Noventa e três mastocitomas cutâneos caninos, presentes em 74 animais, tiveram seus históricos clínicos investigados. Destes, 25 casos foram submetidos à análise morfométrica nuclear em lâminas de citopatologia nas colorações Panótico rápido e H&E; e 28 casos à graduação histopatológica e à imunoistoquímica para detecção das expressões de KIT, BAX, Ki-67 e PCNA, de modo a identificar os melhores indicadores prognósticos para a neoplasia. Os parâmetros citomorfométricos nucleares investigados foram: área, diâmetro-médio e perímetro. Os tumores foram classificados em graus histopatológicos I (bem diferenciados), II (de diferenciação intermediária) ou III (pobremente diferenciados), segundo a proposta de Patnaik, Ehler e MacEwen (1984). Os resultados obtidos através dos indicadores investigados foram comparados às graduações oferecidas pelo exame histopatológico, bem como aos tempos de sobrevida pós-cirúrgica e à mortalidade em função da doença. A citomorfometria obteve correlação com a mortalidade e o tempo de sobrevida dos animais em ambas as colorações testadas. Entre os marcadores imunoistoquímicos, as expressões de Ki-67 e BAX mostraram-se eficientes na previsão da sobrevida e mortalidade para o tumor, enquanto que as de KIT e PCNA não foram indicadores prognósticos para a neoplasia. Quanto maiores os parâmetros citomorfométricos e as porcentagens de núcleos positivos para Ki-67 e BAX, maiores as taxas de mortalidade e menores os períodos de sobrevida pós-cirúrgica. Finalmente, a graduação histopatológica foi o método que obteve os resultados mais expressivos na histopatologia, demonstrando ainda ser o método mais confiável deste painel. Nossos resultados apresentam um novo método de classificação para os mastocitomas cutâneos caninos, a partir da análise morfométrica aliada ao exame citopatológico, e confirmam que as avaliações imunoistoquímicas de Ki-67 e BAX, podem auxiliar na graduação dos mastocitomas, contribuindo com o estabelecimento de prognósticos e tratamentos mais precisos. / Ninety-three canine cutaneous mast cell tumors, of 74 dogs, had their clinical history investigated. Among these, 25 cases were submitted to nuclear morphometric analysis on cytopathology slides stained by panoptic method and hematoxylin & eosin; and 28 cases were submitted to histopathologic grading and immunohistochemistry to evaluate KIT, BAX, Ki-67 e PCNA expressions, in order to identify the best prognostic indicators for this neoplasm. The nuclear cytomorphometric parameters used in the present study were: area, average-diameter and perimeter. The tumors were histologically classified as grades I (well differentiated), II (of intermediate differentiation) or III (poorly differentiated), as proposed by Patnaik, Ehler and MacEwen (1984). The results obtained by the indicators investigated were compared to the histopathologic grading, as well as to post-surgical survival times and mortality due to mast cell disease. Cytomorphometry was correlated with mortality and survival times in both staining methods. Among the immunohistochemical markers, Ki-67 and BAX expressions were efficient in predicting survival times and mortality due to mast cell tumors. KIT and PCNA expressions were not prognostic indicators for this tumor. The increases observed in the cytomorphometric parameters, and Ki-67 and BAX positive nuclei percentages were correlated with higher mortality rates and reduced post-surgical survival times. Finally, using the histopathologic grading system, we obtained the best results on histopathology, being this the most trustworhty method of this diagnostic panel. Our results presents a new grading system for canine cutaneous mast cell tumors, based on morphometric analysis on cytopathology slides, and confirm that immunohistochemical evaluation of Ki-67 and BAX expression may contribute to mast cell grading, leading to more accurate prognosis and treatment.

Aproximately 4500 new cases of sarcoma are reported in USA each year. Seventy five percent will develop lung metastases and at majority which the only site. The best resulta of surgical treatment do not excced 32% of the cases. The use of adjuvant chemotherapy has reduced but not eliminated the incidence of pulmonary metastases. The perfusion of one organ with more drug and without systemic effects of chemotherapy can change the natural history of thia diaease. We present the result of the first phase which ia to set up successfully the model of perfusion and of the aecond phase, which ia to develop the pulmonary sarcoma metaatases. We were able to perfuse the left lung of 14 rata and to follow them up, at least for 10 days. The overall mortal i ty r ate was 28,5%. Wi th improvement of the technique, we had 10% of mortality. In the second phase, we were able to produce aarcoma metastases in 32 days, following intravenous injection of sarcoma cella auspenaion. Therefore, the first lung perfusion model in successfully. phase, to implement the single the rat, has been accomplished The aecond phase ahowed us that the pulmonary aarcoma metastases model ia confirmed. The CT scan can be used instead of sacrificing the animais to follow up the metastases.

Made available in DSpace on 2015-04-14T14:51:17Z (GMT). No. of bitstreams: 1 438254.pdf: 475511 bytes, checksum: 55b686575a0c7ed0d039db86576374f6 (MD5) Previous issue date: 2012-03-30 / Ewing?s sarcoma is a highly aggressive tumor of bone and soft tissues. It affects mainly children and young adults. In about 88% to 95% there is the occurrence of a translocation between the EWS gene (22q12 locus) and two members of ETS transcription factors family: FLI1 (11q24 locus) or ERG (21q22). The most common translocation involves gene EWS and FLI1. This translocation leads to the formation of EWS/FLI1chimeric aberrant transcription factor. The EWS/FLI1 regulates the NR0B1 gene promoter through a direct binding to GGAA microsatellites sequences. Our objective was to identify and describe the molecular structure of GGAA motifs in NR0B1 promoter in unrelated Ewing's Sarcoma patients and healthy subjects from South Brazilian population. Were identified 21 different alleles in the 224 subjects. All alleles had at least 4 to 5 consecutive GGAA motifs. The 24.2, corresponding to (GGAA)7A(GGAA)7A(GGAA)10 sequence, was the most frequent in our population, being present in 50.4% of subjects. Allele 24.2 could be associated to Ewing's sarcoma development since it was significantly more frequent among patients. Differences in the global configuration of NR0B1 promoter GGAA microsatellites (size, sequence, amount and position of GGAA repeats and single 'A' base insertions) could result in differences in Ewing's sarcoma susceptibility. These results would provide insights for the understanding of tumorigenesis. / O sarcoma de Ewing ? um tumor altamente agressivo que afeta ossos e tecidos moles. Ele acomete principalmente crian?as e adultos jovens. Em torno de 88% a 95% dos casos verifica-se a ocorr?ncia de uma transloca??o entre o gene EWS (l?cus 22q12) e dois membros da fam?lia do fator de transcri??o ETS: o FLI1 (11q24) ou o ERG (21q22). A transloca??o mais frequente envolve os genes EWS e FLI1. Esta transloca??o leva ? forma??o do fator de transcri??o quim?rico aberrante EWS/FLI1. O fator EWS/FLI1 regula o promotor do gene NR0B1 atrav?s de uma liga??o direta a sequ?ncias de microssat?lites GGAA. Nosso objetivo foi identificar e descrever a estrutura molecular de motivos GGAA no promotor de NR0B1 em pacientes com Sarcoma de Ewing n?o relacionados e n?o afetados da popula??o sul brasileira. Foram identificados 21 alelos diferentes em 224 indiv?duos estudados. Todos os alelos tiveram, pelo menos, de 4 a 5 motivos consecutivos GGAA. O alelo 24.2, correspondente a sequ?ncia (GGAA)7A(GGAA)7A(GGAA)10, foi o mais freq?ente em nossa popula??o, estando presente em 50,4% de todos os indiv?duos. O alelo 24.2 pode estar associado ao desenvolvimento do Sarcoma de Ewing, dado que sua frequ?ncia foi significativamente mais alta entre afetados. Diferen?as na configura??o global dos microssat?lites GGAA no promotor de NR0B1 (em rela??o ? tamanho, sequ?ncia, quantidade e posi??o das repeti??es GGAA e inser??es de base ?nica A ) podem resultar em diferente susceptibilidade ao sarcoma de Ewing. Tais resultados poder?o fornecer subs?dios para uma melhor compreens?o da tumorig?nese.

Noventa e três mastocitomas cutâneos caninos, presentes em 74 animais, tiveram seus históricos clínicos investigados. Destes, 25 casos foram submetidos à análise morfométrica nuclear em lâminas de citopatologia nas colorações Panótico rápido e H&E; e 28 casos à graduação histopatológica e à imunoistoquímica para detecção das expressões de KIT, BAX, Ki-67 e PCNA, de modo a identificar os melhores indicadores prognósticos para a neoplasia. Os parâmetros citomorfométricos nucleares investigados foram: área, diâmetro-médio e perímetro. Os tumores foram classificados em graus histopatológicos I (bem diferenciados), II (de diferenciação intermediária) ou III (pobremente diferenciados), segundo a proposta de Patnaik, Ehler e MacEwen (1984). Os resultados obtidos através dos indicadores investigados foram comparados às graduações oferecidas pelo exame histopatológico, bem como aos tempos de sobrevida pós-cirúrgica e à mortalidade em função da doença. A citomorfometria obteve correlação com a mortalidade e o tempo de sobrevida dos animais em ambas as colorações testadas. Entre os marcadores imunoistoquímicos, as expressões de Ki-67 e BAX mostraram-se eficientes na previsão da sobrevida e mortalidade para o tumor, enquanto que as de KIT e PCNA não foram indicadores prognósticos para a neoplasia. Quanto maiores os parâmetros citomorfométricos e as porcentagens de núcleos positivos para Ki-67 e BAX, maiores as taxas de mortalidade e menores os períodos de sobrevida pós-cirúrgica. Finalmente, a graduação histopatológica foi o método que obteve os resultados mais expressivos na histopatologia, demonstrando ainda ser o método mais confiável deste painel. Nossos resultados apresentam um novo método de classificação para os mastocitomas cutâneos caninos, a partir da análise morfométrica aliada ao exame citopatológico, e confirmam que as avaliações imunoistoquímicas de Ki-67 e BAX, podem auxiliar na graduação dos mastocitomas, contribuindo com o estabelecimento de prognósticos e tratamentos mais precisos. / Ninety-three canine cutaneous mast cell tumors, of 74 dogs, had their clinical history investigated. Among these, 25 cases were submitted to nuclear morphometric analysis on cytopathology slides stained by panoptic method and hematoxylin & eosin; and 28 cases were submitted to histopathologic grading and immunohistochemistry to evaluate KIT, BAX, Ki-67 e PCNA expressions, in order to identify the best prognostic indicators for this neoplasm. The nuclear cytomorphometric parameters used in the present study were: area, average-diameter and perimeter. The tumors were histologically classified as grades I (well differentiated), II (of intermediate differentiation) or III (poorly differentiated), as proposed by Patnaik, Ehler and MacEwen (1984). The results obtained by the indicators investigated were compared to the histopathologic grading, as well as to post-surgical survival times and mortality due to mast cell disease. Cytomorphometry was correlated with mortality and survival times in both staining methods. Among the immunohistochemical markers, Ki-67 and BAX expressions were efficient in predicting survival times and mortality due to mast cell tumors. KIT and PCNA expressions were not prognostic indicators for this tumor. The increases observed in the cytomorphometric parameters, and Ki-67 and BAX positive nuclei percentages were correlated with higher mortality rates and reduced post-surgical survival times. Finally, using the histopathologic grading system, we obtained the best results on histopathology, being this the most trustworhty method of this diagnostic panel. Our results presents a new grading system for canine cutaneous mast cell tumors, based on morphometric analysis on cytopathology slides, and confirm that immunohistochemical evaluation of Ki-67 and BAX expression may contribute to mast cell grading, leading to more accurate prognosis and treatment.

The EWSR1-FLI1 t(11;22)(q24;q12) translocation is the pathognomonic genomic alteration in 85% of the Ewing Family of Tumors (EWFT) a malignancy of the bone and the surrounding tissue, predominantly affecting children and adolescents. This translocation results in the formation of a chimeric oncoprotein which acts as an aberrant transcription factor that is currently not pharmaceutically druggable, driving the need for more effective targeted therapies. The EWSR1-FLI1 translocation induces a variety of changes including dysregulation of the epigenome and altered gene expression to drive tumorigenesis, and consequently contributes to the hypersensitivity of EWFT to several classes of chemotherapeutics. We sought to exploit these intrinsic sensitivities by employing a matched pair of cell lines derived from the same patient with Ewing sarcoma prior to and following chemotherapy, a panel of Ewing sarcoma cell lines, and several patient-derived xenografts (PDX) collected at the time of relapse or autopsy, which led us to the development of two novel combination targeted therapies for EWFT. In our matched pair of EWFT cell lines, we found sensitivity to the Poly(ADP-ribose Polymerase (PARP) inhibitor olaparib was diminished following chemotherapy, despite a predicted sensitivity. In addition, we discovered increased expression of the antiapoptotic protein BCL-2 in the chemotherapy-resistant cells, conferring apoptotic resistance to olaparib. We found that EWS-FLI1 increases BCL-2 expression; however, inhibition of BCL-2 alone is insufficient to sensitize EWFT cells to olaparib, revealing a dual necessity for BCL-2 and BCL-XL (BCL2L1) in EWFT survival. These data reveal BCL-2 and BCL-XL act together to drive olaparib mediated apoptotic resistance in Ewing sarcoma and identify a novel, rational combination therapy using olaparib and the BCL-2/BCL-XL inhibitor navitoclax. In addition, using high throughput drug screening we have identified a novel epigenetic susceptibility in EWFT to GSK-J4 (GlaxoSmithKline), an inhibitor of lysine 27 of histone 3 (H3K27) demethylases: ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX) and Jumonji D3 (JMJD3). Treatment with GSK-J4 leads to a decrease in H3K27 acetylation (H3K27ac) and ultimately, the silencing of EWS-FLI1 gene targets. We sought to sensitize GSK-J4-mediated inhibition of EWS-FLI1 targets by blocking RNA polymerase II activity using the Cyclin Dependent Kinase 7 (CDK7) inhibitor THZ1. By targeting CDK7-mediated transcription we were able to sensitize EWFTs to H3K27 demethylase inhibition. We therefore propose co-targeting of H3K27 demethylases and CDK7 acts as a surrogate EWS-FLI1 inhibitor. Given the difficulties targeting EWS-FLI1, these strategies may present viable clinical therapies.

Abstract not available

Despite improvements in the clinical management of soft tissue sarcomas (STS), 50% of patients will die of metastatic disease that is largely unresponsive to conventional chemotherapeutic agents. The aims of this study were to identify genes and pathways that are dysregulated in progressive and metastatic STS. In addition to this, cell lines from fresh tumours were initiated and established, thus increasing the repository of cell lines available for functional studies. Recent advances in the understanding of the molecular biology of STS have thus far not resulted in the use of molecular markers for clinical prognostication. Identifying novel genes and pathways will lead to molecular diagnostic methods to better stratify prognostic groups and could identify cellular targets for more efficacious treatments. Gene expression profiling of sarcoma cell lines of increasing metastatic potential revealed over-expression of genes involved in the epidermal growth factor (EGF) and transforming growth factor beta (TGFb) pathways. Factors involved in invasion and metastasis such as integrins and MMPs were over-expressed in the cell lines with higher metastatic potential. The developmental Notch pathway and cell cycle regulators were also dysregulated. NDRG1 was significantly over-expressed in the high grade sarcoma cell line, a novel finding in sarcomas. The expression of EGFR, NDRG1 and other genes from the above pathways was validated using quantitative RT-PCR in real time (qRT-PCR). A tissue microarray (TMA) comprising STS of varying tumour grades was constructed for high throughput assessment of target proteins. EGFR, its activated form and its signal transducers were investigated using immunohistochemistry (IHC). Activated EGFR (HR 2.228, p < 0.001) and phosphorylated Akt (HR 2.032, p = 0.003) were found to be independent predictors of overall survival and both correlated with tumour grade. Of the several STS cultures initiated and maintained, two of these cell lines were fully characterised in terms of cytogenetics, telomerase and alternate lengthening of 5 telomeres (ALT) status, KIT and TP53 mutation and the expression of certain biomarkers using both qRT-PCR and IHC. In summary, transcript profiling identified several potential biomarkers of tumour progression and metastasis in STS. Crucially, activated EGFR and pAkt were found in a cohort of STS samples to correlate with clinical outcome, identifying them as potential diagnostic and therapeutic targets in the treatment of STS. Activated EGFR can be used as a diagnostic marker for patient selection, as well as for target effect monitoring. Furthermore, the cell lines established in this project will serve as valuable tools in future preclinical studies.

Integrase catalyzes insertion of a retroviral genome into the host chromosome. Following reverse transcription, integrase binds specifically to the ends of the duplex retroviral DNA, endonucleolytically cleaves two nucleotides from each 3'-end (the processing activity), and inserts these ends into the host DNA (the joining activity) in a concerted manner. Additionally, it has been observed that integrase can catalyze the removal of inserted viral ends (the disintegration activity) in vitro. Presteady-state experiments were performed using synapsed substrates to probe the processing reaction and a disintegration substrate to determine the number of protomers in a functional multimeric complex. In single-turnover studies, a novel "splicing" reaction was observed that revealed complications with accurate quantification of enzymatic activity using the synapsed substrates. The splicing reaction was further used to gain insight into the selection of nucleophiles and electrophiles at the binding site. To reduce the complexity introduced by the integrase-catalyzed splicing reaction, 5'-5' reverse-polarity synapsed substrates were designed that were not susceptible to the splicing reaction and that allowed direct comparison of LTR ends simultaneously bound at the active site. Analysis of the presteady-state assays using these reverse-polarity substrates revealed that the concurrent binding of the biologically relevant U3/U5 combination of viral ends facilitates maximal activity of the processing reaction. A disintegration substrate was used in presteady-state active site titrations to determine a reaction stoichiometry of four integrase protomers per one substrate molecule for the disintegration reaction. A tetrameric active complex was then confirmed using atomic force microscopy to image integrase-DNA complexes during the first catalytic turnover. The observed increase of the tetramer population in the presence of substrate DNA demonstrates that the binding of the disintegration substrate induces assembly of the active tetramer and suggests that tetramer assembly may be an integral and dynamic component of the catalytic pathway. / Graduation date: 2003

Regulation of growth is of fundamental importance for development of the organism and to maintain health. The induction of cell proliferation and matrix production are influenced by several different signaling systems, most importantly by growth factors. The human HER-family of growth factor ligands and receptors is one of the most studied and, at present, one of the most complex including 4 tyrosine kinase receptors and at least 11 different ligands cooperating in the transfer of signals. The HER-family growth responses are also influenced by other intercellular and extracellular signals, including matrix components, cytokines and hormones mediating e.g. inflammation. HER-1 (EGFR) is one of the best known and most extensively studied growth factor receptors. TGF-alpha is possibly the most potent HER-1 ligand and influences wound healing, epidermal maintenance, gastrointestinal function, lactation, pulmonary function and more. Several studies have shown important regulatory functions for some inflammatory cytokines on TGF-alpha production in white blood cells. HER-1 is widespread in epithelial cells but also in mesenchymal cells such as fibroblasts, osteogenic and chondrogenic cells. Consequently, many tumors arising from these cell types express HER family members and often show TGF-alpha and/or HER activation. Indeed, mammary cancer development has been shown when over expressing both TGF-alpha and HER-2 in mouse mammary cells in vivo. In recent years the first HER-1 and HER-2 inhibitors have come into clinical practice for treatment of breast cancer, lung cancer and gastrointestinal cancers, sometimes with great success. However, more knowledge is needed concerning the inflammatory regulation of HER-family expression including where and how the ligands and receptors cooperate. Therefore we were interested in studying the role of TGF-alpha in normal and abnormal growth. First we showed that the acute inflammatory cytokine IL-6 regulates TGF-alpha expression in U-937-1 monocytoid cells. Secondly, we detected a possible long-term enhancing influence of singledose UVR on HER-1 expression in normal human melanocytes. We continued thirdly by revealing TGF-alpha production concomitant with HER-2 in normal human synovia and release of soluble TGF-alpha into the synovial fluid. Both TGF-alpha and HER-2 production were significantly increased in inflammatory joint conditions, e.g. RA. Fourthly, we demonstrated expression of TGF-alpha, HER-1 and HER-2 in synovial sarcoma cells in culture; the observed HER-2 phosphorylation was dependent on ligand induced HER-1 activation. The presented results indicate that TGF-alpha expression can be enhanced by acute inflammatory cytokine IL-6, possibly contributing to growth stimulatory effects assigned to IL-6 itself. The acute effects of UVR on melanocytes mediate up-regulated steady-state expression of HER-1, constituting a potential target for locally produced TGF-alpha that may induce melanocyte proliferation. TGF-alpha and HER-2 seem to have a role in the maintenance of synovial joint tissues. Upregulation of TGF-alpha and HER-2 in inflammatory joint conditions, e.g. RA, represents a novel mechanism for synovial proliferation contributing to joint deterioration. TGF-alpha, HER1 and HER-2 may have a role in synovial sarcoma proliferation; further investigation is needed to evaluate HER-family inhibitors as a possible treatment alternative in this type of cancer.

Bartonella, a group of Gram negative facultative intracellular bacteria, are known to cause diseases, such as Cat Scratch Disease, Trench Fever and Carrion’s Disease, that involve angiogenesis during the infective cycle. B. bacilliformis, the etiological agent of Carrion’s Disease, causes a bi-phasic infection resulting in the formation of blood-filled angiogenic proliferative cutaneous nodules called verruga peruana. The work presented here was undertaken to characterize the mechanism by which these nodules are produced. Previous work in our laboratory suggested that the Bartonella henselae genome contains a homologue to the virB operon, a set of genes coding for a Type IV Secretion System (TFSS) that has been implicated in the pathogenesis of other α-2-proteobacteria. We identified virB operons in two additional Bartonella pathogens, B. quintana and B. clarridgeiae. No corresponding operon sequences were detected in B. bacilliformis DNA, however. This finding suggests that virB gene products are not required for verruga peruana formation. To continue our search for factors involved in B. bacilliformis-induced angiogenesis, we conducted a microarray analysis of differential gene expression in infected and uninfected endothelial cells. The results suggest similarities between later stage (36 hours) B. bacilliformis infection and that of HHV-8, the causative agent of Kaposi’s Sarcoma, particularly in relation to the host immune response. Finally, our research focused on the secreted factors that B. bacilliformis produces during its host infective cycle. Our data suggest that the B. bacilliformis homologue to the molecular chaperone GroEL not only induces angiogenesis in endothelial cells, but also protects endothelial cell tubule from the degradation seen when these cells are in the presence of live B. bacilliformis. In summary, the induction of verruga peruana nodules via B. bacilliformis may be the result of multiple factors over the course of persistent infection. Early infection may cause vascular damage, which induces VEGF and hypoxia factors. As infection persists, bacterial secretion of a unique GroEL may result in continued angiogenesis and the ensuing activation of immune cells, producing a localized environment of continual incomplete angiogenesis in areas of cutaneous infection.