Dechamps Christophe (2008). Etude fonctionnelle dun oncogène humain impliqué dans le Sarcome dEwing, oncTre210, homologue à deux gènes levuriens, MSB3 et MSB4 (thèse de doctorat). Gembloux, Faculté Universitaire des Sciences Agronomiques, 173 p., 12 tabl., 40 fig. Résumé : Le produit de l'oncogène humain oncTre210 est apparenté, par sa structure primaire, aux protéines Ypt/Rab GAP (GTPase Activating Proteins spécifiques des Ypt/Rab GTPases). En effet, sa région N-terminale, qui est fortement homologue aux deux GAP de Saccharomyces cerevisiae Msb3p et Msb4p, renferme le domaine catalytique TBC, hautement conservé, des Ypt/Rab GAP. Les protéines Msb3p et Msb4p de Saccharomyces cerevisiae font partie de la famille des GTPase activating protein (GAP) spécifique aux Ypt/Rab GTPase. Elles sont primordiales au trafic vésiculaire et sont impliquées dans la régulation de l'exocytose et dans l'organisation du cytosquelette d'actine. Mais, leurs rôles biologiques exacts nont jamais été déterminés. La délétion simultanée des 2 gènes MSB3 et MSB4 dans la levure S. cerevisiae induit une inhibition de croissance de la levure sur un milieu de culture contenant du DMSO et/ou de la caféine, perturbe lorganisation du cytosquelette dactine, produit une anomalie de bourgeonnement dans la levure diploïde et affecte la ségrégation des noyaux. Pour trouver des composants qui interagissent génétiquement avec les produits des gènes MSB3 et MSB4, nous avons criblé une banque génomique pour des suppresseurs homologues extragéniques multicopies restaurant la croissance du double mutant levurien msb3 msb4 en présence de DMSO et/ou de caféine. Sept gènes ont ainsi été identifiés après une série de vérifications. Ces 7 suppresseurs peuvent être classés pour la fonction biologique de leurs produits en plusieurs classes : transport vésiculaire, cycle cellulaire, chaperon moléculaire, protéasome et ARN ribosomial. Ces résultats nous permettent d'identifier les voies physiologiques où les deux protéines Msb3p et Msb4p seraient impliquées. Le produit de l'oncogène oncTre210 est impliqué dans différents cancers humains dont le sarcome d'Ewing. Pour létude des partenaires interagissant avec lune ou lautre des deux parties de la protéine de fusion oncTre210p, nous avons utilisé le système double-hybride en levure en utilisant différentes banques d'expression. Un grand nombre de partenaires protéiques a été isolé comme interagissant avec l'oncoprotéine. Deux protéines impliquées dans l'organisation et la structure du cytosquelette ont été choisies parmi les partenaires de l'oncoprotéine pour être étudiées. L'interaction de ces deux protéines avec la partie GAP de oncTre210p a été confirmée par les techniques de GST pulldown, de co-immunoprécipitation et de co-localisation. Ces protéines identifiées comme interagissant avec la partie GAP sont la chaîne légère régulatrice de la myosine II (Myl2) et LOC91256, protéine contenant des motifs ankyrine. A partir de ces observations un nouveau rôle de l'oncoprotéine oncTre210p a été suggéré. L'ensemble de nos résultats ainsi que des données expérimentales acquises par d'autres équipes internationales nous a permis de proposer un modèle d'action pour l'oncoprotéine oncTre210p. Dechamps Christophe (2008). Functional study of an oncogene implicated in Ewing's sarcoma, oncTre210, a human homologue of two yeast genes, MSB3 and MSB4 (Thesis in French). Gembloux, Belgium, Gembloux Agricultural University, 173 p., 12 tabl., 40 fig. Summary : The oncTre210 oncogene product is structurally related to the Ypt/Rab GTPase-Activating Proteins (Ypt/Rab GAPs). Particularly, the N-terminal region of the oncoprotein shares with the yeast proteins Msb3p and Msb4p the highly conserved TBC domain, forming the catalytically active domain of Ypt/Rab GAPs. The Msb3p and Msb4p proteins of Saccharomyces cerevisiae are members of the Ypt/Rab-specific GTPase activating protein (GAP) family. They are important to vesicular trafficking and involved in the regulation of exocytosis and in the organization of the actin cytoskeleton, but their exact biological roles have yet to be determined. The msb3 msb4 yeast double mutation causes growth inhibition in the presence of DMSO and/or caffeine, affects the organization of the actin cytoskeleton, produces a random budding pattern in diploid cells, and affects segregation of the nucleus. To find cell components that interact genetically with the products of the MSB3 and MSB4 genes, we screened a genomic library for multicopy suppressor genes restoring normal growth of the double mutant in the presence of DMSO and caffeine. Six genes were identified, and the extent to which each gene corrects specific growth defects of the msb3 msb4 mutant is described. The encoded suppressors were classified on the basis of functional features into five groups: vesicular transport, cell division, molecular chaperon, proteasome and ribosomal RNA. These results allow us to identify the physiologic ways where the Msb3p and Msb4p proteins are implicated. The product of the oncTre210 oncogene is involved in various human cancers, including Ewings sarcoma. In order to identify proteins interacting with the two parts of this protein, we performed yeast two-hybrid screening of various libraries. A large number of proteins was identified to be partners of the oncogene product. Two components of the cytoskeleton were chosen to be studied, whose interaction with the GAP region was confirmed by GST-pulldown, co-immunoprecipitation, and colocalisation experiments. The proteins found to interact with the GAP region are the light regulatory chain of myosin II (Myl2) and LOC91256, a protein containing ankyrin repeats. From these observations a new role for the oncTre210p oncoprotein in cytokinesis was suggested. Our results and data from other international teams allow us to propose a model for the action of the oncTre210 oncogene product.

Como otros tumores sólidos, el sarcoma de Ewing (SE) requiere de un aporte vascular adecuado que permita nutrir y oxigenar a las células tumorales. En el SE, la vascularización tumoral se encuentra caracterizada principalmente por los procesos de vasculogenesis, mimetismo vascular y angiogénesis. Considerando la participación de CAV1 en el aporte vascular, nos planteamos demostrar que CAV1 juega un papel importante en el desarrollo vascular de la enfermedad. Estableciendo tres nuevos modelos de baja expresión de CAV1 en células se SE (RDES, SKES1 y TC71), fue posible observar la reducción en el desarrollo tumoral en xenoinjertos, y a su vez esta reducción fue relacionada con la disminución en la densidad micro-vascular. Este trabajo demuestra el efecto directo del silenciamiento de CAV1 sobre el proceso angiogénico en el SE, regulando la respuesta de la célula endotelial ante las señales producidas por la célula tumoral. Nuestros resultados mostraron que las células endoteliales presentan mayor migración ante el estímulo de las señales producidas por la célula tumoral. Mediante el análisis de expresión de diferentes factores utilizando RT-PCR, fue posible determinar que la expresión de bFGF era afectada en los modelos analizados como efecto del silenciamiento de CAV1. Para determinar el mecanismo molecular a través del cual CAV1 regula la angiogénesis en el sarcoma de Ewing, partimos del análisis de expresión de 3 miembros de la familia Eph descritos como pieza clave en el desarrollo vascular. La expresión del receptor EphA2 en los modelos de baja expresión de CAV1 mostró una disminución en los clones, donde el silenciamiento de CAV1 conlleva la redistribución y reducción EphA2. Los análisis mostraron además una interacción entre ambas proteínas. Establecida la relación EphA2/CAV1, analizamos el efecto de la estimulación de EphA2 a partir de EfnA1 y confirmamos que las células con baja expresión de CAV1 presentaban una respuesta menor. La estimulación resultó en el incremento de la fosforilación de AKT, sugiriendo que los efectos podrían depender de la actividad quinasa del receptor. Para respaldar esto, las células RDES y TC71 fueron transfectadas con un dominante negativo de EphA2 que no modificaba la expresión de CAV1 (EphA2-Kd). Los modelos EphA2-Kd reprodujeron los resultados observados en los modelos de baja expresión de CAV1. La estimulación del modelo TC71-EphA2-Kd con la proteína recombinante EfnA1, mostró tanto la activación de AKT como la sobreexpresión de bFGF, reproduciendo en cierta medida lo observado por el silenciamiento de CAV1 y destacando la importancia de la actividad dependiente de quinasa de EphA2 en el proceso angiogénico de la enfermedad. Considerando que CAV1 es necesario para el correcto funcionamiento de las rutas de señalización controladas por EphA2 en el SE, decidimos determinar la implicación de CAV1 y EphA2 en el mimetismo vascular de la enfermedad. Se decidió analizar el papel independiente de quinasa del receptor EphA2 como una posible pieza clave en el proceso de mimetismo vascular en el SE. Nuestros resultados mostraron que el silenciamiento de CAV1 reduce el desarrollo de vasos miméticos in vivo e impide la formación tubular in vitro. Además, el efecto de bloquear la actividad quinasadependiente del receptor EphA2 mostró que la actividad quinasa del receptor no afecta al proceso de mimetismo vascular en esta entidad tumoral. Para confirmar la participación de EphA2 en el proceso de formación de vasos miméticos, se transfectó un mutante que carece de toda la región citoplasmática del receptor EphA2 (ΔCyto), eliminando así sus funciones dependientes e independientes de quinasa. Los resultados mostraron que el bloqueo total del receptor tenía un efecto negativo en la formación de estructuras tubulares. Estos resultados confirman la importancia de la actividad prooncogénica del receptor EphA2 en el sarcoma de Ewing. / We established low CAV1 expressing models by knocking down CAV1 in TC71, RDES and SKES1 Ewing sarcoma (ES) cells by stably transfecting a previously validated shRNA construct. In vivo studies showed a decrease in tumor volume from the CAV1 knocked-down clones when compared with controls. This correlated with a reduction in microvascular density (MVD) and higher levels of necrosis. Conditioned media from CAV1 knocked-down cells showed reduced capability to promote migration of endothelial cells with no changes in proliferation. Different pro-angiogenic factors were analyzed by RT-PCR in the models and, downregulation of bFGF was observed in all four. Results suggested that CAV1 was indirectly affecting bFGF expression. EphA2 expression was observed in ES cell lines and tumor samples. CAV1 knocked-down cells showed a reduction in EphA2 phosphorylation as well as a displacement from the membrane to the cytoplasm. Furthermore, we showed that the CAV1/EphA2 interaction was necessary for Eph-mediated signaling. To further support these results, RDES and TC71 cells were stably transfected with a dominant negative of EphA2 that did not alter the expression of CAV1 (EphAh2-kd). EphA2-Kd models were able to replicate the effects observed in the CAV1 knocked-down models, where the stimulation of the TC71-EphA2-Kd model with the recombinant protein EfnA1, showed both the activation of AKT and the overexpression of bFGF. These results replicate the effect of silencing CAV1 and highlighted the importance of the kinase-dependent activity of EphA2 in the angiogenic process in ES. We decided to analyze the kinaseindependent role of EphA2 as a possible key in the process of vascular mimicry in ES. Our results showed that the silencing of CAV1 reduces the development of mimetic vessels in vivo and prevents the tubular formation in vitro. In addition, our results showed that the kinase-dependent activity of the receptor does not affect the process of vascular mimicry in ES. Moreover, we stably transfected a mutant that lacks all the cytoplasmic region of EphA2, blocking its kinase-dependent and –independent activities. Results showed a negative effect on the formation of tubular structures. These results confirm the importance of the pro-oncogenic activity of EphA2 in ES.

My thesis explores how female writers of the Golden Age of children’s literature used their domestic stories to convey their visions of a more desirable society to their child readers, and thus to widen their influence beyond the homely sphere. My first chapter reconsiders the nineteenth-century historical circumstances wherein the woman and the child came to be constructed and enshrined as the domestic woman and the Romantic child within the home, and excluded from the public discourses. I then consider how in domestic stories women writers tried to overcome this shared deprivation of autonomy with the child, focusing on the works of Charlotte Yonge, Juliana Ewing, and Mary Louisa Molesworth. It emerges that these women writers were all keen to encourage their young readers to question the boundaries that separate home from the public realm, and to imagine a society wherein these dividing lines would be mitigated and even be extinguished. The thesis argues that these female writers’ literary efforts to exhaust the potential of the domestic story, and that their motivation to provide their child readers a sense of agency were integral in the development of Golden Age children’s literature. Charlotte Yonge’s technique of evoking sympathy for the child characters forged a more intimate relationship between adult author and young reader, and initiated the unsettling of the hierarchy between old and young, and author and reader. Juliana Ewing’s experiments with child narrators and her mingling of adventure and fantasy stories with domestic stories showed successive writers the various directions the domestic story could go. Mary Louisa Molesworth’s nursery stories realized the purpose of Ewing’s literary experiments, as her stories’ natural interweaving of quotidian nursery and fairy tale elements not only alleviated the hierarchy between fantasy and domestic realism, but also opened an era in which the blending of these two modes would become one of the most popular genres in children’s literature.

Introduction: Little is known about the aetiology of cancer in teenagers and young adults (TYA) aged 15-24 years, although in England, cancer is the most common cause of disease-related mortality in this age group. The most common cancers at this age are lymphomas, central nervous system (CNS) tumours and germ cell tumours (GCT). The commonest carcinomas seen at older ages including lung, breast, large bowel and prostate account for only 3-4% of TYA cancers. In this thesis I describe the incidence patterns of selected cancers in TYA and the variation seen with geography, time and in population subgroups. The focus is on CNS tumours, GCT and bone tumours as they either peak in incidence in TYA and/or contribute disproportionately to cancer related mortality in TYA. This will allow formulation of hypotheses regarding aetiology of cancer in this age group which can then be tested by further research. Methods: For the majority of the analysis, anonymised national cancer registration data from England on individual patients of all ages with newly diagnosed cancer between 1979 and 2003 were used. To contrast the incidence patterns in England with that of India, data from five Indian urban population based cancer registries were used for part of the analysis. Age, sex, site and histology specific incidence rates were calculated and expressed per million person years. All rates, where appropriate, were adjusted to the world standard population using direct methods. To explore the link of growth with development of osteosarcoma and Ewing sarcoma, a random-effects meta-analysis was undertaken on studies which investigated an association of these tumours with height at diagnosis. Results: The incidence of cancer in TYA overall in England exceeded that of India. This was also true for most individual sites including epithelial cancers of lung, colon/rectum, breast, ovary and cervix, and non-epithelial cancers including melanoma, Hodgkin lymphoma and testicular cancer. Notable exceptions to this pattern were cancers of the mouth, gall bladder and stomach (females only) where incidence was higher in India. In England, CNS tumours in TYA were a composite of pilocytic astrocytomas and embryonal tumours (representing tail end of childhood CNS tumours), pituitary tumours, nerve sheath tumours, high grade astrocytomas and meningiomas (representing early-onset of CNS tumours that peak in incidence in the 6th and 7th decade of life), and of CNS GCTs, pleomorphic xanthoastrocytomas and neurocytomas which show a peak incidence in TYA. Irrespective of site or histology, GCT in England showed a peak in incidence between ages of 10 to 39 years which was more marked in males. This however varied by site and the peak incidence was seen at 10 to 14 years in the CNS, 15 to 19 years in ovary, 25 to 29 in mediastinum & thorax and abdomen & pelvis, and 30 to 34 years in testicular tumours. Osteosarcoma and Ewing sarcoma were the predominant bone tumours in TYA in England and showed a distinct peak of incidence at 10 to 14 years age in females and a larger peak at 15 to 19 years age in males. The peak incidence of osteosarcoma of long bones of the lower limb was six times more than that at any other site while the peak incidence of Ewing sarcomas located in the bones of the central axis exceeded those in long bones of the lower limb. The average height of patients with osteosarcoma at diagnosis was found to be significantly above the average height of the reference population, at the 95% level. The association of greater height at diagnosis with Ewing sarcoma was also significant at the 95% level but much weaker. Conclusion: In this thesis I have explored the epidemiology of cancer in TYA using some of the established methodologies which have previously been used in advancing our knowledge of childhood and older adult cancers. These studies provide some clues to aetiology. Variation in environmental exposures and lifestyle factors between England and India can explain the majority of the differences in incidence patterns observed. Genetic predisposition to cancer along with carcinogen exposure could lead to early onset of some cancers generally seen in older adults. Regardless of site, the similarity in age-incidence patterns of GCT, suggests a common initiation of these tumours in embryonic/foetal life with variable rates of tumour progression as a result of local factors or events during postnatal and pubertal period. The incidence patterns of osteosarcoma along with the strong and consistent association with a greater height at diagnosis indicate that bone growth is important in the development of this tumour while different biological pathways which may be unrelated to growth could also be relevant for Ewing sarcoma.

Introduction: Genetic rearrangements in Ewing sarcoma, prostate, and leukemia cells result in activation of oncogenic ETS transcription factor fusions. Mithramycin (MTM) has been identified as an inhibitor of EWS-FLI1 transcription factor, a gene fusion product responsible for oncogenesis in Ewing sarcoma. Despite preclinical success, a phase I/II clinical trial testing MTM therapy in refractory Ewing sarcoma was terminated. Liver and blood toxicities resulted in dose de-escalation and sub-therapeutic exposures. However, the promise of selectively targeting oncogenic ETS transcription factors like EWS-FLI1 prompted us to undertake the discovery of more selective, less toxic analogues of MTM. MTM is a potent inhibitor of ubiquitous SP1 transcription factor, likely inducing non-specific toxicity. In collaboration with two medicinal chemistry groups, two semi-synthetic efforts were implemented to develop novel analogues of MTM. The first effort utilized the biosynthetic product mithramycin SA (MTMSA) to modify C3-side chain. The second effort utilized an oxime linker directly formed on MTM’s C3-side chain (MTM-oxime; MTMox). Here I present the pharmacological assessment of over 75 novel MTM analogues towards selectively targeting oncogenic ETS transcription factors, like EWS-FLI1, over ubiquitous transcription factors, like SP1. Methods: Novel MTM analogues were evaluated for selective cytotoxicity against ETS fusion-dependent cell lines. Selectively cytotoxic analogues were evaluated for inhibitory effects on several gene promoters in TC-32 reporter cell lines, a Ewing sarcoma cell line dependent on EWS-FLI1, transfected with luciferase reporter vector. Cloned reporter vectors incorporated NR0B1 (EWS-FLI1 binding), β-actin (SP1 binding) and CMV (non-specific) gene promoters. Furthermore, gene (mRNA) and protein expression changes of EWS-FLI1 and SP1, as well as regulated target genes, namely NR0B1, VEGFA and BCL-2 were evaluated with MTM analogue treatments. The MTM analogues with most selective activity in vitro were administered to mice by intravenous bolus dose for pharmacokinetic analysis. The MTM analogues with highest systemic exposure from each semi-synthetic effort, namely MTMSA-Trp-A10 and MTMox-24, were further evaluated. Metabolic stabilities in whole blood, plasma, and tumor cell matrices, and across multiple species were compared with MTM. Moreover, intrinsic hepatic clearances were estimated using mouse liver microsomes. Tumor and liver distributions were estimated in tumor bearing mice. Additionally, the effect of organic anionic transporter polypeptides (OATP) on distribution of MTM was investigated. Maximum tolerated doses were evaluated for lead MTM analogues, having both selective activities in vitro and high systemic exposure, compared to MTM. Complete blood cell counts and plasma alanine aminotransferase activity were measured to evaluate dose-dependent blood and liver toxicities, respectively. ETS fusion-dependent and non-dependent cell lines were implanted subcutaneously into immunocompromised mice for efficacy studies. Average tumor volumes and survival were tracked for mice receiving treatment, compared to MTM and vehicle treatment. Results: Evaluation of MTM analogues from both semi-synthetic efforts revealed that conjugation of MTM C3-side chain with tryptophan (Trp) and/or phenylalanine (Phe) improved selective cytotoxicity against ETS fusion-dependent cell lines. This was highlighted by MTMSA-Trp-A2 (also refer to as MTMSA-Phe-Trp) and MTMSA-Trp-A10 (also refer to as MTMSA-Trp-Trp), with selective indices of 19.1 and 15.6, respectively, compared to MTM (1.5). Similarly, MTMox-23 (also refer to as MTMox-Phe-Trp) and MTMox-20 (also refer to as MTMox-Trp) had selectivity indices of 4.6 and 4.5, respectively. These selectively cytotoxic MTM analogues inhibited EWS-FLI1-mediated transcription 10-fold more effectively than both non-specific CMV-mediated and SP1-mediated (via β-actin promoter) transcription in TC-32 reporter cell lines. Moreover, gene (mRNA) and protein expression of EWS-FLI1 and regulated gene, NR0B1, were inhibited with MTM analogue treatment (GI50, 6-hour) in TC-32 cells. Similarly, SP1 and target genes, VEGFA and BCL-2, gene (mRNA) and protein expressions were also inhibited with MTM analogue treatment (GI50, 6-hour) in TC-32 cells. Conjugation of Trp and/or Phe to C3-side chain of MTM increased systemic exposure in vivo. Most impressively, the addition of two Trp residues, namely MTMSA-Trp-A10 and MTMox-24 (also refer to as MTMox-Trp-Trp), resulted in systemic exposure increases of 218- and 42-fold, respectively, after intravenous (IV) bolus dose. Metabolically, tryptophan/phenylalanine conjugated MTM analogues are liable to esterase activity on carboxy-methyl functional group. Very rapid de-methylation in biological matrix was observed with MTMox-24, compared to MTMSA-Trp-A10, suggesting a regiospecific effect. However, esterase activity was limited to rodent matrices and demethylation occurred at significantly diminished rates in non-human primate and human plasma. MTM analogues were not susceptible to p450-mediated metabolism, with negligible loss in mouse liver microsome assay compared to verapamil control. MTM (1mg/kg) and MTMox-24 (6mg/kg) were detected in subcutaneously implanted (flank) LL2 tumors and liver homogenates after IV bolus dose. Interestingly, MTMSA-Trp-A10 (2mg/kg) was not. Despite a 3-fold increase in systemic exposure with rifampin oral pretreatment, an OATP inhibitor, exposure of MTM was unaffected in Oatp knockout mouse model. Exposure of MTM in liver tissue was 8.4-fold higher compared to tumor tissue with low tissue clearance. This agrees with the lack of metabolism observed in liver microsomes and may provide a mechanism for clinically observed liver toxicity. MTMSATrp-A10 had a single maximum tolerated dose (MTD) of 0.75mg/kg, compared to 1mg/kg for MTM, administered by IV bolus. In contrast, MTM-oxime analogues (MTMox-20, -23, -24 and -25) had single maximum tolerated doses of 20 – 25mg/kg. These increased tolerances are the result of additive differences in whole blood stability, cytotoxicity and systemic exposure. At a dose of 0.75mg/kg, administered every 3 days, MTMSA-Trp-A10 did not result in an efficacious result in tumor xenograft studies. These studies remain under further investigation, but the result may indicate high plasma protein binding of MTMSA-Trp-A10 and lack of free fraction available within tumor. The most selective MTM-oxime analogue in vitro, MTMox-23, significantly inhibited TC-32 (EWS-FLI1+) tumor xenograft growth (p=0.0025, day 16, one-way ANOVA multiple comparisons test) compared to MTM (p=0.1174, day 16) and extending survival for 17 days out of 48 days on study (p=0.0003, Log Rank (Mantel-Cox) single comparison test) with treatment at MTD every 3 days, compared to vehicle. Additionally, the MTM-oxime analogue with highest systemic exposure, MTMox-24, also significantly inhibited TC-32 (EWS-FLI1+) tumor xenograft growth (p=0.0003, day 21, one-way ANOVA multiple comparisons test) compared to MTM (p=0.032, day 21) and extending survival for 12 days out of 37 days on study (p=0.0004, Log Rank (Mantel-Cox) single comparison test) with treatment, compared to vehicle. Conclusion: These studies in whole highlight the importance of exposure (pharmacokinetics; PK), toxicity and efficacy (pharmacodynamics; PD) relationships. The cytotoxicity and high systemic exposure of MTMSA-Trp-A10 directly contributes to its lower tolerated dose. However, despite a similar tolerated dose to MTM, systemic exposure remains 163-fold higher at the MTD. High systemic exposure may be attributed to high plasma protein binding, but also reduces the exposure of free MTMSA-Trp-A10 within the tumor tissue, which drives the efficacious response. In contrast, the less cytotoxic and rapidly de-methylated MTM-oxime analogues allow for 25-fold higher tolerances in mice. This unique metabolism and clearance may prevent exposures required to induced systemic blood and liver toxicities induced by MTM. Moreover, at these highly tolerated doses, the initial systemic exposure at MTD is highest among analogues tested, which resulted in an efficacious response with MTMox-23 and MTMox-24 treatment in tumor xenograft models. It remains to be determined if these PK/PD relationships can be reproduced in additional animal models, including human, without inducing toxicity. Nonetheless, these initial studies in mice demonstrate that a more selective, more tolerated analogue of MTM has potential for clinical success in treating ETS fusion-dependent tumors.

The 1952 Presidential campaign ushered into national prominence the Democratic nominee, Adlai Ewing Stevenson. His sudden and dramatic emergence as an important factor in world politics was due in part to his unique oratory. Dynamic in style and content, his speeches commanded the rapt attention of the American people for three intense months during which he delivered over two hundred and fifty. Of interest and importance is the new insight into national problems that he gave to the American voter as a result of these orations. The problem is to analyze, through his public addresses before and during the 1952 campaign, the power of his oratory with respect to the enforcement of ideas, and to more fully understand his personality and philosophy.

Le sarcome de Ewing est la seconde tumeur pédiatrique de l’os la plus fréquente. Elle est caractérisée par une translocation chromosomique résultant à la fusion de EWSR1 avec un membre de la famille ETS. Chez 85% des patients, cette fusion conduit à l’expression de la protéine chimérique EWS-FLI1 qui est l’oncogène majeur de ce sarcome. Ce dernier agit principalement par son action transcriptionelle sur des cibles qui lui sont propres. Au niveau thérapeutique, le sarcome d’Ewing est traité par chimiothérapie, chirurgie locale et par radiothérapie. La survie à long terme des patients est de l’ordre de 70%, mais beaucoup plus basse pour les patients métastatiques et quasi nulle lors d’une récidive. Parmi maintes caractéristiques, certains cancers présentent une dérégulation énergétique. L’influence d’EWS-FLI1 sur cet aspect n’a fait l’objet d’aucune étude dans le contexte du sarcome d’Ewing. Nous avons donc étudié par profilage métabolomique des cellules de sarcome d’Ewing en présence ou en absence d’EWS-FLI1. En comparant ces deux conditions, des modulations du profil énergétique relatif au cycle de Krebs, des précurseurs de le glycosylation ainsi que des métabolites de la voie de la méthionine et du tryptophane ont été observés. En parallèle, grâce à un crible de banque de shRNAs réalisé dans des conditions expérimentales similaires à l’étude métabolomique (lignée d’Ewing avec ou sans EWS-FLI1), nous avons pu identifier des gènes présentant des caractéristiques « synthétique létales », c'est-à-dire tuant uniquement les cellules du sarcome d’Ewing en présence de son oncogène. / Ewing sarcoma, the second most commonly occurring pediatric bone tumor, is most often characterized by a chromosomal translocation between EWSR1 and FLI1. The gene fusion EWS-FLI1 accounts for 85% of all Ewing sarcoma and is considered the major oncogene and master regulator of Ewing sarcoma. EWS-FLI1 is a transcriptional modulator of targets, both directly and indirectly. Ewing sarcoma is aggressively treated with chemotherapy, localized surgery and radiation and has an overall survival of about 70%, however, survival for metastasis or relapsed cases remains low. One of the cancer hallmarks, metabolic deregulation, is most likely partly dependent on EWS-FLI1 in Ewing sarcoma cells. In order to get a better understanding of Ewing sarcoma biology and oncogenesis, it might be of high interest to investigate the influence of EWS-FLI1 in Ewing sarcoma cells. We therefore performed a global metabolic profiling of Ewing sarcoma cells with or without inhibition of EWS-FLI1. Several changes in the energy metabolism were observed throughout this study; the observed changes were consistent with an energy profile that moved from a cancer cell energy metabolism towards the energy metabolism of a more normal cell upon EWS-FLI1 inhibition, primarily based on the TCA cycle. Levels of TCA intermediates, glycosylation precursors, methionine pathway metabolites and amino acids, especially changes in the tryptophan metabolic pathway, were altered upon EWS-FLI1 inhibition. Parallel to this study, we performed a high-throughput synthetic lethality screen, in order to not only identify essential genes for cell survival and proliferation, but also to identify new synthetic lethal targets that could specifically target Ewing sarcoma cells carrying the EWS-FLI1 fusion gene.

O câncer pediátrico é raro em números absolutos, porém quando comparado às incidências em adultos vem apresentando aumento nas taxas de incidência, exigindo um preparo do sistema de saúde para acompanhamento. Este acompanhamento não pode ser igual ao do adulto, visto que crianças possuem diferenças fisiológicas nas diferentes faixas etárias pediátricas. Sendo assim, estudos de utilização de medicamentos são importantes nesta população, a fim de promover o uso racional dos mesmos, bem como, garantir seu uso seguro e uma terapêutica eficaz. O presente estudo tem por objetivo avaliar as prescrições de antineoplásicos, na unidade de oncologia pediátrica de um hospital universitário de Porto Alegre, identificando os protocolos mais utilizados, e posteriormente contextualizando-os com o preconizado no referencial teórico. Foi realizado um estudo transversal prospectivo envolvendo internações realizadas na unidade de oncologia pediátrica do Hospital de Clínicas de Porto Alegre (HCPA). Foram analisadas 274 internações, das quais 40 eram primeira internação e 234 reinternações. A maioria dos pacientes tinha idade de 0 a 10 anos, uma discreta prevalência do sexo masculino, de raça/cor branca, residentes na mesorregião metropolitana. A principal forma de financiamento das internações foi o público, e a principal causa de internação foi o tratamento, sendo o mais frequente o quimioterápico. Foram analisados os protocolos utilizados durante as internações de pacientes com diagnóstico de Leucemia Linfocítica Aguda, Retinoblastoma e Sarcoma de Ewing. Os protocolos são conjunto de regras criadas a partir de grupos cooperativos ou da indústria farmacêutica, permitindo um maior conhecimento sobre os medicamentos utilizados na pediatria. A oncologia pediátrica com sua particularidade de ser uma doença culturalmente ligada ao óbito possui maior facilidade de estudos deste porte. Porém, ainda são necessários mais estudos de utilização destes medicamentos, a fim de agregar conhecimento aos protocolos de tratamento. / Pediatric cancer is rare in absolute numbers, but when compared to the incidence in adults has shown an increase in incidence rates, requiring a prepared system health for following. Pediatrics treatment can’t be the same as that of adults, because children have different physiological differences in pediatric age groups. Studies of medication use in this population are very important in order to promote the rational use of drugs, as well as to guarantee their safe use and effective therapy. The present study aims to evaluate the anticancer prescriptions in pediatric oncology unit of a university hospital in Porto Alegre, identifying the most used protocols, and contextualizing them later with the recommendations in the theoretical framework. A prospective cross-sectional study involving hospital admissions in the pediatric oncology unit of the Hospital de Clínicas de Porto Alegre (HCPA) was performed. We analyzed 274 admissions, which 40 were first admission and 234 were readmissions. These admissions most patients were aged 0 to 10 years, white race, a slight male prevalence, residing in Metropolitan Mesoregion. The financing the hospital admissions was public, and the maining cause of hospitalization was treatment, the most frequent being the chemotherapy. We analyzed the protocols used during the admissions of patients diagnosed with Acute Lymphocytic Leukemia, Retinoblastoma and Ewing's Sarcoma. Protocols are studies that allow a greater knowledge about medicines for pediatric use. The pediatric oncology with its characteristic of being a disease has culturally linked with death ease of studies of this size. However, more studies of medication use are still needed to use these drugs in order to knowledge to the treatment protocols.

O câncer pediátrico é raro em números absolutos, porém quando comparado às incidências em adultos vem apresentando aumento nas taxas de incidência, exigindo um preparo do sistema de saúde para acompanhamento. Este acompanhamento não pode ser igual ao do adulto, visto que crianças possuem diferenças fisiológicas nas diferentes faixas etárias pediátricas. Sendo assim, estudos de utilização de medicamentos são importantes nesta população, a fim de promover o uso racional dos mesmos, bem como, garantir seu uso seguro e uma terapêutica eficaz. O presente estudo tem por objetivo avaliar as prescrições de antineoplásicos, na unidade de oncologia pediátrica de um hospital universitário de Porto Alegre, identificando os protocolos mais utilizados, e posteriormente contextualizando-os com o preconizado no referencial teórico. Foi realizado um estudo transversal prospectivo envolvendo internações realizadas na unidade de oncologia pediátrica do Hospital de Clínicas de Porto Alegre (HCPA). Foram analisadas 274 internações, das quais 40 eram primeira internação e 234 reinternações. A maioria dos pacientes tinha idade de 0 a 10 anos, uma discreta prevalência do sexo masculino, de raça/cor branca, residentes na mesorregião metropolitana. A principal forma de financiamento das internações foi o público, e a principal causa de internação foi o tratamento, sendo o mais frequente o quimioterápico. Foram analisados os protocolos utilizados durante as internações de pacientes com diagnóstico de Leucemia Linfocítica Aguda, Retinoblastoma e Sarcoma de Ewing. Os protocolos são conjunto de regras criadas a partir de grupos cooperativos ou da indústria farmacêutica, permitindo um maior conhecimento sobre os medicamentos utilizados na pediatria. A oncologia pediátrica com sua particularidade de ser uma doença culturalmente ligada ao óbito possui maior facilidade de estudos deste porte. Porém, ainda são necessários mais estudos de utilização destes medicamentos, a fim de agregar conhecimento aos protocolos de tratamento. / Pediatric cancer is rare in absolute numbers, but when compared to the incidence in adults has shown an increase in incidence rates, requiring a prepared system health for following. Pediatrics treatment can’t be the same as that of adults, because children have different physiological differences in pediatric age groups. Studies of medication use in this population are very important in order to promote the rational use of drugs, as well as to guarantee their safe use and effective therapy. The present study aims to evaluate the anticancer prescriptions in pediatric oncology unit of a university hospital in Porto Alegre, identifying the most used protocols, and contextualizing them later with the recommendations in the theoretical framework. A prospective cross-sectional study involving hospital admissions in the pediatric oncology unit of the Hospital de Clínicas de Porto Alegre (HCPA) was performed. We analyzed 274 admissions, which 40 were first admission and 234 were readmissions. These admissions most patients were aged 0 to 10 years, white race, a slight male prevalence, residing in Metropolitan Mesoregion. The financing the hospital admissions was public, and the maining cause of hospitalization was treatment, the most frequent being the chemotherapy. We analyzed the protocols used during the admissions of patients diagnosed with Acute Lymphocytic Leukemia, Retinoblastoma and Ewing's Sarcoma. Protocols are studies that allow a greater knowledge about medicines for pediatric use. The pediatric oncology with its characteristic of being a disease has culturally linked with death ease of studies of this size. However, more studies of medication use are still needed to use these drugs in order to knowledge to the treatment protocols.

O câncer pediátrico é raro em números absolutos, porém quando comparado às incidências em adultos vem apresentando aumento nas taxas de incidência, exigindo um preparo do sistema de saúde para acompanhamento. Este acompanhamento não pode ser igual ao do adulto, visto que crianças possuem diferenças fisiológicas nas diferentes faixas etárias pediátricas. Sendo assim, estudos de utilização de medicamentos são importantes nesta população, a fim de promover o uso racional dos mesmos, bem como, garantir seu uso seguro e uma terapêutica eficaz. O presente estudo tem por objetivo avaliar as prescrições de antineoplásicos, na unidade de oncologia pediátrica de um hospital universitário de Porto Alegre, identificando os protocolos mais utilizados, e posteriormente contextualizando-os com o preconizado no referencial teórico. Foi realizado um estudo transversal prospectivo envolvendo internações realizadas na unidade de oncologia pediátrica do Hospital de Clínicas de Porto Alegre (HCPA). Foram analisadas 274 internações, das quais 40 eram primeira internação e 234 reinternações. A maioria dos pacientes tinha idade de 0 a 10 anos, uma discreta prevalência do sexo masculino, de raça/cor branca, residentes na mesorregião metropolitana. A principal forma de financiamento das internações foi o público, e a principal causa de internação foi o tratamento, sendo o mais frequente o quimioterápico. Foram analisados os protocolos utilizados durante as internações de pacientes com diagnóstico de Leucemia Linfocítica Aguda, Retinoblastoma e Sarcoma de Ewing. Os protocolos são conjunto de regras criadas a partir de grupos cooperativos ou da indústria farmacêutica, permitindo um maior conhecimento sobre os medicamentos utilizados na pediatria. A oncologia pediátrica com sua particularidade de ser uma doença culturalmente ligada ao óbito possui maior facilidade de estudos deste porte. Porém, ainda são necessários mais estudos de utilização destes medicamentos, a fim de agregar conhecimento aos protocolos de tratamento. / Pediatric cancer is rare in absolute numbers, but when compared to the incidence in adults has shown an increase in incidence rates, requiring a prepared system health for following. Pediatrics treatment can’t be the same as that of adults, because children have different physiological differences in pediatric age groups. Studies of medication use in this population are very important in order to promote the rational use of drugs, as well as to guarantee their safe use and effective therapy. The present study aims to evaluate the anticancer prescriptions in pediatric oncology unit of a university hospital in Porto Alegre, identifying the most used protocols, and contextualizing them later with the recommendations in the theoretical framework. A prospective cross-sectional study involving hospital admissions in the pediatric oncology unit of the Hospital de Clínicas de Porto Alegre (HCPA) was performed. We analyzed 274 admissions, which 40 were first admission and 234 were readmissions. These admissions most patients were aged 0 to 10 years, white race, a slight male prevalence, residing in Metropolitan Mesoregion. The financing the hospital admissions was public, and the maining cause of hospitalization was treatment, the most frequent being the chemotherapy. We analyzed the protocols used during the admissions of patients diagnosed with Acute Lymphocytic Leukemia, Retinoblastoma and Ewing's Sarcoma. Protocols are studies that allow a greater knowledge about medicines for pediatric use. The pediatric oncology with its characteristic of being a disease has culturally linked with death ease of studies of this size. However, more studies of medication use are still needed to use these drugs in order to knowledge to the treatment protocols.