O glioblastoma multiforme (GBM) é a forma mais maligna de tumor cerebral, originado de células astrocíticas e caracterizado pela intensa proliferação, angiogênese e invasão celular pelo parênquima cerebral normal. O ácido gama-linolênico (GLA) mostrou ter ações anti-tumorais, nos processos de proliferação, migração e angiogênese. Utilizou-se o modelo ortotópico de GBM de rato (C6) e o modelo ex vivo tratados com GLA para análise de migração e proliferação celular. Foi observada uma redução da imunomarcação do fator de crescimento para endotélio vascular (VEGF), seu receptor Flt-1 e da metaloproteinase-2 de matriz, com consequente diminuição de vasos após o tratamento com GLA. No modelo ex vivo observou que o GLA reduziu a distância de migração e a mitose das células tumorais e também causou aumento das células tumorais em processo de apoptose. Os resultados revelaram que o GLA foi capaz de modular a expressão de algumas proteínas envolvidas nos processos angiogênico, migratório e proliferativo do GBM, o que sugere a sua utilização no tratamento desta patologia. / Glioblastoma multiforme (GBM) is the most malignant form of brain tumour originating from astrocytes and is characterized by intense proliferation, angiogenesis and cell invasion through the normal brain parenchyma. Gamma-linolenic acid (GLA) has anti-tumour activities in the processes of proliferation, migration and angiogenesis. This study used the orthotopic GBM rat model (C6) and ex vivo model treated with GLA to analyze cell migration and proliferation. Decreased immunostaining was observed for vascular endothelial growth factor (VEGF), its receptor Flt-1 and matrix metalloproteinase-2, with consequent reduction of blood vessels after treatment with GLA. In the ex vivo model GLA reduced the migration distance and mitosis of tumor cells and increased tumour cell apoptosis. The results revealed that GLA was able to modulate the expression of several proteins involved in angiogenesis, migration and proliferation in GBM, supporting the use of GLA in the treatment of this disease.

Thesis (MMed (Biomedical Sciences. Anatomy and Histology))--University of Stellenbosch, 1993. / Glial fibrillary acidic protein (GFAP) is found in varying amounts in the cytoplasm of most normal and neoplastic cells of astroglial origin. Though not glial specific, immunoelectron microscopy has shown that vimentin and GFAP are coexpressed as monomers of glial intermediate filaments. These structures display irreversible assembly and a slow metabolic turnover. Although currently applied as astroglial markers, these intermediate filament proteins may reflect the functional and developmental differentiation status of the cells in which they are expressed. Some authors have tried to apply these aspects as diagnostic parameters for grades of malignancy and anaplasia whilst other workers have indicated variable concentrations of GFAP in different astroglial cell types and entities. Different processing protocols, including the use of epoxy and acrylic resins, omission of osmium tetroxide and variations in concentration and incubation time of primary fixatives, were evaluated to find a compromise between antigen availability and acceptable ultrastructure. Thin sections were labelled on grid for GFAP (Dako A561) and vimentin (Dako M725) by means of the indirect immunogold method. For semi- quantification of relative antigen concentrations, a novel method was devised to calculate the labelling density, percentage heterogeneity of the particle distribution and the surface area investigated. This allowed expression of labelling results as a three figure unit. Standardized post-embedding immunoelectron microscopy was performed on 11 normal and neoplastic human tissue specimens. The tissue was exposed to conventional immersion fixation in glutaraldehyde and osmium tetroxide prior to modified embedding in LR White resin. The validity of these results was verified by correlation with conventional histopathological, immunohistochemical and clinical data obtained for each specimen. The presence of epoxy resin in thin sections was shown to reduce antigen availability to such an extent that very low to negative labelling was encountered. Acrylic LR White resin allowed more acceptable immunodetection, but at the cost of inferior ultrastructure and greater instability of thin sections in the electron beam. This masked the effects of glutaraldehyde fixation on the density of the tissuefixative matrix which included destruction of the vimentin and some GFAP associated epitopes. Although osmium tetroxide was required for acceptable ultrastructure, it reduced the labelling sensitivity by 20% and was responsible for premature curing of acrylic resin during impregnation of tissue. Despite superior resolution gained by electron microscopy and the advantage of semi-quantification of labeling results, the labelling sensitivity of this technique is lesser than that of light microscopical immunohistochemistry. Immunoelectron microscopy confirmed the association between GFAP and glial intermediate filaments in almost all the glial tumours studied, correlating well with GFAP expression in matching specimens demonstrated at light microscopical level. In the absence of intermediate filaments, no positivity for GFAP or vimentin was found in oligodendroglial components of mixed tumours. GFAP positivity in astrocytomas was demonstrated by between 17 and 126 particles / µm2, whilst lower figures were obtained for the glioblastoma (PD = 8) and some of the mixed gliomas (Pd = 6). Rosenthal fibres showed both peripheral and central positive labelling for GFAP, thus providing more evidence for their hypothetical degenerative, astroglial nature. The meningioma studied, was GFAP negative, but produced low density positivity for vimentin. Coexpression of GFAP and vimentin was demonstrated in an astroblastoma and degenerative infant brain tissue, thus supporting the presence of both these proteins development of glial structures. Although sites of likely glial intermediate filament synthesis were found, the antigen availability for vimentin was too low to allow a reliable assessment of specific vimentin localization and determination of the GFAP : vimentin ratio in individual intermediate filaments and/or astroglial fibres. Variations in particle densities (PD) which demonstrated GFAP in the various astroglial entities studied, were considered to be a result of variable technical and tissue processing factors rather than truly significant differences in expression of GFAP in individual intermediate filaments. This lead to the conclusion that the GFAP concentration / glial intermediate filament area is likely to be constant for mature glial intermediate filaments and therefore cannot be used to distinguish between different astroglial cells or entities. Whether each cell has a different number of glial intermediate filaments, has not been established satisfactorily. Following complementary conventional immunohistochemistry and careful orientation of biopsy material, the procedure can be applied to suitable specimens for the electron microscopical localization of high concentrations of aldehyde resistant, cytoplasmic antigens.

This thesis is about new and improved computational methods to analyze complex biological data produced by advanced biotechnologies. Such data is not only very large but it also is characterized by very high numbers of features. Addressing these needs, we developed a set of methods and tools that are suitable to analyze large sets of data, including next generation sequencing data, and built transparent models that may be interpreted by researchers not necessarily expert in computing. We focused on brain related diseases. The first aim of the thesis was to employ the meta-server approach to finding peaks in ChIP-seq data. Taking existing peak finders we created an algorithm that produces consensus results better than any single peak finder. The second aim was to use supervised machine learning to identify features that are significant in predictive diagnosis of Alzheimer disease in patients with mild cognitive impairment. This experience led to a development of a better feature selection method for rough sets, a machine learning method.  The third aim was to deepen the understanding of the role that STAT3 transcription factor plays in gliomas. Interestingly, we found that STAT3 in addition to being an activator is also a repressor in certain glioma rat and human models. This was achieved by analyzing STAT3 binding sites in combination with epigenetic marks. STAT3 regulation was determined using expression data of untreated cells and cells after JAK2/STAT3 inhibition. The four papers constituting the thesis are preceded by an exposition of the biological, biotechnological and computational background that provides foundations for the papers. The overall results of this thesis are witness of the mutually beneficial relationship played by Bioinformatics in modern Life Sciences and Computer Science.

Les gliomes sont des tumeurs agressives de mauvais pronostic, très angiogéniques et infiltrantes ce qui rend leur exérèse particulièrement difficile. Vu les limites des techniques actuelles d’imagerie, nous avons proposé la spectro-imagerie Infrarouge à Transformée de Fourier (IRTF), d’une résolution spatiale de 6 µm, pour apporter une information moléculaire à l’examen histologique actuel des gliomes. Nos travaux ont été fondés sur la recherche de paramètres moléculaires des vaisseaux sanguins, notamment sur la base des contenus de leur membrane basale. Celle-ci subit des altérations dûes au stress angiogénique tumoral. Nous avons mis en évidence des altérations de la structure secondaire des protéines (tels les collagènes) des vaisseaux sanguins au cours de la croissance de la tumeur. Nous avons aussi évalué les modifications des chaines d’acides gras des phospholipides membranaires, qui révélent un degré d’insaturation plus important pour les vaisseaux tumoraux. Ensuite, sur un modèle de gliome murin, nous avons établi une méthode efficace de classification des capillaires sanguins sur la base d’absorptions de leurs contenus glucidiques et lipidiques, permettant de discriminer totalement les capillaires sains et tumoraux. La combinaison de ces paramètres a été mise à profit pour assurer une histopathologie moléculaire des gliomes humains. Nos résultats ont démontré qu’il est possible de différencier entre la vasculature saine et tumorale sur ces gliomes humains, ce qui permet une bonne délimitation des zones tissulaires correspondantes. Cette technique pourrait devenir un outil analytique fiable, rapide d’une durée compatible avec la chirurgie et donc très utile pour les neurochirurgiens. / Malignant gliomas are very aggressive tumors with poor prognosis, highly angiogenic and invasive into the surrounding brain parenchyma, making their resection very difficult. Regarding the limits of current imaging techniques, we have proposed Fourier Transform Infrared (FTIR) spectro-imaging, with a spatial resolution of 6 µm, to provide molecular information for the histological examination of gliomas. Our work was based on the research of molecular parameters of blood vessels, notably on the basis of the contents of their basement membrane, which undergoes changes due to tumor angiogenic stress. We have identified alterations of the secondary structure of proteins (such as collagen) in blood vessels during tumor growth. We have also assessed the changes in fatty acyl chains of membrane phospholipids, which revealed a higher unsaturation level in tumor vessels. Then, on a murine glioma model, we have established an efficient method of blood vessels classification based on their carbohydrates and fats contents, allowing the differentiation between healthy and tumor blood vessels. The combination of these parameters was used to provide a molecular histopathology for the study of human gliomas. Our results have demonstrated the feasibility of differentiating between healthy and tumor vasculature in these human gliomas, which help delimitating areas of corresponding tissue. This technique could become a reliable and fast analytical tool, with duration compatible with the surgery and thus very useful for neurosurgeons.

Les gliomes de bas-grade représentent la tumeur cérébrale la plus fréquente chez l’enfant. Elles sont caractérisées par un large spectre de sous-types tumoraux, très hétérogènes. Leur définition actuelle est principalement basée sur des critères histologiques ce qui représente une limite importante car ces classifications souffrent d’un manque de précision. Les progrès récents de la génomique nous permettent d’approfondir considérablement les connaissances sur la biologie de ces tumeurs afin d’enrichir leur classification actuelle. Ce travail présente une analyse approfondie des altérations génomiques de l’ADN et l’ARN des gliomes de bas-grade pédiatriques. Le premier niveau d’analyse se base sur l’analyse du séquençage à haut débit de 169 gliomes de bas-grade de l’enfant. Bien que les mutations des gènes BRAF et FGFR1 sont les plus fréquemment décrites dans ces tumeurs, nous avons identifié pour la première fois le réarrangement chromosomique MYB-QKI majoritairement associé aux gliomes angiocentriques. Dans un deuxième temps ce travail décrit l’analyse du transcriptome de 151 gliomes de bas grade extraits à partir de tissu conservé en paraffine. Nous avons observé des différences moléculaires en fonction de leur sous-type histologique, de la localisation tumorale et de leur statut BRAF. Dans le dernier volet de ce travail, nous avons testé la faisabilité d’isoler par cytométrie en flux une cellule unique en les distinguant selon un marqueur de différenciation glial (A2B5+ et A2B5-) et d’effectuer une analyse transcriptomique à haut-débit en séquençant l’ARN à l’échelle d’une cellule unique. Cette technique nous a permis de décrire des différences moléculaires intéressantes entre des cellules A2B5+ et A2B5-. Ces résultats soulignent l’intérêt d’exploiter des nouvelles technologies de pointe pour servir de base à l’étude des caractéristiques biologiques des cellules tumorales. / Low-grade gliomas represent the most frequent brain tumor arising during childhood. They are characterized by a broad spectrum of tumor types.The definition of low-grade gliomas has been mainly based on morphology. This histological classification of pediatric low-grade gliomas (PLGG), suffers from the lack of reproducibility. The recent progress in molecular biology and genetics has brought new insights in the biology of those tumors and allows better understanding of their biology. This work provides a comprehensive analysis of two different genetic approaches in PLGGs. The first part is based on the description of somatic genetic alterations of the DNA. Using a large PLGG cohort, we have dissect the genome of those tumors and draw the landscape of their genetic alteration. Although BRAF and FGFR1 alterations are predominantly altered, we have discovered a new translocation, MYB-QKI, that is almost exclusively present in a specific histological subgroup; angiocentric gliomasThe second part of the thesis describes transcriptomic analysis of bulk PLGGs. This work describes molecular differences between PLGGs from distinct histologies and arising from different locations in the brain as well as different BRAF mutation status.We were also able to test single-cell expression analyses in three pilocytic astrocytomas (PAs) using RNA-sequencing. In this experimental work we have successfully tested the hypothesis that we can isolate single-cells from fresh PLGG tumors in order to analyze the trasncriptome at a large scale. We observed that single-cells expressing A2B5, a glial progenitor marker, isolated in pediatric PAs are characterized as a distinct biological population. These results underline the importance to improve the precision of the transcriptomic studies to capture the molecular signal of tumor cells and further understand the different pattern between normal cells and tumor cells.

Os hormônios glicocorticóides (GCs) têm sido amplamente empregados como agentes antiinflamatórios e anti-tumorais. Sua ação ocorre via receptores nucleares (GR) sendo dependente da remodelação da estrutura da cromatina. As proteínas Brm e BRG1, componentes essenciais de um complexo regulador global da transcrição (SWI/SNF), por remodelamento da cromatina, exercem um papel-chave na ação de GR. Para estudar o mecanismo de ação de GCs, foram utilizadas as linhagens celulares ST1 e P7, derivadas da linhagem celular C6, de glioma de rato. P7 é insensível ao tratamento com GC, enquanto ST1 apresenta reversão fenotípica tumoral→normal, gerando um bloqueio específico na fase G1. Um anti-soro policlonal específico para Brm e BRG1, foi gerado através da inoculaçâo de coelha com a proteína hBRG1 recombinante. Este antisoro foi utilizado para análisar os níveis destas proteínas nas duas linhagens celulares, sob ação de GC. Enquanto em ST1, Brm é induzida por GC, em células P7, o nível basal de Brm é relativamente alto, mantendo-se inalterado na presença de GC. A possíbilidade de existirem mutações no gene brm de células P7, foi investigada através de amplificação do DNA, por PCR, e seqüenciamento. A superexpressão de brm e BRG1 em células P7 mostrou que clones isolados apresentavam, de um modo geral, achatamento celular, diminuição da taxa de crescimento e da eficiência de plaqueamento em substrato sólido e semi-sólido. Alguns destes clones passaram a responder ao tratamento com GC, porém não tão drasticamente como as células ST1. Co-imunonoprecipitação mostrou algumas diferenças entre os complexos SWI/SNF de células ST1 e P7. / Glucocorticoid hormones (GCs) have been used as anti-inflammatory and anti-tumor agents, acting via nuclear receptors (GR) and being dependent on remodeling of the chromatin structure. As components of the global chromatin remodeling transcription complex (SWI/SNF), Brm and BRG-1 proteins play a key role in the action of GR. In order to study the mechanisms of action of GCs, we have been using the ST1 and P7 cell lines, derived from the C6, a rat glioma cell line. P7 is insensitive to the GC treatment, while ST1 displays a complete phenotypic reversion from tumoral to normal, including a G1-specific block in the cell cycle. A Brm and BRG1-specific polyclonal antiserum was generated, in rabbit, using recombinant hBRG1 protein as antigen. This antiserum was used to analyze the levels of Brm and BRG1 in these two cell lines, under GC treatment. While Brm is induced by GC, in ST1 cells, the basal level of Brm, in P7 cells, is relatively high, remaining unchanged under GC treatment. The possibility of brm mutations occurring in the P7 cells, was analyzed by DNA sequencing. Overexpression of brm and BRG1 in P7 cells led to morphological alterations (cell flattening) and decreased colony formation in agarose suspension and in solid substrate. Some of these clones became partially responsive to GC, when compared to the ST1 cell line. Co-immunoprecipitation assays revealed some differences in the SWI/SNF complex between ST1 and P7 cells.

Atualmente os gliomas representam cerca de 81% dos tumores cerebrais malignos, com aumento na incidência tanto em crianças, como em adultos acima dos 45 anos. Um número elevado de receptores neuroquinina tipo 1 (NK-1) estão expressos em células de glioma, estando a ligação da Substância P (SP) a esses receptores, envolvida no desenvolvimento e progressão desse tipo de tumor. A SP quelada ao DOTA (SP-DOTA), radiomarcada, vem sendo testada para utilização na terapia de gliomas, sendo o lutécio-177 (177Lu), devido a seu menor alcance tecidual, o radioisótopo mais indicado para tumores localizados em áreas críticas do cérebro. No entanto, estudos indicam a necessidade da adição de um excesso de metionina para prevenção da oxidação peptídica da SP-DOTA-177Lu, visando aumentar a estabilidade e a capacidade de ligação às células tumorais. Para superar esse desafio, surge a perspectiva da utilização de um novo análogo da SP, com estrutura modificada, para prevenir a oxidação peptídica. Neste contexto, o objetivo desse trabalho foi estudar a marcação de um novo análogo da SP com 177Lu e caracterizar suas propriedades in vitro e in vivo, visando a obtenção de um radiofármaco inédito e com potencial aplicação na terapia de tumores cerebrais e realizar estudos preliminares de marcação deste novo análogo com Ítrio-90 (90Y). O novo análogo foi obtido pela troca do aminoácido metionina (Met) pelo aminoácido norleucina (Nle) na posição 11 da cadeia peptídica da SP, sendo esses peptídeos denominados respectivamente SP(Met11)-DOTA e SP(Nle11)-DOTA. Após análise da oxidação peptídica dos dois peptídeos, os parâmetros da radiomarcação da SP(Nle11)-DOTA, com 177LuCl3, foram estudados para determinar a melhor condição de marcação. As estabilidades in vitro da SP(Nle11)-DOTA-177Lu sob refrigeração (2-8°C), no freezer (-20°C) e em soro humano (37°C) foram determinadas após radiomarcação com alta atividade, quanto ao uso de agentes estabilizantes e após diluição. A SP(Nle11)-DOTA também foi radiomarcada com 90Y, utilizando-se a condição padrão determinada, sendo a estabilidade in vitro da SP(Nle11)-DOTA-90Y sob refrigeração (2-8°C) e em freezer (-20°C), avaliada após radiomarcação com alta atividade e quanto a utilização de agente estabilizante. A capacidade de ligação in vitro às células tumorais (U-87 MG e M059J) e a biodistribuição in vivo em camundongos BALB/c sadios foram determinadas para a SP(Nle11)-DOTA-177Lu e comparadas à SP(Met11)-DOTA-177Lu. A ligação às proteínas plasmáticas e a biodistribuição em camundongos Nude com modelo tumoral também foram avaliadas. Os resultados obtidos na análise da oxidação peptídica comprovaram a importância da adição de excesso de metionina para prevenção da oxidação peptídica e indicaram uma alta estabilidade da SP(Nle11)-DOTA, durante e após o processo de radiomarcação. A adição de 148 MBq (4 mCi) da solução de 177LuCl3 em HCl 0,05N à 10 μg de SP(Nle11)-DOTA diluída em tampão acetato de sódio 0,4 M pH 4,5 seguida pela incubação a uma temperatura de 90ºC por 30 minutos, sob agitação de 350 rpm foi definida com condição padrão de marcação. O congelamento (-20°C), o uso de agentes estabilizantes e a diluição apresentaram-se como métodos efetivos para garantir uma alta estabilidade in vitro da SP(Nle11)-DOTA-177Lu, após a marcação com alta atividade. Bons resultados também foram observados para a marcação da SP(Nle11)-DOTA com 90YCl3 e para a estabilidade in vitro da SP(Nle11)-DOTA-90Y, após congelamento (-20°C) e quando utilizado ácido gentísico como estabilizante. A SP(Nle11)-DOTA-177Lu apresentou uma boa especificidade pelas células tumorais, principalmente pelas células de glioma humano M059J, sugerindo que a substituição do aminoácido metionina por norleucina na posição 11 não compromete a capacidade de ligação da SP(Nle11) às células tumorais. Uma baixa porcentagem de ligação às proteínas plasmáticas e um rápido clareamento sanguíneo foram observados para a SP(Nle11)-DOTA-177Lu, sendo esse radiofármaco eliminado preferencialmente por via renal. A SP(Nle11)-DOTA-177Lu apresentou uma boa estabilidade in vivo e se mostrou incapaz de atravessar a barreira hematoencefálica, sendo seu uso indicado por injeção intratumoral ou intracavitária. O estudo de biodistribuição em animais com modelo tumoral, mostrou que esse radiofármaco se liga às células tumorais por ligações receptor específicas. Com base nesse dados conclui-se que a SP(Nle11)-DOTA-177Lu, apresenta-se como um radiofármaco inédito que devido às suas propriedades in vitro e in vivo favoráveis, apresenta potencial aplicação na terapia de tumores cerebrais, representando uma nova possibilidade dentro do limitado arsenal terapêutico para esse tipo de tumor. / Currently gliomas represent about 81% of malignant brain tumors with increased incidence in children and in adults over 45 years. A large number of type 1 neurokinin receptor (NK-1) are expressed in glioma cells, being the binding of substance P (SP) to these receptors, involved in the development and progression of this tumor type. The SP conjugated at DOTA chelator (SP-DOTA), radiolabeled, have been tested for use in the treatment of gliomas, and the lutetium-177 (177 Lu), due to its lower tissue range, has been the most suitable radioisotope for tumors located in critical areas brain. However, studies indicate the necessity of adding an excess of methionine to prevent the peptide SP-DOTA-177Lu oxidation in order to increase the stability and capacity to bind to tumor cells. To overcome this challenge, there is the prospect of using a new analog of SP with a modified structure, to prevent peptide oxidation. In this context, the aim of this work was study the labeling of a new analog of SP with 177Lu and characterize their properties in vitro and in vivo, in order to obtain a novel radiopharmaceutical with potential application in brain tumor therapy, and perform preliminary studies labeling of this new analog with yttrium-90 (90Y). The new analog was obtained by replacement of the amino acid methionine (Met) by the amino acid norleucine (Nle) at position 11 of the peptide chain of SP, and these peptides were called SP(Met11)-DOTA and SP(Nle11)-DOTA respectively. After analysis of the oxidation for the two peptides, the radiolabeling parameters of the SP(Nle11)-DOTA with 177LuCl3 were studied to determine the best labeling condition. The SP(Nle11)-DOTA was also radiolabeled with 90Y, using standard condition, and the stability in vitro of the SP(Nle11)-DOTA-90Y assessed under refrigeration (2-8 °C) and under freezing (-20° C), after radiolabeling with high activity and use of stabilizing agent. The stabilities in vitro of the SP (Nle11)-DOTA-177Lu under refrigeration (2-8 °C), under freezing (-20 °C) and in human serum (37 °C) were determined after radiolabeling with high activity, with use of stabilizing agents and after dilution. The ability of in vitro binding to tumor cells (U-87 MG and M059J) and the biodistribution in vivo in healthy BALB/c mice were determined for the 177Lu-DOTA-SP(Nle11) and compared to 177Lu-DOTA-SP(Met11). The plasma protein binding and biodistribution in Nude mice with tumor model were also evaluated. The results obtained from analysis of oxidation for the two peptides confirmed the importance of adding excess methionine to prevent peptide oxidation and indicated a high stability of the DOTA- SP(Nle11), during and after the radiolabeling process. The addition of 148 MBq (4 mCi) of 177LuCl3 solution in 0.05N HCl at 10 μg DOTA-SP(Nle11) diluted in 0.4 M sodium acetate buffer pH 4.5 followed by incubation at a temperature of 90 °C for 30 minutes under constant agitation to 350 rpm was defined as standard labeling condition. The freezing (-20 °C), the use of stabilizing agents and the dilution were presented as effective methods to ensure high stability in vitro 177Lu-DOTA-SP(Nle11), after labeling with high activity. Good results were also observed for labeling DOTA-SP(Nle11) with 90YCl3 and for stability in vitro of the 90Y-DOTA-SP(Nle11) after freezing (-20 °C) and when gentisic acid was used as a stabilizer. The 177Lu-DOTA-SP(Nle11) showed good specificity to tumor cells, particularly human glioma cells (M059J), suggesting that substitution of the amino acid norleucine for methionine at position 11 does not compromise the capacity of SP(Nle11) binding to tumor cells. A low percentage of plasma protein binding and rapid blood clearance were observed for the 177Lu-DOTA-SP(Nle11), being this radiopharmaceutical preferably eliminated by the kidney. The 177Lu-DOTA-SP(Nle11) showed good stability in vivo and inability to cross the blood brain barrier, being its use indicated through intratumoral or intracavitary injection. The biodistribution studies in animals with tumor model showed that the radiopharmaceutical binds to the tumor cells by specific receptor binding. Based on this data was concluded that the 177Lu-DOTA-SP(Nle11), can be presented as a novel radiopharmaceutical that due to its favorable properties in vitro and in vivo, presents a potential application in the therapy of brain tumors, representing a new possibility within the limited therapeutic options for this type of tumor.

Apesar das evidências apontarem à participação da amilóide sérica A (SAA) em processos que favorecem a carcinogênese e metástase, associado à proposta da SAA como um marcador de progressão tumoral, não há ainda nenhum estudo avaliando a atividade direta da SAA em células tumorais. Este estudo examinou o efeito direto da SAA em duas linhagens de glioma humano. Gliomas são tumores cerebrais primários mais comuns em adultos e as linhagens deste estudo, A172 e T98G, representam carcinomas humanos caracterizados por um comportamento biológico altamente agressivo e quase sempre fatais, sendo classificados como grau IV. As linhagens A172 e T98G possuem algumas diferenças importantes em relação à invasividade; elas são respectivamente menos invasiva e mais invasiva. Para este estudo, avaliamos o efeito de SAA sobre a síntese de compostos representativos de algumas das diferentes classes de substâncias que estão envolvidas na progressão do tumor; entre elas estão a citocina IL-8, IL-6, TNF-α, a molécula mensageira NO, as metaloproteases, MMP2 e MMP9 e o gene RECK. Além disso, nos perguntamos se SAA pode estar envolvida nos processos de proliferação, migração e invasão celular. SAA induziu a produção de NO em ambas as linhagens de glioma. Em relação a IL-8 observamos que sua produção basal é bastante diferente dependendo da linhagem, sendo pouco produzida pela linhagem A172 que foi a única que respondeu à SAA. IL-6 e TNF-α não foram produzidas pelos gliomas quando estimulados com SAA. O tratamento das células com SAA aumentou a expressão das MMP-2 e MMP-9 e diminuiu a de RECK em ambas linhagens. SAA mostrou ser um estímulo mitogênico para os gliomas T98G e A172. SAA aumentou a migração e invasão da linhagem T98G e inibiu estes mesmos parâmetros nas células A172. SAA é constitutivamente expressa e produzida por ambas linhagens, sendo que a isoforma SAA1 é a predominante. A expressão gênica de todas as isoformas, SAA1, SAA2, SAA4, e a síntese protéica das SAA foram aumentadas pela adição de IFN-γ. Nossos resultados com base em ensaios in vitro suportam uma contribuição direta da SAA para a progressão e metástase do tumor, dependendo do tipo de célula e concentração da SAA. O fato de que IFN-γ é um indutor de SAA nos gliomas aponta que SAA pode exercer tanto um efeito intrácrino quanto autócrino ou parácrino nos tumores. / In spite of the evidences sustaining the participation of SAA in processes that favor carcinogenesis and metastasis, and the proposal of SAA as a marker of tumor progression, no studies have yet addressed a potential direct activity of SAA on tumor cells. This study examined the direct effect of SAA on two human glioma lineages. Gliomas are primary brain tumors more common in adults and the lineages of this study, A172 and T98G, represent human carcinomas characterized by a highly aggressive biological behaviour and almost always fatal, classified as grade IV. A172 and T98G have some important differences in the invasiveness, they are less invasive and more invasive, respectively. For this study, we evaluated the effect of SAA on the synthesis of compounds representing different classes of substances that are somehow involved in tumor progression, among them we can cite the cytokine IL-8, the messenger molecule NO, the metalloproteinases MMP2 and MMP9 and RECK gene. Furthermore, we wonder if SAA was involved in the processes of proliferation, migration and cell invasion. SAA stimulated the production of IL-8 in lineage A172, while T98G produced high amounts of IL-8 that were not modified by SAA addition. SAA did not stimulate the production of IL-6 and TNF-α. SAA induced the production of NO, increased the expression of MMP-2 and MPP-9 and decreased the regulator of the expression of the MMPs; gene RECK. Moreover, we observed that SAA was a mitogenic stimulus, but it had a dual effect on migration and invasiveness behavior depending on cell lineage. For T98G SAA increased migration and invasion, and for A172 SAA inhibited migration and invasion. SAA was constitutively expressed and produced by both strains, and the isoform SAA1 predominated. The gene expression of all isoforms, SAA1, SAA2, SAA4, and protein synthesis of SAA were increased by the addition of INF-γ. Our findings based on in vitro assays support a direct contribution of SAA to tumor development, progression and metastasis depending on the cell type and concentration of SAA. Besides the role of SAA on tumor growth during an acute phase, the fact that SAA was expressed in tumor cells suggests an intracrine or an autocrine action of SAA.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais / A radioterapia é uma das principais abordagens terapêuticas utilizadas no tratamento do câncer e é indicada, principalmente, em casos onde as lesões são inoperáveis. No entanto, uma de suas limitações advém dos próprios efeitos biológicos da radiação, além de outros fatores tais como a radiorresistência inerente a alguns tipos tumorais, tais como os cerebrais. Então, a aplicação concomitante de agentes antineoplásicos com radioterapia vem sendo praticada na clínica de modo a maximizar o efeito benéfico do último e ao mesmo tempo minimizar os efeitos colaterais da exposição à radiação ionizante. Dentro deste contexto geral é importante a pesquisa de novos compostos que possam ser selecionados como protótipos para o desenvolvimento de agentes que possuam os menores efeitos adversos possíveis. As tiossemicarbazonas são uma classe de compostos sintéticos que apresenta um amplo perfil farmacológico e já demonstraram atividade antitumoral. Também já foi relatado que a complexação destes compostos com cátions metálicos pode ser capaz de torná-los mais eficazes. O objetivo do presente trabalho foi avaliar o potencial radiossensibilizador de uma tiossemicarbazona derivada de N(4)-metil-toluil-2-acetilpiridina e seu complexo com cobre sobre linhagens celulares de glioblastoma multiforme, o qual de todos os tumores cerebrais é o que apresenta a maior agressividade e o maior índice de morbidade. Foi avaliada a sensibilidade de linhagens celulares de glioblastoma com diferentes status de p53 RT2 e U87 (p53 selvagem) e T98 (p53 mutante) à radiação gama de uma fonte de 60Co. Os resultados de citotoxicidade indicaram que as linhagens em questão apresentam uma radiossensibilidade similar, p53-independente. Os efeitos citotóxicos da tiossemicarbazona Lac e seu complexo CuLac foram avaliados e os resultados indicaram que ambas possuem excelente efeito citotóxico na ordem de 10-8 M em todas as linhagens avaliadas, portanto menores que drogas como a hidroxiuréia, cisplatina e etoposídeo (IC50 entre 10-4 e 10-6 M em média). O tratamento com as tiossemicarbazonas seguido de 6 Gy de radiação gama se mostrou mais eficiente que o tratamento com radiação isolada em todas as linhagens. A complexação com cobre não alterou de modo significativo o efeito antitumoral da tiossemicarbazona livre sobre as linhagens testadas. Análises de fotomicrografias ópticas indicaram que todos os tratamentos ocasionaram alterações morfológicas, tais como arredondamento celular, redução do volume citoplasmático e surgimento de vesículas na membrana citoplasmática. O conjunto de dados indicam que a tiossemicarbazona Lac e seu complexo de cobre possuem potente efeito antitumoral e também induzem radiossensibilização nas linhagens testadas. / Radiation therapy is one of the main therapeutical approaches used for the treatment of cancer and is indicated, mostly, in cases which the lesions are inoperable. However, one of its limitations comes from its own biological effects, besides other factors such as the radioresistance inherent to some types of tumors like the cerebral ones. Therefore, the concurrent aplication of antineoplasic agentes with the radiation therapy has been used in the clinical pratice with the objective of maximize the benefical effects of the latter and at the same time minimize the side effects of the exposure to ionizing radiation. In this general context is important the research for new compounds that can be selected as prototypes for the development of agents that possess the least adverse effects as possible. The thiosemicarbazones are a class of synthetic compounds that present a broad pharmacological profile and have demonstrated antitumoral activity. Also has been reported that the coordination of these compounds to metallic cations may be capable of make them more effective. The objective of the present work was to evaluate the radiosensitizing potential of a thisemicarbazone derived from N(4)-methyl-tolyl-2-acetylpyridine and its copper complex against glioblastoma multiforme cell lines, which of all brain tumors types, are the most agressive and have the highest morbidity. The sensitivity of glioblastoma cell lines with different p53 status RT2 and U87 (p53 wild type) and T98 (p53 mutant) to gamma radiation from a 60Co source was assessed. Citotoxicity results indicated that the cell lines in study presented a similar radiosensitivity, p53-independent. The citotoxic effects of the thiosemicarbazone Lac and its complex CuLac were assayed and the results indicated that both possess na excelent effect at the order of 10-8 M in all cell lines evaluated, therefore smaller than drugs such as hydroxyurea, cisplatin and etoposide (IC50 between 10-4 and 10-6 M). The treatment with the thiosemicarbazones followed by a 6 Gy gamma radiation dose showed to be more effective than the radiation alone in all cell lines. Coordination to Cooper had not changed significantly the antitumoral effect of the free thiosemicarbazone agains the cell lines tested. Morphological analysis of optical photomicrographies indicated that all treatment caused alterations, such as cell rounding, diminishing of the cytoplasmic volume and rising of vesicles at the cytoplasmic membrane. Together these data indicate that the thiosemicarbazone Lac and its metallic complex possess potent antitimoral effect amd also induce radiosensitivity in the tested cell lines.

Numerous studies have suggested rapid weight gain following diagnosis and initial treatment of childhood craniopharyngioma (CP) due to the damage sustained by the hypothalamus. Hypothalamic lesions formed by the treatment of the tumor and/or by invasiveness of the tumor itself are known to cause intractable weight gain, known as hypothalamic obesity. In contrast, hypothalamic obesity manifested in pediatric low-grade glioma (PLGG) patients is not as prominently addressed in literature; likely due to the expansive set of histological tumor subtypes that makes generalization challenging. Specifically, there is a lack of analysis that examines the difference in treatment, endocrinopathies, and weight gain between CP and PLGG patients. The purpose of this study was to compare hypothalamic damage in subjects diagnosed with pediatric hypothalamic low-grade glioma versus subjects diagnosed with childhood craniopharyngioma. We hypothesized that CP patients will have a more rapid post diagnosis weight gain and a greater degree of obesity compared with PLGG patients due to the more invasive nature of the tumor and the aggressive surgical treatments involved. We performed a retrospective review of the clinical records of patients who received a diagnosis of childhood craniopharyngioma or pediatric low-grade glioma at Dana-Farber Cancer Institute between 1980 and 2009. We identified 45 patients, who met criteria for evaluation, 28 were previously diagnosed with childhood craniopharyngioma and 17 were diagnosed with hypothalamic pediatric low-grade glioma. We analyzed the impact of treatment, the presence of endocrinopathies, and weight gain after diagnosis. We concluded that there was no statistically significant difference in the rate or magnitude of post diagnosis weight gain, disproving our initial hypotheses.