Demonstration of helicase activity in the nonstructural protein, NSs, of the negative-sense RNA virus, Groundnut bud necrosis virus

The nonstructural protein NSs, encoded by the S RNA of groundnut bud necrosis virus (GBNV) (genus Tospovirus, family Bunyaviridae) has earlier been shown to possess nucleic-acid-stimulated NTPase and 50 a phosphatase activity. ATP hydrolysis is an essential function of a true helicase. Therefore, NSs was tested for DNA helicase activity. The results demonstrated that GBNV NSs possesses bidirectional DNA helicase activity. An alanine mutation in the Walker A motif (K189A rNSs) decreased DNA helicase activity substantially, whereas a mutation in the Walker B motif resulted in a marginal decrease in this activity. The parallel loss of the helicase and ATPase activity in the K189A mutant confirms that NSs acts as a non-canonical DNA helicase. Furthermore, both the wild-type and K189A NSs could function as RNA silencing suppressors, demonstrating that the suppressor activity of NSs is independent of its helicase or ATPase activity. This is the first report of a true helicase from a negative-sense RNA virus.

The beta hairpin structure within ribosomal protein S5 mediates interplay between domains II and IV and regulates HCV IRES function

Translation initiation in Hepatitis C Virus (HCV) is mediated by Internal Ribosome Entry Site (IRES), which is independent of cap-structure and uses a limited number of canonical initiation factors. During translation initiation IRES-40S complex formation depends on high affinity interaction of IRES with ribosomal proteins. Earlier, it has been shown that ribosomal protein S5 (RPS5) interacts with HCV IRES. Here, we have extensively characterized the HCV IRES-RPS5 interaction and demonstrated its role in IRES function. Computational modelling and RNA-protein interaction studies demonstrated that the beta hairpin structure within RPS5 is critically required for the binding with domains II and IV. Mutations disrupting IRES-RPS5 interaction drastically reduced the 80S complex formation and the corresponding IRES activity. Computational analysis and UV cross-linking experiments using various IRES-mutants revealed interplay between domains II and IV mediated by RPS5. In addition, present study demonstrated that RPS5 interaction is unique to HCV IRES and is not involved in 40S-3 ` UTR interaction. Further, partial silencing of RPS5 resulted in preferential inhibition of HCV RNA translation. However, global translation was marginally affected by partial silencing of RPS5. Taken together, results provide novel molecular insights into IRES-RPS5 interaction and unravel its functional significance in mediating internal initiation of translation.

Bolaamphiphiles as carriers for siRNA delivery: From chemical syntheses to practical applications

In this study we have investigated a new class of cationic lipids - ``bolaamphiphiles'' or ``bolas'' - for their ability to efficiently deliver small interfering RNAs (siRNAs) to cancer cells. The bolas of this study consist of a hydrophobic chain with one or more positively charged head groups at each end. Recently, we reported that micelles of the bolas GLH-19 and GLH-20 (derived from vernonia oil) efficiently deliver siRNAs, while having relatively low toxicities in vitro and in vivo. Our previous studies validated that; bolaamphiphiles can be designed to vary the magnitude of siRNA shielding, its delivery, and its subsequent release. To further understand the structural features of bolas critical for siRNAs delivery, new structurally related bolas (GLH-58 and GLH-60) were designed and synthesized from jojoba oil. Both bolas have similar hydrophobic domains and contain either one, in GLH-58, or two, in GLH-60 positively charged head groups at each end of the hydrophobic core. We have computationally predicted and experimentally validated that GLH-58 formed more stable nano sized micelles than GLH-60 and performed significantly better in comparison to GLH-60 for siRNA delivery. GLH-58/siRNA complexes demonstrated better efficiency in silencing the expression of the GFP gene in human breast cancer cells at concentrations of 5 mu g/mL, well below the toxic dose. Moreover, delivery of multiple different siRNAs targeting the HIV genome demonstrated further inhibition of virus production. Published by Elsevier B.V.

STX13 regulates cargo delivery from recycling endosomes during melanosome biogenesis

Melanosomes are a class of lysosome-related organelles produced by melanocytes. Biogenesis of melanosomes requires the transport of melanin-synthesizing enzymes from tubular recycling endosomes to maturing melanosomes. The SNARE proteins involved in these transport or fusion steps have been poorly studied. We found that depletion of syntaxin 13 (STX13, also known as STX12), a recycling endosomal Qa-SNARE, inhibits pigment granule maturation in melanocytes by rerouting the melanosomal proteins such as TYR and TYRP1 to lysosomes. Furthermore, live-cell imaging and electron microscopy studies showed that STX13 co-distributed with melanosomal cargo in the tubular-vesicular endosomes that are closely associated with the maturing melanosomes. STX family proteins contain an N-terminal regulatory domain, and deletion of this domain in STX13 increases both the SNARE activity in vivo and melanosome cargo transport and pigmentation, suggesting that STX13 acts as a fusion SNARE in melanosomal trafficking pathways. In addition, STX13-dependent cargo transport requires the melanosomal R-SNARE VAMP7, and its silencing blocks the melanosome maturation, reflecting a defect in endosome-melanosome fusion. Moreover, we show mutual dependency between STX13 and VAMP7 in regulating their localization for efficient cargo delivery to melanosomes.

Glioblastoma-derived Macrophage Colony-stimulating Factor (MCSF) Induces Microglial Release of Insulin-like Growth Factor-binding Protein 1 (IGFBP1) to Promote Angiogenesis

Glioblastoma (grade IV glioma/GBM) is the most common primary adult malignant brain tumor with poor prognosis. To characterize molecular determinants of tumor-stroma interaction in GBM, we profiled 48 serum cytokines and identified macrophage colony-stimulating factor (MCSF) as one of the elevated cytokines in sera from GBM patients. Both MCSF transcript and protein were up-regulated in GBM tissue samples through a spleen tyrosine kinase (SYK)-dependent activation of the PI3K-NF kappa B pathway. Ectopic overexpression and silencing experiments revealed that glioma-secreted MCSF has no role in autocrine functions and M2 polarization of macrophages. In contrast, silencing expression of MCSF in glioma cells prevented tube formation of human umbilical vein endothelial cells elicited by the supernatant from monocytes/microglial cells treated with conditioned medium from glioma cells. Quantitative proteomics based on stable isotope labeling by amino acids in cell culture showed that glioma-derived MCSF induces changes in microglial secretome and identified insulin-like growth factor-binding protein 1 (IGFBP1) as one of the MCSF-regulated proteins secreted by microglia. Silencing IGFBP1 expression in microglial cells or its neutralization by an antibody reduced the ability of supernatants derived from microglial cells treated with glioma cell-conditioned medium to induce angiogenesis. In conclusion, this study shows up-regulation of MCSF in GBM via a SYK-PI3K-NF kappa B-dependent mechanism and identifies IGFBP1 released by microglial cells as a novel mediator of MCSF-induced angiogenesis, of potential interest for developing targeted therapy to prevent GBM progression.

Definition of a serum marker panel for glioblastoma discrimination and identification of Interleukin 1 beta in the microglial secretome as a novel mediator of endothelial cell survival induced by C-reactive protein

Glioblastoma (GBM) is the most common malignant adult primary brain tumor. We profiled 724 cancer-associated proteins in sera of healthy individuals (n = 27) and GBM (n = 28) using antibody microarray. While 69 proteins exhibited differential abundance in GBM sera, a three-marker panel (LYAM1, BHE40 and CRP) could discriminate GBM sera from that of healthy donors with an accuracy of 89.7% and p < 0.0001. The high abundance of C-reactive protein (CRP) in GBM sera was confirmed in 264 independent samples. High levels of CRP protein was seen in GBM but without a change in transcript levels suggesting a non-tumoral origin. Glioma-secreted Interleukin 6 (IL6) was found to induce hepatocytes to secrete CRP, involving JAK-STAT pathway. The culture supernatant from CRP-treated microglial cells induced endothelial cell survival under nutrient-deprivation condition involving CRP-Fc gamma RIII signaling cascade. Transcript profiling of CRP-treated microglial cells identified Interleukin 1 beta (IL1 beta) present in the microglial secretome as the key mediator of CRP-induced endothelial cell survival. IL1 beta neutralization by antibody-binding or siRNA-mediated silencing in microglial cells reduced the ability of the supernatant from CRP-treated microglial cells to induce endothelial cell survival. Thus our study identifies a serum based three-marker panel for GBM diagnosis and provides leads for developing targeted therapies. Biological significance A complex antibody microarray based serum marker profiling identified a three-marker panel - LYAM1, BHE40 and CRP as an accurate discriminator of glioblastoma sera from that of healthy individuals. CRP protein is seen in high levels without a concomitant increase of CRP transcripts in glioblastoma. Glioma-secreted IL6 induced hepatocytes to produce CRP in a JAK-STAT signaling dependent manner. CRP induced microglial cells to release IL1 beta which in turn promoted endothelial cell survival. This study, besides defining a serum panel for glioblastoma discrimination, identified IL1 beta as a potential candidate for developing targeted therapy. (C) 2015 Elsevier B.V. All rights reserved.

Rab9A is required for delivery of cargo from recycling endosomes to melanosomes

Melanosomes are a type of lysosome-related organelle that is commonly defective in Hermansky-Pudlak syndrome. Biogenesis of melanosomes is regulated by BLOC-1, -2, -3, or AP-1, -3 complexes, which mediate cargo transport from recycling endosomes to melanosomes. Although several Rab GTPases have been shown to regulate these trafficking steps, the precise role of Rab9A remains unknown. Here, we found that a cohort of Rab9A associates with the melanosomes and its knockdown in melanocytes results in hypopigmented melanosomes due to mistargeting of melanosomal proteins to lysosomes. In addition, the Rab9A-depletion phenotype resembles Rab38/ 32-inactivated or BLOC-3-deficient melanocytes, suggesting that Rab9A works in line with BLOC-3 and Rab38/ 32 during melanosome cargo transport. Furthermore, silencing of Rab9A, Rab38/ 32 or its effector VARP, or BLOC-3-deficiency in melanocytes decreased the length of STX13-positive recycling endosomal tubules and targeted the SNARE to lysosomes. This result indicates a defect in directing recycling endosomal tubules to melanosomes. Thus, Rab9A and its co-regulatory GTPases control STX13-mediated cargo delivery to maturing melanosomes.

Accurate sodium bisulfite sequencing in plants.

DNA cytosine methylation is a conserved epigenetic modification frequently correlating with transcriptional silencing in a wide variety of eukaryotic organisms. Sodium bisulfite treatment of DNA converts unmethylated cytosine to uracil, while 5-methylated cytosine is protected. We describe techniques that ensure reliable sequencing data following sodium bisulfite conversion and to avoid common pitfalls such as amplification of unconverted DNA and inclusion of sibling clones.

Engineering nucleic acid mechanisms for regulation and readout of gene expression : conditional Dicer substrate formation and sensitive multiplexed northern blots

Nucleic acids are most commonly associated with the genetic code, transcription and gene expression. Recently, interest has grown in engineering nucleic acids for biological applications such as controlling or detecting gene expression. The natural presence and functionality of nucleic acids within living organisms coupled with their thermodynamic properties of base-pairing make them ideal for interfacing (and possibly altering) biological systems. We use engineered small conditional RNA or DNA (scRNA, scDNA, respectively) molecules to control and detect gene expression. Three novel systems are presented: two for conditional down-regulation of gene expression via RNA interference (RNAi) and a third system for simultaneous sensitive detection of multiple RNAs using labeled scRNAs.

RNAi is a powerful tool to study genetic circuits by knocking down a gene of interest. RNAi executes the logic: If gene Y is detected, silence gene Y. The fact that detection and silencing are restricted to the same gene means that RNAi is constitutively on. This poses a significant limitation when spatiotemporal control is needed. In this work, we engineered small nucleic acid molecules that execute the logic: If mRNA X is detected, form a Dicer substrate that targets independent mRNA Y for silencing. This is a step towards implementing the logic of conditional RNAi: If gene X is detected, silence gene Y. We use scRNAs and scDNAs to engineer signal transduction cascades that produce an RNAi effector molecule in response to hybridization to a nucleic acid target X. The first mechanism is solely based on hybridization cascades and uses scRNAs to produce a double-stranded RNA (dsRNA) Dicer substrate against target gene Y. The second mechanism is based on hybridization of scDNAs to detect a nucleic acid target and produce a template for transcription of a short hairpin RNA (shRNA) Dicer substrate against target gene Y. Test-tube studies for both mechanisms demonstrate that the output Dicer substrate is produced predominantly in the presence of a correct input target and is cleaved by Dicer to produce a small interfering RNA (siRNA). Both output products can lead to gene knockdown in tissue culture. To date, signal transduction is not observed in cells; possible reasons are explored.

Signal transduction cascades are composed of multiple scRNAs (or scDNAs). The need to study multiple molecules simultaneously has motivated the development of a highly sensitive method for multiplexed northern blots. The core technology of our system is the utilization of a hybridization chain reaction (HCR) of scRNAs as the detection signal for a northern blot. To achieve multiplexing (simultaneous detection of multiple genes), we use fluorescently tagged scRNAs. Moreover, by using radioactive labeling of scRNAs, the system exhibits a five-fold increase, compared to the literature, in detection sensitivity. Sensitive multiplexed northern blot detection provides an avenue for exploring the fate of scRNAs and scDNAs in tissue culture.

Towards conditional RNAi : shape and sequence transduction with small conditional RNAs

RNA interference (RNAi) is a powerful biological pathway allowing for sequence-specific knockdown of any gene of interest. While RNAi is a proven tool for probing gene function in biological circuits, it is limited by being constitutively ON and executes the logical operation: silence gene Y. To provide greater control over post-transcriptional gene silencing, we propose engineering a biological logic gate to implement “conditional RNAi.” Such a logic gate would silence gene Y only upon the expression of gene X, a completely unrelated gene, executing the logic: if gene X is transcribed, silence independent gene Y. Silencing of gene Y could be confined to a specific time and/or tissue by appropriately selecting gene X.

To implement the logic of conditional RNAi, we present the design and experimental validation of three nucleic acid self-assembly mechanisms which detect a sub-sequence of mRNA X and produce a Dicer substrate specific to gene Y. We introduce small conditional RNAs (scRNAs) to execute the signal transduction under isothermal conditions. scRNAs are small RNAs which change conformation, leading to both shape and sequence signal transduction, in response to hybridization to an input nucleic acid target. While all three conditional RNAi mechanisms execute the same logical operation, they explore various design alternatives for nucleic acid self-assembly pathways, including the use of duplex and monomer scRNAs, stable versus metastable reactants, multiple methods of nucleation, and 3-way and 4-way branch migration.

We demonstrate the isothermal execution of the conditional RNAi mechanisms in a test tube with recombinant Dicer. These mechanisms execute the logic: if mRNA X is detected, produce a Dicer substrate targeting independent mRNA Y. Only the final Dicer substrate, not the scRNA reactants or intermediates, is efficiently processed by Dicer. Additional work in human whole-cell extracts and a model tissue-culture system delves into both the promise and challenge of implementing conditional RNAi in vivo.

Sexualidade, corpo e saúde masculinos: transformações e permanências nos discursos de homens e da revista Mens Health.

O estudo analisa os discursos de homens e da revista Mens Health acerca do corpo, saúde e sexualidade. Para a construção dos discursos dos homens, realizamos entrevistas semi-estruturadas com 19 homens leitores e nove não leitores. E dois eventos de grupos focais que reuniram 11 homens no total. Foi entrevistado também o editor da revista. Os principais conceitos norteadores deste trabalho foram os de gênero, sexualidade, poder e masculinidades hegemônicas e subalternas. Evidenciou-se que a revista está fortemente atrelada à sociedade de consumo ao estimular a inserção dos homens em um mercado de produtos e serviços até então estranhos a esse gênero. E que suas concepções sobre saúde estão relacionadas a de bem-estar e de individualização que se articulam com os discursos hegemônicos que vêm dando sentido às concepções de saúde e doença atualmente. A publicação investe fortemente na ideia de um corpo musculoso que proporcionará ganhos sociais, sexuais e profissionais aos sujeitos, nem sempre atrelado às questões de saúde. Ela ratifica a heterossexualidade do leitor projetado, expondo o corpo feminino e o sexo heterossexual e silenciando sobre outras formas de sexualidade. Por isso consideramos que a revista se vincula a uma concepção tradicional da masculinidade. Seus discursos, no entanto, não são monolíticos ou isentos de contradição, e também manifestam nuances relativas a um modelo mais contemporâneo de masculinidade, como quando apresenta a ideia de uma nova pedagogia da sexualidade e a valorização dos cuidados estéticos e de saúde com o corpo, aspectos considerados pouco próximos da masculinidade tradicional. Com relação aos discursos dos homens, evidenciou-se que a classe social e a geração são as variáveis mais importantes nas suas concepções sobre corpo, saúde e sexualidade masculina. Que, entre os não leitores, de modo geral, há evidências mais fortes de flexibilização com relação aos padrões mais tradicionais entre os homens mais jovens e/ou de classes mais altas. Enquanto os homens com idade acima dos 30 anos e das classes populares estão mais atrelados às concepções tradicionais. Entre os leitores, observou-se uma grande reflexividade com relação aos discursos da revista demonstrando que eles vêm se apropriando de forma importante dos discursos da revista e ressignificando suas concepções e práticas sobre os três temas da pesquisa a partir desses discursos. E, assim como os discursos da revista, os discursos dos homens, leitores ou não, também apresentaram aspectos contraditórios, ora demonstrando mais afiliação a um novo modelo de masculinidade, ora ao modelo mais tradicional.

DNA-mediated charge transport signaling within the cell

DNA possesses the curious ability to conduct charge longitudinally through the π-stacked base pairs that reside within the interior of the double helix. The rate of charge transport (CT) through DNA has a shallow distance dependence. DNA CT can occur over at least 34 nm, a very long molecular distance. Lastly, DNA CT is exquisitely sensitive to disruptions, such as DNA damage, that affect the dynamics of base-pair stacking. Many DNA repair and DNA-processing enzymes are being found to contain 4Fe-4S clusters. These co-factors have been found in glycosylases, helicases, helicase-nucleases, and even enzymes such as DNA polymerase, RNA polymerase, and primase across the phylogeny. The role of these clusters in these enzymes has remained elusive. Generally, iron-sulfur clusters serve redox roles in nature since, formally, the cluster can exist in multiple oxidation states that can be accessed within a biological context. Taken together, these facts were used as a foundation for the hypothesis that DNA-binding proteins with 4Fe-4S clusters utilize DNA-mediated CT as a means to signal one another to scan the genome as a first step in locating the subtle damage that occurs within a sea of undamaged bases within cells.

Herein we describe a role for 4Fe-4S clusters in DNA-mediated charge transport signaling among EndoIII, MutY, and DinG, which are from distinct repair pathways in E. coli. The DinG helicase is an ATP-dependent helicase that contains a 4Fe-4S cluster. To study the DNA-bound redox properties of DinG, DNA-modified electrochemistry was used to show that the 4Fe-4S cluster of DNA-bound DinG is redox-active at cellular potentials, and shares the 80 mV vs. NHE redox potential of EndoIII and MutY. ATP hydrolysis by DinG increases the DNA-mediated redox signal observed electrochemically, likely reflecting better coupling of the 4Fe-4S cluster to DNA while DinG unwinds DNA, which could have interesting biological implications. Atomic force microscopy experiments demonstrate that DinG and EndoIII cooperate at long range using DNA charge transport to redistribute to regions of DNA damage. Genetics experiments, moreover, reveal that this DNA-mediated signaling among proteins also occurs within the cell and, remarkably, is required for cellular viability under conditions of stress. Knocking out DinG in CC104 cells leads to a decrease in MutY activity that is rescued by EndoIII D138A, but not EndoIII Y82A. DinG, thus, appears to help MutY find its substrate using DNA-mediated CT, but do MutY or EndoIII aid DinG in a similar way? The InvA strain of bacteria was used to observe DinG activity, since DinG activity is required within InvA to maintain normal growth. Silencing the gene encoding EndoIII in InvA results in a significant growth defect that is rescued by the overexpression of RNAseH, a protein that dismantles the substrate of DinG, R-loops. This establishes signaling between DinG and EndoIII. Furthermore, rescue of this growth defect by the expression of EndoIII D138A, the catalytically inactive but CT-proficient mutant of EndoIII, is also observed, but expression of EndoIII Y82A, which is CT-deficient but enzymatically active, does not rescue growth. These results provide strong evidence that DinG and EndoIII utilize DNA-mediated signaling to process DNA damage. This work thus expands the scope of DNA-mediated signaling within the cell, as it indicates that DNA-mediated signaling facilitates the activities of DNA repair enzymes across the genome, even for proteins from distinct repair pathways.

In separate work presented here, it is shown that the UvrC protein from E. coli contains a hitherto undiscovered 4Fe-4S cluster. A broad shoulder at 410 nm, characteristic of 4Fe-4S clusters, is observed in the UV-visible absorbance spectrum of UvrC. Electron paramagnetic resonance spectroscopy of UvrC incubated with sodium dithionite, reveals a spectrum with the signature features of a reduced, [4Fe-4S]+1, cluster. DNA-modified electrodes were used to show that UvrC has the same DNA-bound redox potential, of ~80 mV vs. NHE, as EndoIII, DinG, and MutY. Again, this means that these proteins are capable of performing inter-protein electron transfer reactions. Does UvrC use DNA-mediated signaling to facilitate the repair of its substrates?

UvrC is part of the nucleotide excision repair (NER) pathway in E. coli and is the protein within the pathway that performs the chemistry required to repair bulky DNA lesions, such as cyclopyrimidine dimers, that form as a product of UV irradiation. We tested if UvrC utilizes DNA-mediated signaling to facilitate the efficient repair of UV-induced DNA damage products by helping UvrC locate DNA damage. The UV sensitivity of E. coli cells lacking DinG, a putative signaling partner of UvrC, was examined. Knocking out DinG in E. coli leads to a sensitivity of the cells to UV irradiation. A 5-10 fold reduction in the amount of cells that survive after irradiation with 90 J/m2 of UV light is observed. This is consistent with the hypothesis that UvrC and DinG are signaling partners, but is this signaling due to DNA-mediated CT? Complementing the knockout cells with EndoIII D138A, which can also serve as a DNA CT signaling partner, rescues cells lacking DinG from UV irradiation, while complementing the cells with EndoIII Y82A shows no rescue of viability. These results indicate that there is cross-talk between the NER pathway and DinG via DNA-mediated signaling. Perhaps more importantly, this work also establishes that DinG, EndoIII, MutY, and UvrC comprise a signaling network that seems to be unified by the ability of these proteins to perform long range DNA-mediated CT signaling via their 4Fe-4S clusters.

Gramáticas pós-NGB: do discurso oficial a outros discursos (im)possíveis

A implementação da Nomenclatura Gramatical Brasileira (NGB) em 1959 e a assunção da Linguística no Brasil na década de 60 são acontecimentos que provocaram profundas mudanças no fazer gramatical. A NGB, enquanto acontecimento discursivo, ao evidenciar determinados termos e silenciar outros, reestrutura a memória do discurso gramatical brasileiro, regulando a relação do sujeito com o dizível e instaurando uma nova formação discursiva dominante, a qual se sobrepôs às formações discursivas anteriores. No presente trabalho, partimos do pressuposto de que, apesar do efeito da censura imposta pela terminologia oficial, o discurso gramatical produzido após a sua instituição é constitutivamente da ordem do heterogêneo. Assim sendo, com base no aporte teórico da Análise de Discurso de Pêcheux e Orlandi e nos estudos do projeto História das Ideias Linguísticas, investigamos o funcionamento do discurso legitimado pela NGB nas gramáticas cuja publicação a sucederam, mais especificamente em sete gramáticas publicadas entre 1959 e 1969. Interessa-nos, portanto, com vistas a depreender a forma como se materializa a tensão entre os sentidos oficiais e os sentidos censurados, desnaturalizar o processo de (re)significação dos termos acolhidos pela NGB, depreendendo, assim, como os sentidos silenciados se fizeram significar na materialidade linguística das gramáticas

O papel da proteína supressora de tumor p53 na indução de ciclooxigenase-2 em Carcinoma de Pulmão de Células Não Pequenas: aspectos moleculares e clínicos

O Carcinoma de Pulmão de Células Não Pequenas (NSCLC) é uma doença freqüentemente letal e altamente resistente à terapia oncológica convencional, como por exemplo, o tratamento quimioterápico com cisplatina e paclitaxel. A superexpressão de Ciclooxigenase-2 (COX-2) é constantemente observada em pacientes com NSCLC, estando associada ao prognóstico ruim destes pacientes. Acredita-se que a alta expressão de COX-2 produz efeitos anti-apoptóticos, porém pouco é conhecido sobre os mecanismos de regulação desta enzima. Muitos sinais capazes de ativar COX-2 também induzem a proteína supressora de tumor p53, conhecida pelo seu papel fundamental no controle da proliferação celular e apoptose. Dados recentes indicam que a proteína p53 é um importante regulador da expressão de COX-2. O objetivo desta dissertação foi avaliar os efeitos da quimioterapia na expressão da enzima COX-2 em linhagens celulares com diferente status do gene TP53, e ainda, correlacionar a expressão de COX-2 e o status mutacional de TP53, com as características clínico-patológicas de pacientes com NSCLC. Como ferramentas experimentais foram usadas técnicas de biologia celular e molecular como interferência de RNA, PCR em tempo real, análise mutacional e imuno-histoquímica. Com os resultados obtidos, observamos que as linhagens celulares de câncer de pulmão que apresentam p53 na sua forma selvagem, quando expostas ao tratamento com cisplatina, apresentaram indução da expressão de COX-2 (RNAm e proteína), em adição ao aumento da síntese de Prostaglandina E2 (PGE2). Em contrapartida, a expressão de COX-2 não foi alterada após o tratamento com cisplatina nas linhagens celulares que apresentavam mutação no gene TP53. Ao avaliar o tratamento com paclitaxel, foi observado um aumento da expressão de COX-2 nas linhagens A549 e H460 (linhagens celulares do tipo selvagem para p53), entretanto não foi observada alteração nos níveis de PGE2. Em adição, o tratamento com paclitaxel induziu um aumento da expressão de COX-2 na linhagem com deleção em TP53, ACC LC-319. Em seguida, após silenciamento de p53 na linhagem celular A549, por interferência de RNA, a cisplatina passou a não ser mais capaz de induzir o aumento da expressão de COX-2. No tratamento com paclitaxel, o silenciamento de TP53 não mudou a expressão de COX-2, indicando assim um efeito independente de p53. Dessa maneira, sugerimos que a indução de COX-2, por cisplatina, em linhagens celulares NSCLC é dependente de p53. Na análise dos pacientes NSCLC, os resultados demonstram que 54% dos pacientes apresentam expressão positiva de COX-2. Mutações em TP53 foram observadas em 57% dos pacientes, incluindo 56% de fumantes correntes e 37% de ex-fumantes. Uma associação entre a expressão de COX-2 e o status selvagem de TP53 foi observada, entre os pacientes que apresentaram expressão positiva de COX-2, 80% apresentaram TP53 selvagem. Um número maior de pacientes é necessário para aumentar o poder estatístico e confirmar as tendências observadas nesse estudo

Gerando eus, tecendo redes e trançando nós: ditos e não ditos das professoras e estudantes negras nos cotidianos do curso de pedagogia

O presente trabalho objetiva compreender como a temática afrobrasileira era abordada nos cotidianos do Curso de Pedagogia, campus I, da Universidade Estadual da Paraíba. A questão norteadora da pesquisa foi descobrir como professoras e estudantes negras se sentem e enfocam essa temática nas suas redes de conhecimentos, práticas e relações. Para tanto, foi preciso adentrar na história do povo negro no Brasil a partir da luta dos movimentos sociais negros buscando entender as noções de raça, racismo, identidade e os limites da educação do/a negro/a ao longo da história brasileira. Neste percurso se dialoga com diversos estudiosos/as como: Santos (2002), Viana (2007), Oliveira (2003, 2006, 2007, 2008), Fernandes (2007), Domingues (2009), Munanga (2002, 2006, 2009), Veiga (2007, 2008)), Pereira (2008, 2009), Gomes (2003, 2006, 2008), Morin (2000), Hall (2003), Moreira e Câmara (2008), dentre outros. Exemplificando a luta e a resistência de mulheres negras são apresentadas as histórias de vida de quatro professoras e quatro estudantes negras, praticantes (CERTEAU, 2007), sujeitos da pesquisa, que expõem o processo de tessitura de suas identidades. Suas narrativas evidenciam indícios de racismo e discriminação que marcaram suas trajetórias, desde o seio familiar, fortalecendo-se na escola, na academia e, para algumas, chegando até o ambiente de trabalho. Uma das professoras é a pesquisadora que, à medida que narra a história de vida das praticantes, tece sua própria história, como mulheres costurando uma colcha de retalhos em que cada estampa retrata os momentos vividos. A metodologia de pesquisa nos/dos/com os cotidianos (ALVES, 2008) foi o caminho percorrido. Além da observação dos cotidianos, foram realizadas conversas (LARROSA, 2003) sobre as histórias de vida (BOSI, 2003; PORTELLI, 1997) e tecidas as narrativas a partir dessas vivências. Estas revelaram que tais mulheres, ao longo de suas histórias, viveram/vivem processos de afirmaçãonegação, visto que suas identidades não são fixas, mas negociações e renegociações (MUNANGA, 2010) que geram alegrias e conflitos. O entendimento do que é Ser Negra é experimentado por cada uma, sem um modelo padrão para suas existências. Elas foram se gerando nas relações com o/a outro/a, em cada contexto familiar, escolar, acadêmico e profissional. São senhoras de suas vidas e mesmo as mais jovens buscam fazer suas histórias com autonomia. São praticantes porque lutaram/lutam para conquistar um lugar na vida, utilizando astúcias e táticas para fabricar (CERTEAU, 2007) meios de enfrentamento das adversidades. As táticas do silêncio, do estudo e do trabalho revelaram que essas mulheres não ficaram invisíveis nem se colocaram como vítimas no espaço social. Assim, urge delinear a superação da visão da pessoa negra a partir dos traços físicos e reconhecer as raízes do povo brasileiro para compreender a história da negritude. A partir da ancestralidade, serão identificadas as origens do povo negro, que poderão trazer o passado de resistência e luta por liberdade, dignidade, cidadania que produzem o orgulho de ser negra. Orgulho que recupera a autoestima e a capacidade de organização e mobilização para combater o racismo e as desigualdades raciais. Os cursos de formação docente precisam abordar essa temática para capacitar os/as futuros/as professores/as nessa tarefa. Essa abordagem envolve o ensinaraprender, em que professores/as e estudantes assumem compromisso com uma educação crítica e inclusiva. A reflexão entre os/as docentes formadores/as nessa perspectiva plural e intercultural poderia ajudar na superação do eurocentrismo ainda presente nos conteúdos e nas mentalidades. Sob essa ótica, tecer um processo de formação docente no Curso de Pedagogia, comprometido com uma educação inclusiva, considera um contínuo fazerdesfazer, na tessitura de um diálogo permanente entre a práticateoriaprática, num movimento de pesquisaintervenção. Isso implica a reflexão da trajetória pessoal e coletiva e a articulação da ação pedagógica com um projeto político de transformação da sociedade excludente em sociedade plural e solidária. Os não ditos necessitam de aprofundamento em trabalhos posteriores, pois os silenciamentos se referem ao passado próximo e as relações interraciais na família, na academia e também nos ambientes de trabalho. Será que não existe mais racismo? Ou este foi naturalizado dificultando sua identificação nos cotidianos? Os limites do processo de introjeção do racismo, que provocam a invisibilidade de algumas praticantes, são novos desafios para estudos futuros. Assim, percebo que colcha de retalhos tecida ao longo desta trajetória não é justaposição como usualmente é entendida. Para além disto, significa a socialização das experiências vividas nos cotidianos das praticantes, a fim de inspirar atitudes de combate ao racismo que se ampliem para o contexto social.