bovine oviductal epithelial cells (OEC; n=38), or in explanted immature mouse oviducts (n=54). Of the embryos that cleaved once, 52.6, and 30.4 and 0.0% developed to morulae/blastocysts after culture in OEC, TCM-199, and explanted mouse oviducts. Mean cell number for embryos cultured in OEC (24.5) was higher than for embryos cultured in TCM-199 (12.8) or in explanted mouse oviducts (5.9; P<.05). Bovine morulae were subjected to bisection (n=44; 20 to 30 cells) or biopsy (n=50; 8 to 12 cells) to assess embryo development in vitro and compare the efficiency of PCR amplification of an endogenous 18S rRNA. Mean development scores (l1=degenerate, 2=morula, 3=blastocyst) and mean cell number after microsurgery and 48 h of culture did not differ between treatments (P>.05; 2.4 ± .1 and 41.8 ± 2.5 versus 2.3 ± .1 and 48.8 ± 2.9, respectively). Frequency of the 18S rRNA amplifications was similar (P>.05) for demi-morulae (78%; 32/41) and biopsies (81%; 39/48). In the third experiment, in vivo fertilized one- (n=155), two- (n=57) and four-cell (n=62) bovine embryos were collected for pronuclear and nuclear DNA microinjection. Approximately 70% of the embryos were injected with DNA and 30% served as controls. Injection did not affect (P>.05) mean development scores after 144 h of cultured in TCM-199 with OEC. Sixty-five (34%) of the DNA injected embryos developed to morulae and were bisected. Injected DNA was amplified by PCR in 29% (19/65) of the demi-morulae. Frequency of DNA detection was more frequent (P<.01) in morulae injected at the 1-cell stage (50%: 16/32) than at the 2-cell (10%; 1/10) or 4-cell (9%: 2/23) stage. Production and selection of transgenic bovine morulae was most successful when one-cell bovine embryos were microinjected. / Ph. D.

Durante o desenvolvimento deste projeto adotou-se como estratégia o sequenciamento de ESTs, com a finalidade de encontrar mensagens relacionadas com desenvolvimento, metabolismo e principalmente amplificação/politenização em glândulas salivares de Rhynchosciara americana, um díptero (Sciarídeo) que apresenta cromossomos politênicos e amplificação gênica rigidamente regulada ao longo do desenvolvimento larval, tanto neste tecido quanto em outros. Um total de 8193 ESTs foi gerado, estas foram anotadas e categorizadas segundo os termos do Gene Ontology Consortium, proporcionando uma visão geral do status metabólico, como em um Northern eletrônico, de um ponto importante no desenvolvimento desta espécie, quando surgem amplificações gênicas específicas e a glândula salivar necessita secretar as proteínas do casulo. Outros frutos deste seqüenciamento foram a determinação de 91 polimorfismos e a criação de uma tabela de códon usage. Diversos ESTs foram identificados com potencial envolvimento com os endociclos observados neste tecido, destes, RaDup e RaMCM5 foram selecionados para estudo. Suas regiões genômicas foram isoladas e suas localizações cromossômicas foram identificadas, em relação a RaDup, toda a porção codificante de seu mensageiro e 12kb de DNA genômico contendo seu gene foram seqüenciados, revelando sua estrutura gênica. Anticorpos foram produzidos para detectar esta proteína, gerando evidências de sua participação tanto na replicação mitótica como nos endociclos presentes nas glândulas salivares. A localização cromossômica de RaDup é um dado muito interessante, pois pela primeira vez um pufe amplificado é relacionado com um gene regulatório. / In this work EST sequencing was used as strategy to find messages related to development, metabolism and polyteny/amplification in salivary glands of Rhynchosciara americana, a dipteran (Sciaridae) that shows in this tissue giant polytene chromosomes and gene amplification tightly regulated throughout development of the larvae. A total of 8193 EST sequences were generated, annotated and categorized using Gene Ontology Consortium terms, providing a general view of the metabolic status, like an electronic Northern, of an important point in development of the larvae, that shows where specific genes are amplified and the salivary gland needs to secrete the proteins to form the cocoon. Other data include determination of 91 SNPs and a statistic of codon usage. Several ESTs were identified with potential connection to endocicles, from these RaDup and RaMCM5 were selected for further studies. Both chromosomal loci were identified and genomic regions isolated, for RaDup the coding region of its mRNA and 12kb of genomic region were completely sequenced, revealing its gene structure, and antibodies were raised against this protein, making evident data about its involvement in replication in mitotic cells and in endocicles in salivary glands. About the chromosomal locus of RaDup, it becomes very interesting, because for the first time one amplified puff can be related to a regulatory gene.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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Submitted by VICTOR BERNARDES BARROSO DA COSTA null (victorbernardes_@hotmail.com) on 2016-03-16T04:08:27Z No. of bitstreams: 1 Dissertação - VICTOR (Versão Final).pdf: 20193346 bytes, checksum: f7da64e79358575f01cf61a5054c4587 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-03-18T12:52:04Z (GMT) No. of bitstreams: 1 costa_vbb_me_sjc.pdf: 20193346 bytes, checksum: f7da64e79358575f01cf61a5054c4587 (MD5) / Made available in DSpace on 2016-03-18T12:52:04Z (GMT). No. of bitstreams: 1 costa_vbb_me_sjc.pdf: 20193346 bytes, checksum: f7da64e79358575f01cf61a5054c4587 (MD5) Previous issue date: 2016-01-21 / Submitted by VICTOR BERNARDES BARROSO DA COSTA null (victorbernardes_@hotmail.com) on 2016-01-26T01:08:29Z No. of bitstreams: 1 Dissertação - VICTOR (Versão Final).pdf: 20193437 bytes, checksum: c041b8e5fa02ed4f74d445c5838128fd (MD5) / Approved for entry into archive by Sandra Manzano de Almeida (smanzano@marilia.unesp.br) on 2016-01-26T13:39:40Z (GMT) No. of bitstreams: 1 costa_vbb_me_sjc.pdf: 20193437 bytes, checksum: c041b8e5fa02ed4f74d445c5838128fd (MD5) / Made available in DSpace on 2016-01-26T13:39:40Z (GMT). No. of bitstreams: 1 costa_vbb_me_sjc.pdf: 20193437 bytes, checksum: c041b8e5fa02ed4f74d445c5838128fd (MD5) Previous issue date: 2016-01-21 / Item merged in doublecheck by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-03-21T13:12:44Z Item was identical to item(s): 135509, 132061 at handle(s): http://hdl.handle.net/11449/136305, http://hdl.handle.net/11449/132945 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM) / O carcinoma de células escamosas (CEC) de boca é uma neoplasia incomum em pacientes jovens. Na literatura de língua inglesa não há relatos de estudos que investiguem a amplificação do EGFR e a expressão desta proteína neste grupo etário. O objetivo deste estudo foi investigar a amplificação do EGFR através da técnica de hibridização por fluorescência in situ (FISH) e correlacionar os resultados obtidos através da imunomarcação da proteína EGFR com a epidemiologia e com o prognóstico dos pacientes avaliados. Ao final dos testes de FISH e imuno- histoquímicos, 21 amostras do grupo teste (pacientes ≤ 40 anos) e 39 amostras do grupo controle (pacientes ≥ 50 anos) foram consideradas adequadas para avaliação. As variáveis clínicas e anatomopatológicas foram comparadas pelos testes Qui-Quadrado ou exato de Fisher. A expressão do marcador EGFR e do método de FISH foi comparada entre os grupos por meio do teste não paramétrico de Mann-Whitney. As curvas de sobrevida foram calculadas utilizando o método de Kaplan-Meier e suas curvas foram comparadas através do teste de log-rank. Houve maior número de pacientes do sexo masculino, leucodermas, tabagistas e etilistas. A amplificação do EGFR foi maior no grupo teste (p = 0,018). A amplificação do EGFR associou-se estatisticamente com a variável estadiamento clínico avançado (p = 0,013), independente do grupo. A expressão da proteína EGFR correlacionou-se com tumores bem diferenciados (p = 0,011) e a presença de metástase (p = 0,035), independente da idade. A presença de amplificação foi mais frequente no grupo tese. Alguns casos de pacientes ≥40 anos de idade podem ser adequados ao emprego da terapia anti-EGFR, devido à amplificação do EGFR. / Oral squamous cell carcinoma (OSCC) is uncommon neoplasia in young patients. In the English literature, there are no reports of studies that investigate the amplification of EGFR and expression of this protein in this age group. The aim of this study was to investigate the amplification of EGFR by fluorescence in situ hybridization (FISH) and to correlate the results by immunostaining of EGFR protein with clinicopathological features and prognosis. After FISH and immunohistochemistry, 21 samples of the test group (≤ 40 years) and 39 samples of control group (≥ 50 years) were suitable for evaluating. Categorical variables were compared by the Pearson chi-square test or Fisher's exact test. Associations between protein levels and FISH results with clinicopathological characteristics of the patients were analyzed by Mann-Whitney U test. Survival rates were calculated using the Kaplan-Meier method and the curves were compared by the log-rank test. There was predominance for male patients, leucoderma, smoking and alcohol consumption. The EGFR amplification was higher in the test group (p = 0.018) and it was associated statistically with advanced clinical stage (p = 0.013), independent of the group. The expression of EGFR protein was correlated to well differentiated tumors (p = 0.011) and presence of metastasis (p = 0.035), regardless of age. Presence of EGFR amplification and/or expression. Some cases of patients ≥40 years old might be suitable for anti-EGFR therapy because of EGFR amplification. / FAPEAM: 254/2014

Resumo: A maioria dos casos de fraturas ósseas é utilizada nas cirurgias, implantes ortopédicos de osteossíntese (placa-parafuso e parafusos) confeccionados em aço inoxidável austeníticos, devido à sua baixa relação custo-benefício. As infecções associadas aos implantes de osteossintese estão relacionadas com o crescimento de microrganismos em biofilmes, resultando em um processo infeccioso de difícil erradicação. O objetivo do estudo foi identificar os microrganismos recuperados do conjunto metálico placaparafuso e parafusos após remoção cirúrgica, avaliar a capacidade de aderência dos microrganismos, verificar a viabilidade celular, caracterizar genotipicamente os isolados Gram-positivos e detectar a resistência aos antimicrobianos. Os conjuntos placa-parafuso e parafusos de aço inoxidável austenítico ASTM F138/F139 e ISO NBR 5832-1/9 foram transportados em bolsa de polietileno para o Laboratório de Microbiologia Clínica. Os implantes foram lavados em solução tampão fosfato-salino, armazenados em bolsa contendo solução Ringer e submetidos ao banho ultrassônico em freqüência de 40±2 kHz por 5 minutos. O fluido sonicado foi transferido para tubos Falcon e centrifugado a 3.000g durante 20 minutos. O sedimento foi resuspendido em nova solução Ringer, homogeneizado por vortex e 10μL semeados em ágar Sangue de carneiro 5%, MacConkey e Sabouraud. Os meios foram incubados em diferentes condições de temperatura por 7 a 14 dias para recuperação de microrganismos. O perfil de resistência dos isolados foi obtido de acordo com CLSI 2011. Para análise da viabilidade celular foi utilizado o kit Live/Dead e microscópio de fluorescência... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The majority of cases of bone fractures is used in surgery, internal fixation of orthopedic implants (screw-plate and screws) manufactured in austenitic stainless steel due to its low cost-benefit. Infections associated with implant fixation are related to the growth of microorganisms in biofilms, resulting in an infection difficult to eradicate. The objective of study was to identify the microorganisms recovered from the set screwplate and screws metallic after surgical removal, evaluate the capacity of adherence of microorganisms, check the cell viability and characterize the isolates genotypically Gram-positive and detect antimicrobial resistance. The sets screw-plate and screws of austenitic stainless steel ASTM F138/F139 and ISO NBR 5832-1/9 were transported in polyethylene bag to the Laboratory of Clinical Microbiology. The implants were washed in phosphate buffered saline solution, stored in bags containing Ringer's solution and submitted to ultrasonic bath at a frequency of 40 kHz ± 2 for 5 minutes. The sonicate fluid was transferred to falcon tubes and centrifuged at 3.000g for 20 minutes The new sediment was re-suspended in Ringer's solution, homogenized by vortex and 10μL seeded agar 5% sheep blood, MacConkey and Sabouraud. The media were incubated at different temperatures for up to 7 days for growth of CFU mL-1. The resistance profile of the isolates was obtained according to CLSI 2011. For analysis of cell viability kit was used for Live/Dead and fluorescence microscope. After microbiological analysis the isolation was observed in some samples of polymicrobial implants. The data obtained showed that bacteria were the most isolated coagulase-negative... (Complete abstract click electronic access below) / Orientador: Clarice Queico Fujimura Leite / Coorientador: Elisabeth Loshchagin Pizzolitto / Banca: Eliana Aparecida Varanda / Banca: Beatriz Maria Machado de Medeiros / Banca: Denise Crispin Tavares / Mestre

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Abstract Human prostate-specific antigen (hPSA, KLK3) and glandular kallikrein 2 (hK2, KLK2), two members of a large human tissue kallikrein enzyme family, were produced as recombinant mature proteins for the first time and characterized. Furthermore, their association with prostate cancer was studied. Both proteins were produced with baculovirus expression vector system in pilot-scale using bioreactors. Recombinant hPSA was either active with chymotrypsin-like activity or inactive with incorrect processing of N-terminus. The molecular weight of active recombinant hPSA was 31 kD and it formed stable complexes with serine protease inhibitors, α1-antichymotrypsin (ACT) and α2-macroglobulin (2αM). Two polymorphic forms of KLK2, Arg226hK2 and Trp226hK2, were found. The recombinant Arg226hK2 had trypsin-like activity, while recombinant Trp226hK2 was inactive. The Arg226hK2 was labile with low production yields. The molecular weights of hK2 polymorphic forms were 33 kD. hPSA isoforms secreted by prostate cancer cells, LNCaP, were isolated and characterized. These proteins were N-terminally heterogeneous: 10-60% of LNCaP-PSAs were correctly processed. Molecular modeling suggested that the additions or deletions of two or four N-terminal amino acids could affect the three-dimensional structure and reduce the activity of LNCaP-PSA. Active isoforms had chymotrypsin-like activity and formed stable complexes with ACT and 2αM. The expression of hPSA and hK2 was studied with in situ hybridization and immunohistochemistry techniques in benign and cancerous prostate tissue. hK2 mRNA was expressed at a significantly higher level in prostate cancer tissue than in benign prostate tissue (P &lt; 0.0005). The hPSA mRNA expression levels were reversed (P = 0.06). In benign tissue, the mean level of hK2 mRNA was 82% of the respective value of hPSA (P &lt; 0.003), whereas in tumor tissue the mean hK2 expression level was 21% higher than that of hPSA (P &lt; 0.01). The results at protein level supported the mRNA findings. There was a correlation between hPSA and hK2 mRNA levels in both benign (r = 0.735; P &lt; 0.01) and malignant (r = 0.767; P &lt; 0.01) prostate tissue. It was shown with competitively differential PCR that the KLK2 gene was amplified in prostate tumor tissue, while the KLK3 gene was not. These results suggest that hK2 and hPSA have a diverse value in the diagnosis of prostate cancer.

Durante o desenvolvimento deste projeto adotou-se como estratégia o sequenciamento de ESTs, com a finalidade de encontrar mensagens relacionadas com desenvolvimento, metabolismo e principalmente amplificação/politenização em glândulas salivares de Rhynchosciara americana, um díptero (Sciarídeo) que apresenta cromossomos politênicos e amplificação gênica rigidamente regulada ao longo do desenvolvimento larval, tanto neste tecido quanto em outros. Um total de 8193 ESTs foi gerado, estas foram anotadas e categorizadas segundo os termos do Gene Ontology Consortium, proporcionando uma visão geral do status metabólico, como em um Northern eletrônico, de um ponto importante no desenvolvimento desta espécie, quando surgem amplificações gênicas específicas e a glândula salivar necessita secretar as proteínas do casulo. Outros frutos deste seqüenciamento foram a determinação de 91 polimorfismos e a criação de uma tabela de códon usage. Diversos ESTs foram identificados com potencial envolvimento com os endociclos observados neste tecido, destes, RaDup e RaMCM5 foram selecionados para estudo. Suas regiões genômicas foram isoladas e suas localizações cromossômicas foram identificadas, em relação a RaDup, toda a porção codificante de seu mensageiro e 12kb de DNA genômico contendo seu gene foram seqüenciados, revelando sua estrutura gênica. Anticorpos foram produzidos para detectar esta proteína, gerando evidências de sua participação tanto na replicação mitótica como nos endociclos presentes nas glândulas salivares. A localização cromossômica de RaDup é um dado muito interessante, pois pela primeira vez um pufe amplificado é relacionado com um gene regulatório. / In this work EST sequencing was used as strategy to find messages related to development, metabolism and polyteny/amplification in salivary glands of Rhynchosciara americana, a dipteran (Sciaridae) that shows in this tissue giant polytene chromosomes and gene amplification tightly regulated throughout development of the larvae. A total of 8193 EST sequences were generated, annotated and categorized using Gene Ontology Consortium terms, providing a general view of the metabolic status, like an electronic Northern, of an important point in development of the larvae, that shows where specific genes are amplified and the salivary gland needs to secrete the proteins to form the cocoon. Other data include determination of 91 SNPs and a statistic of codon usage. Several ESTs were identified with potential connection to endocicles, from these RaDup and RaMCM5 were selected for further studies. Both chromosomal loci were identified and genomic regions isolated, for RaDup the coding region of its mRNA and 12kb of genomic region were completely sequenced, revealing its gene structure, and antibodies were raised against this protein, making evident data about its involvement in replication in mitotic cells and in endocicles in salivary glands. About the chromosomal locus of RaDup, it becomes very interesting, because for the first time one amplified puff can be related to a regulatory gene.

O Receptor do Fator de Crescimento Epidérmico (do inglês, EGFR) é uma proteína de membrana celular que consiste em um domínio extracelular para o acoplamento do ligante e em um domínio intracelular apresentando sítio catalítico de tirosinoquinase. Em ~40% dos GBMs primários é observada a amplificação de EGFR resultando na sua hiperexpressão, o que raramente ocorre em GBM secundário. Mais da metade dos casos de GBM com amplificação do receptor está associado com rearranjo do gene, uma forma deletada de EGFR (EGFRvIII). Adicionalmente, o receptor de interleucina 13 alfa-2 (IL-13R 2), apresenta-se abundante e especificamente hiperexpresso em gliomas de alto grau, em particular, GBM. O objetivo do presente estudo é analisar a expressão, amplificação, e deleção de EGFR em astrocitomas, bem como a coexpressão entre esse gene e o da IL-13R 2. Foram analisadas 145 astrocitomas (22 astrocitomas pilocíticos (AP); 22 astrocitomas grau II (AGII); 17 astrocitomas anaplásico (AA); e 84 GBM) e 17 tecidos cerebrais não tumorais provenientes de cirurgia de epilepsia. A deleção EGFRvIII foi analisada por RT-PCR, e confirmada por PCR em tempo real (RQ-PCR). A expressão relativa de EGFR e IL-13R 2 foi estudada por RQ-PCR utilizando-se o método SYBR Green, comparado ao tecido não tumoral, e normalizado para os genes de referência endógena, HPRT e Gus- . A amplificação de EGFR foi também determinada por RQ-PCR com relação ao gene da beta-hemoglobina, descrito como um gene de cópia única. Foi realizada imunohistoquímica para analisar a expressão da proteína EGFR. A deleção EGFRvIII foi somente encontrada em GBM (19/84, 23%), demonstrando a exclusividade dessa alteração num grau tumoral de maior malignidade e uma diminuição da sobrevida desses pacientes (p = 0,030). A hiperexpressão de EGFR foi encontrada em 88 casos (61%), correspondendo a 50% de GBM, 88% de AA, e interessantemente em 77% de AGII e 64% de AP. Da mesma maneira, a hiperexpressão de IL-13R 2 foi encontrada em 62 casos (43%), correspondendo a 48% de GBM, 29% de AA, 18% de AGII e, surpreendentemente em 59% de AP. Embora tenha havido um aumento de expressão de ambos os genes em todos os graus de astrocitomas, não houve coexpressão dos mesmos. A amplificação de EGFR foi observada em 29 casos (20%) correspondendo a 31% de GBM e ainda um caso para cada um dos demais graus de astrocitomas, sendo que dos 29 casos amplificados, 21 pacientes eram mais velhos que 45 anos (p < 0,001) e 50% dos casos de GBM com amplificação de EGFR, apresentaram simultaneamente EGFRvIII. Adicionalmente, o acúmulo citoplasmático da proteína foi detectado em 74 casos (51%), correspondendo a 55% de GBM, 47% de AA, 54,5% de AGII e 37% de AP, além de um acúmulo nuclear detectado em 15% dos casos de astrocitomas difusamente infiltrativos. Assim, a alta freqüência de hiperexpressão dos genes estudados em todos os graus de astrocitomas, principalmente a amplificação de EGFR e a presença da deleção EGFRvIII foram observados entre os astrocitomas de alto grau, especialmente em GBM. Os resultados apresentados contribuem para um melhor direcionamento no futuro em métodos terapêuticos específicos, salientando a importância da análise de expressão molecular, protéica e das alterações mutacionais que envolvem genes candidatos, em particular, EGFR e IL-13R 2 / Epidermal Growth Factor Receptor, EGFR is a transmembrane protein consisting of an extracellular EGF-binding domain and an intracellular domain with ligand-activated tyrosine kinase activity. In ~40% of primary GBM is observed amplification leading to overexpression of EGFR, but rarely in secondary GBM. Over the half of primary GBM with EGFR amplification is associated to gene rearrangement, a deleted form of EGFR (EGFRvIII). Additionally, the interleukin-13 alpha 2 receptor (IL13R 2) is abundant and specifically overexpressed in high-grade gliomas, particularly in GBM. The aim of the present study is to analyze the EGFR expression, amplification, and deletion in astrocytomas as well as its coexpression with IL13R 2. We have analyzed 145 surgical astrocytoma samples (22 pilocytic astrocytomas (PA); 22 low-grade astrocytomas (LGA); 17 anaplastic astrocytomas (AA); and 84 GBM) and 17 non-neoplastic brain tissue from epilepsy surgery. EGFRvIII deletion was analyzed by RT-PCR, and also confirmed by real time PCR (RQ-PCR). The relative EGFR and IL-13R 2 expression was studied by RQ-PCR using SYBR Green method, compared to non-neoplastic tissue, normalized for HPRT and Gus- genes. The EGFR amplification was also determined by RQ-PCR relative to the hemoglobin beta gene, described as a single copy gene. Immunohistochemistry was performed to analyze the protein expression in tumor samples. The EGFRvIII deletion was found only in GBM cases (19/84, 23%) demonstrating the exclusivity of this alteration in higher tumor grade and survival was decreased in these patients (p = 0.030). EGFR overexpression was found in 88 cases (61%), corresponding to 50% of GBM, 88% of AA, and interestingly in 77% of LGA and 64% of PA. In the same way, the overexpression of IL-13R 2 was found in 62 cases (43%), corresponding to 48% of GBM, 29% of AA, 18% of LGA and, surprising in 59% of PA. Although increased expression of both genes was demonstrated in all astrocytoma grades, it was no coexpression of the genes. The amplification was observed in 29 cases (20%) corresponding to 31% of GBM and only one case each of PA, LGA and AA. Among 29 cases with EGFR amplification, 21 patients were older than 45 years (p < 0.001) and 50% of GBM with EGFR amplification presented simultaneously the EGFRvIII. Moreover, the EGFR cytoplasmic accumulation was detected in 74 cases (51%), corresponding to 55% of GBM, 47% of AA, 54.5% of LGA and 37% of PA, and nuclear accumulation was detected in 15% of diffusely infiltrative astrocytomas. Thus, the high overexpression frequency of the genes studied in all grades of astrocytomas, mainly the EGFR amplification and presence of EGFRvIII deletion were observed among high-grade astrocytomas, mainly in GBM. The present results contribute to better tailoring specific future therapeutical approach in patients with astrocytomas, pointing out the importance of the molecular, protein expressions and mutational analyses of candidates genes, in particular, EGFR and IL-13R 2