Este trabalho avaliou o efeito de fibroblastos cardíacos (FC) modificados geneticamente para produzir VEGF (vascular endothelial growth factor) e/ou em conjunto com IGF-1(insulin -like growth factor) associados a um biopolímero de fibrina na indução de angiogênese, vasculogênese e melhora de função em miocárdio isquêmico. Em experimentos preliminares demonstramos que 106 FC modificados pelo AdRSVLacZ expressam o transgene na parede livre do ventrículo esquerdo de ratos Lewis por até 45 dias. Primeiro, o tratamento dos diversos grupos foi feito e 7 dias após, os animais foram submetidos à isquemia por 45 minutos seguida de reperfusão. 21 dias depois, a proteína humana VEGF e a densidade capilar apresentaram aumento no grupo VEGF (proteína humana VEGF: 1209,6±11,4 vs. Veículo 123,1±5,2; Célula 104,2±7,4 e Null 73,2±2,4 células positivas/campo, p< 0,01 e densidade capilar: 543,8 ± 52,1 vs. 349,2 ± 0,9, 288± 19,0 e 245 ± 2,6 capilares/mm2, p< 0,01). A imunofluorescência dupla-marcação para detecção de células endoteliais e células musculares lisas apresentou aumento no grupo VEGF sugerindo formação de vasos estruturados (45±3 vs. 10±2, 8±1 e 16±3, p<0,001) e a área de infarto foi reduzida no VEGF vs. VEÍCULO (3,0 ± 1,3% vs. 8,0 ± 0,8%, p< 0,05. Para testar o efeito terapêutico desta intervenção, um segundo estudo foi realizado com os grupos: VEÍCULO= controle, POLÍMERO = biopolímero de fibrina, CÉLULA, NULL, IGF-1, VEGF e IGF-1+VEGF. Os tratamentos foram realizados 24 horas após os animais terem sido submetidos à isquemia por ligadura permanente. Um mês depois, as proteínas humanas VEGF e IGF 1 apresentaram aumento significativo nos grupos VEGF, IGF-1 e IGF-1+VEGF, com *p=0,0001. Da mesma maneira, somente os grupos que receberam VEGF isoladamente ou associados a IGF-1 tiveram aumento do número de capilares e da densidade vascular e redução da porcentagem de colágeno (35,12 ± 7,05 vs. 31,28 ± 5,03 vs. 30,07 ± 6,21 vs. 25,89 ± 2,92 vs. 15,43 ± 2,02* vs. 16,07 ± 1,83%*, *p<0,05, para os grupos Veículo, Polímero, Célula, Null, IGF-1, VEGF, IGF-1+VEGF, respectivamente). Os índices cardíacos basais morfológicos e funcionais permaneceram inalterados na avaliação direta e pelo ECO entre os grupos enquanto que as medidas diretas de função cardíaca sob estresse farmacológico com a fenilefrina mostraram aumentos significativos no trabalho cardíaco e volume sistólico e diminuição na pressão diastólica final somente nos animais que receberam terapia celular que incluía VEGF. Em conjunto, os dados mostram que a terapia celular combinada com o aumento da expressão de fator angiogênico (VEGF) ou em combinação com fator de crescimento (IGF-1) tem efeito benéfico, uma vez que estes fatores estimularam a proliferação capilar e vascular podendo contribuir para o aumento da circulação colateral, reduzindo o tamanho do infarto e promovendo melhora cardíaca funcional. / The effect of modified cardiac fibroblasts (CF) expressing VEGF (vascular endothelial growth factor) and/or IGF-1 (insulin-like growth factor) associated to fibrin biopolymer to induce angiogenesis, vasculogenesis and improve cardiac function in ischemic cardiac tissue was tested. The direct injection of 106 CF genetically modified to express the reporter gene LACZ (AdRSVLACZ) indicated transgene expression up to 45 days. First, all groups were treated and 7 days later the animals were submitted to a 45 min cardiac ischemic injury. Twenty one days later VEGF protein and capillary density increased only in groups that received VEGF the groups: (VEGF protein: 1209.6±11.4 vs. VEHICLE: 123.1±5.2, Cell: 104.2±7.4 and Null: 73.2±2.4 positive cells/field, p< 0.01 and capillary: 543.8 ± 52.1 vs. 349.2 ± 0.9, 288± 19.0 and 245 ± 2.6 capillaries/mm2, p< 0.01). Merged image of immunoassaying for endothelial and smooth muscle cells specific markers, were significantly greater in VEGF group suggesting maturation of newly formed vessels (45±3 vs. 10±2, 8±1 and 16±3, p<0.001) and myocardial scar area was reduced in VEGF vs. VEHICLE (3.0 ± 1.3% vs. 8.0 ± 0.8%, p< 0.05). To test the therapeutic efficacy of this treatment, a second study was performed with groups: VEHICLE= control, POLYMER= fibrin biopolymer, CELL, NULL, IGF-1, VEGF and IGF-1+VEGF. Treatments were performed 24 hs following ligation of the descending coronary artery. After 4 weeks, VEGF and IGF-1 protein increased in IGF-1, VEGF and IGF-1+VEGF groups, p<0.0001. We observed only in VEGF groups an increase in capillary number and vascular density and reduction in myocardial collagen area (35,12 ± 7,05 vs. 31,28 ± 5,03 vs. 30,07 ± 6,21 vs. 25,89 ± 2,92 vs. 15,43 ± 2,02* vs. 16,07 ± 1,83%*, *p<0,05, to groups VEHICLE, POLYMER, CELL, NULL, IGF-1, VEGF, IGF-1+VEGF, respectively). The morphological and functional basal cardiac indices remained unchanged in all groups, however, under pharmacologic stress using phenilephrine, the VEGF groups displayed significant improvement in cardiac work and stroke volume and a reduction in end diastolic pressure. Taken together, these results indicated that cardiac fibroblasts expressing VEGF alone or in combination with IGF-1 can induce agiogenesis and vasculogenesis in ischemic myocardium decreasing myocardial scar area and improving cardiac performance following coronary ligation.

La peau est un organe capable de se régénérer et de cicatriser. Elle constitue la première barrière de protection de notre organisme contre les agressions physico-chimiques extérieures. Depuis plusieurs décennies des recherches ont été menées pour maîtriser la culture du derme pour plusieurs applications telles que la greffe des grands brulés. L’aspect technologique de ce domaine a fait l’objet de plusieurs travaux. Dans les années 2000, l'équipe de notre laboratoire a proposé la méthode de culture du derme associée à des microsystèmes en silicium. C’est l’unique méthode actuelle qui permet la mesure des forces isométrique du derme équivalent lors de sa culture.Dans une première étude, nous avons proposé des nouvelles méthodes de mesure des forces isométriques qui s’exercent dans des peaux reconstruites en culture entre deux lames de silicium afin de fabriquer un bio-dispositif miniaturisé à faible coût. Ainsi, les dimensions optimales ont été calculées et des nouvelles lames ont été fabriquées. L’optimisation que nous avons retenue est relative à l’amélioration de la sensibilité de la mesure des forces. Afin de quantifier le fléchissement de lames due aux forces isométriques appliquées par le derme en culture, nous avons opté pour la mesure des déplacements des lames sous l’effets des forces isométriques, à l’aide d’ondes acoustiques de surface (SAW). Ce choix se justifie par la simplicité de l’intégration des transducteurs interdigités qui génèrent les ondes acoustiques, de la possibilité d’utiliser une interrogation sans fils et la réalisation physique de l’intercorrélation des ondes générées.A l’aide de simulation nous avons identifié les déformations des ondes et les écarts de fréquence qu’elles provoquent. En effet, la dissymétrie de la courbe d’intercorrélation des signaux transmis et reçus, par les transducteurs interdigités, est intiment liée à l’écart de fréquence de l’onde reçue. Nous avons démontré que le fléchissement engendre bien la dissymétrie dans l’axe du temps qui peut être mesurée plus précisément dans les limites d‘échantillonnage. Deux démonstrateurs sont modélisés et fabriqués dans la salle blanche afin de valider l’instrumentation et le principe de transduction d’un signal chirpé avec une onde acoustique de surface. Les résultats obtenus montrent que la méthode de mesure à l’aide d’une onde acoustique nous permet de faire de mesure de force mais dans une gamme d’intensité plus élevée que celle attendue. Par la suite, nous avons étudié la méthode de mesure de forces par les capteurs à base de piézorésistances. Sachant que la technique est basée sur la variation de résistivité du matériau déformé, nous avons décidé de replacer les grilles de lame prévue pour l’accrochage du derme en culture par le matériau piézorésistif implanté sur les micro poutres. Afin d’améliorer la résolution de détection de faibles forces une série de calcul et de simulations de la position et les dimensions du matériau piézorésistif et des micropoutres sont effectués et présentés. Une autre étude que nous avons menée en parallèle concerne le développement d’une instrumentation embarquée permettant de suivre la croissance du derme en culture basé sur un système de vision. Vu les conditions strictes de notre cahier de charges qui exigeait la portabilité et l’autonomie de système final, nous avons prévu le développement d’un système de vision embarqué basé sur un module de caméra et une carte FPGA. La caméra à haute définition montée sur le système de boite de culture finale avec un objectif permet de prendre des images de fluorescences des cellules en culture. / The skin is an organ which can regenerate and heal. It is the first shield of protection of our body against external physico-chemical aggression. For several decades, researches have been conducted to control the dermis culture for several applications such as grafting large burns. The technological aspect of this area has been the subject of several works. In the 2000s, the team of our laboratory proposed the dermis culture method associated with silicon microsystems. This is the only current method that allows the measurement of isometric forces of the equivalent dermis during its culture.In a first stage of study, in order to produce a miniaturized and low-cost bio-device, we proposed new methods to measure isometric forces in reconstructed skins in culture between two silicon beams. Thus, the optimal dimensions were calculated and new beams were fabricated. The chosen optimization is related to improve the sensitivity of the force measurement. To quantify the deflection of the beams due to the isometric forces applied by the dermis in culture, we opted for the measurement of the displacements of the beams under the influence of the isometric forces by using surface acoustic waves (SAW). This choice is justified by the simplicity of the integration of the interdigital transducers (IDT) that generate the SAW, the possibility of using a wireless interrogation and the physical realization of the cross-correlation of the generated waves.Using simulation, we have identified the frequency deviations caused by wave deformations. Indeed, the dissymmetry of the cross-correlation curve of the signals generated and received by IDT is closely related to the frequency deviation of the received wave. We have evidenced that the beam deflection generates the dissymmetry in the time axis which can be measured more precisely within the limits of sampling. Two demonstrators were designed and manufactured in the clean room to validate the instrumentation and the principle of transducing a chirped signal with a SAW. The obtained results show that the proposed SAW-based force measuring method allows us to measure force, but in a higher intensity range than expected. Subsequently, we studied the method of force measurement by piezoresistors. Considering that the technique is based on the variation of resistivity of the deformed material, we decided to replace the silicon grids provided for the attachment of the dermis in culture by the piezoresistive material implanted on the silicon micro-beams. To improve the low-resolution detection, a series of calculations and simulations of the positions and the dimensions of the piezoresistive material and the micro-beams have been carried out and presented. Another study that we conducted in parallel concerns the development of an on-board instrumentation to monitor the growth of the dermis in culture based on a vision system. Considering of the strict conditions of our specifications that required the portability and autonomy of the final system, we developed an embedded vision system based on a camera module and a FPGA card. The high definition camera mounted on the system of final culture box with a lens allows to take fluorescence images of cells in culture.

Gastric cancer (GC) is one of leading causes of death in the world, and peritoneal metastases (PM) are a major site of recurrence. PM from GC implies a poor prognosis, with median overall survival (mOS) approximately 3 months and no survival at five years. The aims of this thesis were to explore the incidence and evaluate prognostic factors for mOS of PM from GC in a defined population; to investigate the outcome of a new multimodal treatment; to analyse the treatment costs, and to investigate differences in drug sensitivity between individual patient samples and between various tumours. The incidence of loco-regional advanced GC was 3.8 per 100,000 person-years. Synchronous loco-regional GC in combination with synchronous distant metastasis was a negative prognostic factor while chemotherapy and good performance status, and radiotherapy plus chemotherapy were positive prognostic factors . There were no significant differences in mOS for the group of patients included during the period 2000-2004 versus 2005-2009, and this lack of improvement in mOS during the past decade justifies new treatment approaches. In a Phase II study of patients treated with neoadjuvant systemic chemotherapy followed by cytoreductive surgery + hyperthermic intraperitoneal chemotherapy, mOS was 14.3 months and for patients with macroscopically radical surgery mOS was 19.1 months. The mean overall cost of the loco-regional treatment was $145,700 compared to $59,300 with systemic chemotherapy treatment. In an ex vivo chemo-sensitivity test, it was determined that GC samples were equivalent to colorectal cancer in chemo-sensitivity to standard drugs and targeted drugs, whereas ovarian cancer samples were more sensitive. The individual GC samples varied considerably in sensitivity to increasing concentrations of the drugs, arguing for individualized drug selection. The incidence of loco-regional advanced GC was more common than previously reported and there were no improvements in mOS over the past decade. The mOS for patients with neoadjuvant systemic chemotherapy followed by macroscopically radical cytoreductive surgery + hyperthermic intraperitoneal chemotherapy was better than in recent reports on treatment with systemic chemotherapy. Treatment of advanced GC patients is costly irrespective of treatment modality. The GC samples varied considerably between individuals in terms of sensitivity to increasing concentrations of the drugs and were comparable to colorectal cancer in chemo-sensitivity.

No description available.

Membrane transport proteins are known to influence the absorption, distribution, metabolism, excretion and toxicity (ADMET) of drugs. At the onset of this thesis work, only a few structure-activity models, in general describing P-glycoprotein (Pgp/ABCB1) interactions, were developed using small datasets with little structural diversity. In this thesis, drug-transport protein interactions were explored using large, diverse datasets representing the chemical space of orally administered registered drugs. Focus was set on the ATP-binding cassette (ABC) transport proteins expressed in the canalicular membrane of human hepatocytes. The inhibition of the ABC transport proteins multidrug-resistance associated protein 2 (MRP2/ABCC2) and bile salt export pump (BSEP/ABCB11) was experimentally investigated using membrane vesicles from cells overexpressing the investigated proteins and sandwich cultured human hepatocytes (SCHH). Several previously unknown inhibitors were identified for both of the proteins and predictive in silico models were developed. Furthermore, a clear association between BSEP inhibition and clinically reported drug induced liver injuries (DILI) was identified. For the first time, an in silico model that described combined inhibition of Pgp, MRP2 and breast cancer resistance protein (BCRP/ABCG2) was developed using a large, structurally diverse dataset. Lipophilic weak bases were more often found to be general ABC inhibitors in comparison to other drugs. In early drug discovery, in silico models can be used as predictive filters in the drug candidate selection process and membrane vesicles as a first experimental screening tool to investigate protein interactions. In summary, the present work has led to an increased understanding of molecular properties important in ABC inhibition as well as the potential influence of ABC proteins in adverse drug reactions. A number of previously unknown ABC inhibitors were identified and predictive computational models were developed.

The liver is the major detoxifying organ, clearing the blood from drugs and other xenobiotics. The extent of hepatic clearance (CL) determines drug exposure and hence, the efficacy and toxicity associated with exposure. Drug-drug interactions (DDIs) that alter the hepatic CL may cause more or less severe outcomes, such as adverse drug reactions. Accurate predictions of drug CL and DDI risk from in vitro data are therefore crucial in drug development. Liver CL depends on several factors including the activities of transporters involved in the hepatic uptake and efflux. The work in this thesis aimed at developing new in vitro and in silico methods to predict hepatic transporter-mediated CL and DDIs in vivo. Particular emphasis was placed on interactions involving the hepatic uptake transporters OATP1B1, OATP1B3, and OATP2B1. These transporters regulate the plasma concentration-time profiles of many drugs including statins. Inhibition of OATP-mediated transport by 225 structurally diverse drugs was investigated in vitro. Several novel inhibitors were identified. The data was used to develop in silico models that could predict OATP inhibitors from molecular structure. Models were developed for static and dynamic predictions of in vivo transporter-mediated drug CL and DDIs. These models rely on a combination of in vitro studies of transport function and mass spectrometry-based quantification of protein expression in the in vitro models and liver tissue. By providing estimations of transporter contributions to the overall hepatic uptake/efflux, the method is expected to improve predictions of transporter-mediated DDIs. Furthermore, proteins of importance for hepatic CL were quantified in liver tissue and isolated hepatocytes. The isolation of hepatocytes from liver tissue was found to be associated with oxidative stress and degradation of transporters and other proteins expressed in the plasma membrane. This has implications for the use of primary hepatocytes as an in vitro model of the liver. Nevertheless, by taking the altered transporter abundance into account using the method developed herein, transport function in hepatocyte experiments can be scaled to the in vivo situation. The concept of protein expression-dependent in vitro-in vivo extrapolations was illustrated using atorvastatin and pitavastatin as model drugs.

Este trabalho avaliou o efeito de fibroblastos cardíacos (FC) modificados geneticamente para produzir VEGF (vascular endothelial growth factor) e/ou em conjunto com IGF-1(insulin -like growth factor) associados a um biopolímero de fibrina na indução de angiogênese, vasculogênese e melhora de função em miocárdio isquêmico. Em experimentos preliminares demonstramos que 106 FC modificados pelo AdRSVLacZ expressam o transgene na parede livre do ventrículo esquerdo de ratos Lewis por até 45 dias. Primeiro, o tratamento dos diversos grupos foi feito e 7 dias após, os animais foram submetidos à isquemia por 45 minutos seguida de reperfusão. 21 dias depois, a proteína humana VEGF e a densidade capilar apresentaram aumento no grupo VEGF (proteína humana VEGF: 1209,6±11,4 vs. Veículo 123,1±5,2; Célula 104,2±7,4 e Null 73,2±2,4 células positivas/campo, p< 0,01 e densidade capilar: 543,8 ± 52,1 vs. 349,2 ± 0,9, 288± 19,0 e 245 ± 2,6 capilares/mm2, p< 0,01). A imunofluorescência dupla-marcação para detecção de células endoteliais e células musculares lisas apresentou aumento no grupo VEGF sugerindo formação de vasos estruturados (45±3 vs. 10±2, 8±1 e 16±3, p<0,001) e a área de infarto foi reduzida no VEGF vs. VEÍCULO (3,0 ± 1,3% vs. 8,0 ± 0,8%, p< 0,05. Para testar o efeito terapêutico desta intervenção, um segundo estudo foi realizado com os grupos: VEÍCULO= controle, POLÍMERO = biopolímero de fibrina, CÉLULA, NULL, IGF-1, VEGF e IGF-1+VEGF. Os tratamentos foram realizados 24 horas após os animais terem sido submetidos à isquemia por ligadura permanente. Um mês depois, as proteínas humanas VEGF e IGF 1 apresentaram aumento significativo nos grupos VEGF, IGF-1 e IGF-1+VEGF, com *p=0,0001. Da mesma maneira, somente os grupos que receberam VEGF isoladamente ou associados a IGF-1 tiveram aumento do número de capilares e da densidade vascular e redução da porcentagem de colágeno (35,12 ± 7,05 vs. 31,28 ± 5,03 vs. 30,07 ± 6,21 vs. 25,89 ± 2,92 vs. 15,43 ± 2,02* vs. 16,07 ± 1,83%*, *p<0,05, para os grupos Veículo, Polímero, Célula, Null, IGF-1, VEGF, IGF-1+VEGF, respectivamente). Os índices cardíacos basais morfológicos e funcionais permaneceram inalterados na avaliação direta e pelo ECO entre os grupos enquanto que as medidas diretas de função cardíaca sob estresse farmacológico com a fenilefrina mostraram aumentos significativos no trabalho cardíaco e volume sistólico e diminuição na pressão diastólica final somente nos animais que receberam terapia celular que incluía VEGF. Em conjunto, os dados mostram que a terapia celular combinada com o aumento da expressão de fator angiogênico (VEGF) ou em combinação com fator de crescimento (IGF-1) tem efeito benéfico, uma vez que estes fatores estimularam a proliferação capilar e vascular podendo contribuir para o aumento da circulação colateral, reduzindo o tamanho do infarto e promovendo melhora cardíaca funcional. / The effect of modified cardiac fibroblasts (CF) expressing VEGF (vascular endothelial growth factor) and/or IGF-1 (insulin-like growth factor) associated to fibrin biopolymer to induce angiogenesis, vasculogenesis and improve cardiac function in ischemic cardiac tissue was tested. The direct injection of 106 CF genetically modified to express the reporter gene LACZ (AdRSVLACZ) indicated transgene expression up to 45 days. First, all groups were treated and 7 days later the animals were submitted to a 45 min cardiac ischemic injury. Twenty one days later VEGF protein and capillary density increased only in groups that received VEGF the groups: (VEGF protein: 1209.6±11.4 vs. VEHICLE: 123.1±5.2, Cell: 104.2±7.4 and Null: 73.2±2.4 positive cells/field, p< 0.01 and capillary: 543.8 ± 52.1 vs. 349.2 ± 0.9, 288± 19.0 and 245 ± 2.6 capillaries/mm2, p< 0.01). Merged image of immunoassaying for endothelial and smooth muscle cells specific markers, were significantly greater in VEGF group suggesting maturation of newly formed vessels (45±3 vs. 10±2, 8±1 and 16±3, p<0.001) and myocardial scar area was reduced in VEGF vs. VEHICLE (3.0 ± 1.3% vs. 8.0 ± 0.8%, p< 0.05). To test the therapeutic efficacy of this treatment, a second study was performed with groups: VEHICLE= control, POLYMER= fibrin biopolymer, CELL, NULL, IGF-1, VEGF and IGF-1+VEGF. Treatments were performed 24 hs following ligation of the descending coronary artery. After 4 weeks, VEGF and IGF-1 protein increased in IGF-1, VEGF and IGF-1+VEGF groups, p<0.0001. We observed only in VEGF groups an increase in capillary number and vascular density and reduction in myocardial collagen area (35,12 ± 7,05 vs. 31,28 ± 5,03 vs. 30,07 ± 6,21 vs. 25,89 ± 2,92 vs. 15,43 ± 2,02* vs. 16,07 ± 1,83%*, *p<0,05, to groups VEHICLE, POLYMER, CELL, NULL, IGF-1, VEGF, IGF-1+VEGF, respectively). The morphological and functional basal cardiac indices remained unchanged in all groups, however, under pharmacologic stress using phenilephrine, the VEGF groups displayed significant improvement in cardiac work and stroke volume and a reduction in end diastolic pressure. Taken together, these results indicated that cardiac fibroblasts expressing VEGF alone or in combination with IGF-1 can induce agiogenesis and vasculogenesis in ischemic myocardium decreasing myocardial scar area and improving cardiac performance following coronary ligation.

Despite the theoretical and practical ramifications arising from sociolinguistic studies in Brazil, the 90s brought the resurgence of a set of reflections on language which, expressed in newspaper columns, school grammars, books “no more mistakes" and writing manuals of major newspapers and public administration bodies, spreads the belief that there is only a "right" way to use language and that is the only acceptable way: the standard norm. All others are "wrong", and should be avoided. In this line of thought, in the state of Sergipe stands the book "Do not bite the Portuguese language", authored by Professor Wilma Ramos, described in its 10th edition as a reference guide for everyday difficulties of the Portuguese language, which brings advice on how to use the language in the light of the grammar of standard variant.This research aims to analyze prescriptions contained in the book “Do not bite the Portuguese language”, which disregard lessons contained in the normative instruments reference of Portuguese and disqualify the several varieties non-standard, as its speakers;discuss lessons that reject typical linguistic phenomena of Brazilian Portuguese, not welcomed by normative instruments reference of the Portuguese language, but widely disseminated in speech or writing from educated urban speakers, and, moreover, to assert the principles underlying the work “Do not bite the Portuguese language”, its contribution to the process of social representation of language and its implications for the process of teaching and learning Portuguese.The theoretical framework will focus in the postulates of sociolinguistics, whose studies have evidenced that natural languages are characterized by the phenomenon of variation and change and that the cultured varieties coexist with the informal varieties in a complex process of interactions, making room for the recognition of the legitimacy of difference, which are a result of the recent publication of the first grammars aimed for variety of Portuguese spoken in Brazil. Based on the norm concept in Coseriu, they seek to clarify the use of the terms "cultured norms", "standard norm", "grammatical norm", "pedagogical norm", "popular norm", characterizing Brazilian linguistic reality as essentially tripartite, formed by a standard norm, a set of prestigious norms and a large range of popular standards. It will set-up the concept of short standard, based on Faraco, to show their materialization in the work under review. It will also discuss the concept of legitimate language, as Bourdieu, aiming to outline the large-scale social factors involved in the construction processof the standard norm,in order to correlate linguistic issues properly with its institutional interface.The process of building the legitimate language, coupled with the sociolinguistic conception of the variation phenomenon, will provide the basis to place the institutional role of the work from Professor Wilma Ramos and discuss their implications for the Portuguese language teaching-learning process. The reference normative instruments of Portuguese language were chosen from grammars and dictionaries in vogue in the second half of the twentieth century, whose authors are or were consecrated philologists. Regarding the dictionaries, works ratified by the National Textbook Program will also be used – Dictionaries. / A despeito dos desdobramentos teóricos e práticos decorrentes dos estudos sociolinguísticos no Brasil, os anos 90 trouxeram o recrudescimento de um conjunto de reflexões sobre a língua que, manifestadas em colunas jornalísticas, gramáticas escolares, livros de “não erre mais” e manuais de redação de grandes jornais e órgãos da Administração Pública, difunde a crença de que só existe uma maneira “certa” de usar a língua e que essa é a única maneira aceitável: a norma-padrão. Todas as outras são “erradas” e devem ser evitadas. Nessa linha de reflexão, no Estado de Sergipe sobressai a obra Não morda a língua portuguesa, de autoria da professora Wilma Ramos, descrita em sua 10ª edição como um manual de consulta para dificuldades corriqueiras da língua portuguesa, que traz conselhos de como usar o idioma à luz da gramática da variante padrão. Esta pesquisa tem por objetivo analisar prescrições contidas na obra Não morda a língua portuguesaque desconsideram lições constantes nos instrumentos normativos de referência da língua e desqualificam as variedades diversas da norma-padrão, assim como seus falantes; discutir lições que rejeitam fenômenos linguísticos típicos do português brasileiro, não acolhidos pelos instrumentos normativos de referência da língua portuguesa, mas amplamente disseminados na fala ou escrita de falantes urbanos escolarizados, além de perquirir os princípios subjacentes à obra Não morda a língua portuguesa, sua contribuição ao processo de representação social da língua e suas implicações para o processo de ensino-aprendizagem da língua portuguesa. O referencial teórico centrarse- á em postulados da Sociolinguística, cujos estudos têm evidenciado que as línguas naturais são caracterizadas pelo fenômeno da variação e mudança e que as variedades cultas convivem com as variedades informais em um complexo processo de interações, abrindo espaço para o reconhecimento da legitimidade da diferença, de que são resultado as recentes publicações das primeiras gramáticas voltadas para a variedade do português falado no Brasil. Com fundamento no conceito de norma em Coseriu, busca-se precisar o emprego dos termos “norma culta”, “norma-padrão”, “norma gramatical”, “norma pedagógica”, “norma popular”, caracterizando a realidade linguística brasileira como essencialmente tripartite, formada por uma norma-padrão, um conjunto de normas prestigiadas e uma gama de normas populares. Definiu-se o conceito de norma curta, com base em Faraco, para mostrar a sua materialização na obra em análise. Discutir-se-á também o conceito de língua legítima, conforme Bourdieu, objetivando delinear os fatores sociais de larga escala envolvidos no processo de construção da norma-padrão, de modo a correlacionar questões linguísticas com sua interface propriamente institucional. O processo de construção da língua legítima, conjugado com a concepção sociolinguística acerca do fenômeno da variação, fornecerá as bases parasituar o papel institucional da obra da professora Wilma Ramos e discutir as suas implicações para o processo de ensino-aprendizagem da língua portuguesa. Os instrumentos normativos de referência da língua portuguesa foram escolhidos entre gramáticas e dicionários em voga na segunda metade do século XX, cujos autores são ou foram filólogos consagrados. No tocante aos dicionários, também foram utilizadas obras referendadas pelo Programa Nacional do Livro Didático – Dicionários.

O complexo esclerose tuberosa (CET) e as doenças renais policísticas autossômica dominante (DRPAD) e autossômica recessiva (DRPAR) são doenças monogênicas associadas a cistogênese renal. Os produtos dos genes mutados nessas enfermidades, respectivamente tuberina e hamartina para CET, policistina-1 (PC1) e policistina-2 para DRPAD, e poliductina/fibrocistina para DRPAR, modulam proliferação, diferenciação, apoptose, crescimento e/ou migração celular. Neste estudo empregamos um sistema tridimensional de cultura de células IMCD para caracterizar os perfis de expressão dessas proteínas durante a tubulogênese. Usando uma matriz de colágeno tipo I/Matrigel e fator de crescimento de hepatócito (HGF), a formação de estruturas alongadas se iniciou dois dias após o plaqueamento in vitro (2 DIV), ao passo que o desenvolvimento de lúmen ocorreu entre 10-14 DIV. A marcação para caspase-3 ativa foi mais intensa nas fases iniciais da tubulogênese, enquanto a marcação para Ki-67 foi uniformemente pronunciada em estágios mais tardios. A tuberina e a hamartina apresentaram expressão citoplasmática e co-localização acentuada em 6 e 12 DIV. A PC1 apresentou maior expressão nas porções ramificadas dos túbulos que nas não ramificadas no 12 DIV, um padrão não verificado para a PC2. Estas proteínas exibiram expressão citoplasmática, assim como expressão ocasional e pontual na membrana plasmática. PD1 também apresentou expressão citoplasmática. Nossos dados sugerem que a apoptose e a ciclagem celular sincrônica durante a tubulogênese in vitro são mais acentuadas, respectivamente, em fases mais precoces e mais tardias da formação tubular. Nossos achados demonstram, além disso, que as proteínas relacionadas ao CET e às DRPs são expressas in vitro durante a tubulogênese, apoiando um papel importante para a interação tuberina-hamartina na formação tubular, e são consistentes com o padrão de expressão diferencial da PC1 observado durante a nefrogênese / Tuberous sclerosis complex (TSC) and autosomal dominant and recessive polycystic kidney diseases (ADPKD and ARPKD) are monogenic diseases associated with renal cystogenesis. The products of the genes mutated in these disorders, respectively tuberin and hamartin for TSC, and polycystin-1 (PC1), polycystin-2 (PC2) and polyductin/fibrocystin (PD1) for PKD, modulate cell proliferation, differentiation, apoptosis, growth and/or migration. We have employed an IMCD tridimensional cell culture system to characterize their expression profiles along tubulogenesis. Using a type I collagen/Matrigel matrix and hepatocyte growth factor (HGF), the formation of elongated structures initiated 2 days after in vitro plating (2 DIV) while lumen developed between 10-14 DIV. Active caspase-3 labeling was more intense in initial phases of tubulogenesis while Ki-67 staining was uniformly pronounced in later stages. Tuberin and hamartin showed cytoplasmic expression and marked co- localization at 6 and 12 DIV. PC1 displayed higher expression in branching than non- branching portions of the tubules at 12 DIV, a pattern not verified for PC2. These proteins presented cytoplasmic and occasional, punctate membrane expression. PD1 also showed cytoplasmic expression. Our data suggest that apoptosis and synchronous cell cycling during in vitro tubulogenesis are more remarkable, respectively, in early and later steps of tubule formation. In addition, our findings demonstrate that the TSC and PKD proteins are expressed in vitro during tubulogenesis, supporting an important role for tuberin-hamartin interaction in tubular formation, and are consistent with the differential PC1 expression pattern observed during nephrogenesis

Sabe-se que TRAIL é capaz de matar células tumorais de forma seletiva e que TRAIL tem sua expressão reduzida em diversos tumores, porém pouco se sabe sobre os mecanismos responsáveis pela sua inibição. Tendo em vista que a expressão de TRAIL pode ser regulada pelo Ácido Retinóico; que PRAME é capaz de inibir a via do ácido retinóico através da proteína EZH2 e que nós observamos anteriormente que a expressão de TRAIL esta diminuída em pacientes com LMC, nós decidimos investigar a associação entre PRAME, EZH2 e TRAIL na LMC. Nós demonstramos que PRAME, mas não EZH2, tem sua expressão aumentada em células BCR-ABL+ e sua expressão está associada com a progressão da LMC. Alem disto, existe uma correlação positiva entre PRAME e BCR-ABL e negativa entre PRAME e TRAIL nestes pacientes. A inibição da expressão de PRAME ou EZH2 por RNAi induziu um aumento da expressão de TRAIL. Estes dados revelam um novo mecanismo de regulação responsável por diminuir a expressão de TRAIL, e geram novos possíveis alvos para a terapia da LMC e, possivelmente, também para outros tumores. / TRAIL was shown to selectively kill tumor cells. Not surprisingly, TRAIL is down-regulated in a variety of tumor cells, but the mechanism responsible for TRAIL inhibition remains elusive. Because TRAIL can be regulate by retinoic acid; PRAME was shown to inhibit transcription of retinoic acid receptor target genes through the polycomb protein EZH2; and we have found that TRAIL is inversely correlated with BCR-ABL in CML patients, we decided to investigate the association of PRAME, EZH2 and TRAIL in BCR-ABL-positive leukemia. Here, we demonstrate that PRAME, but not EZH2, is up-regulated in BCR-ABL cells and is associated with the progression of disease in CML patients. In addition, PRAME expression is positively correlated with BCR-ABL and negatively with TRAIL in these patients. Importantly, knocking down of PRAME or EZH2 by RNA interference restores TRAIL expression. Our data reveal a novel regulatory mechanism responsible for lowering TRAIL expression and provide the basis of alternative targets for combined therapeutic strategies for CML.