Estudo da Toxicidade do Chumbo na Levedura Saccharomyces cerevisiae

O chumbo é um importante poluente ambiental. A levedura Saccharomyces cerevisiae constitui um modelo útil para o estudo dos efeitos tóxicos do chumbo. O conhecimento dos mecanismos de defesa e resistência à presença de metais pesados poderá ser útil em tecnologias de proteção ambiental, nomeadamente no desenvolvimento de novas metodologias para a biorremediação de metais pesados. O presente trabalho teve como objetivo avaliar o impacto do Pb na capacidade proliferativa, na integridade membranar e na produção intracelular de espécies reativas de oxigénio (ROS), na estirpe laboratorial da levedura Saccharomyces cerevisiae BY4741 (estirpe selvagem, WT). Foi também estudado o papel das mitocôndrias, como fonte de ROS induzida por Pb, e o envolvimento da H+-ATPase vacuolar (V-ATPase) e de transportadores vacuolares pertencentes à superfamília ABC (de ATP-binding cassette) na defesa contra a toxicidade do Pb. O estudo cinético do impacto de duas concentrações de Pb na viabilidade das leveduras (avaliado através de um ensaio clonogénico), na integridade da membrana celular (determinada com iodeto de propídio) e na produção intracelular de ROS (o anião superóxido foi detetado com dihidroetídio e o peróxido de hidrogénio com 2’,7’- diclorodihidrofluoresceína), revelou uma perda progressiva da capacidade proliferativa (53 e 17% de células viáveis, após a exposição durante 3h a 250 ou 1000 µmol/l de chumbo, respetivamente), coincidente com a acumulação intracelular de anião superóxido e de peróxido de hidrogénio, na ausência de perda da integridade membranar. A importância das mitocôndrias na produção de ROS, induzida por chumbo, foi levada a cabo usando um mutante deficiente respiratório desprovido de ADN mitocondrial (ƿ0). Quando comparado com a respetiva estirpe parental, o mutante ƿ0 apresentou uma maior resistência ao Pb e uma menor produção de ROS induzida por Pb. A exposição das células da estirpe BY4741 a 250 e 1000 µmol/l de chumbo originou a formação de 49 e 58% de células deficientes respiratórias, respetivamente. A função da V-ATPase, na desintoxicação de chumbo, foi avaliada utilizando mutantes com uma estrutura vacuolar normal mas defetivos em subunidades da VATPase (vma1Δ, vma2Δ, vma3Δ e vph1Δ). Comparativamente às células da estirpe WT, todos os mutantes testados, sem V-ATPase funcional, apresentaram uma maior suscetibilidade ao Pb. O papel dos transportadores vacuolares pertencentes à superfamília ABC, na defesa contra a toxicidade induzida por chumbo, foi levada a cabo utilizando mutantes sem os transportadores Ycf1p ou Vmr1p. Os resultados preliminares mostraram que quando comparadas com as células da estirpe WT, as células das estirpes ycf1Δ ou vmr1Δ não apresentavam uma maior perda da viabilidade. A modificação da morfologia vacuolar, em células expostas a chumbo, foi visualizada utilizando a estirpe Vma2p-GFP. O tratamento das células com Pb originou a fusão dos vacúolos de tamanho médio num único vacúolo de grande dimensão. Em conclusão, os estudos desenvolvidos no presente trabalho, utilizando a estirpe laboratorial BY4741, mostraram que a perda da capacidade proliferativa das leveduras, induzida pelo chumbo, pode ser atribuída à acumulação intracelular do anião superóxido e de peróxido de hidrogénio. As mitocôndrias parecem ser uma das principais fontes de ROS induzido por Pb e, simultaneamente, um dos principais alvos da sua toxicidade. Em S. cerevisiae, o vacúolo desempenha um papel importante na desintoxicação do Pb. A modificação da morfologia vacuolar após exposição ao chumbo poderá ser a consequência da acumulação de Pb no vacúolo. Enquanto os transportadores da superfamília ABC parecem não estar envolvidos na sequestração vacuolar de Pb, é necessária a presença, num estado funcional, da V-ATPase para que ocorra a compartimentação do Pb. Muito provavelmente, a compartimentação do Pb no vacúolo previne a sua acumulação no citosol e o desencadear dos respetivos efeitos tóxicos.

A "Trojan horse" strategy to reverse drug-resistance in brain tumors

Los gliomas malignos representan una de las formas más agresivas de los tumores del sistema nervioso central (SNC). De acuerdo con la clasificación de los tumores cerebrales de la Organización Mundial de la Salud (OMS), los astrocitomas han sido categorizados en cuatro grados, determinados por la patología subyacente. Es así como los gliomas malignos (o de alto grado) incluyen el glioma anaplásico (grado III) así como el glioblastoma multiforme (GBM, grado IV),estos últimos los más agresivos con el peor pronóstico (1). El manejo terapéutico de los tumores del SNC se basa en la cirugía, la radioterapia y la quimioterapia, dependiendo de las características del tumor, el estadio clínico y la edad (2),(3), sin embargo ninguno de los tratamientos estándar es completamente seguro y compatible con una calidad de vida aceptable (3), (4). En general, la quimioterapia es la primera opción en los tumores diseminados, como el glioblastoma invasivo y el meduloblastoma de alto riesgo o con metástasis múltiple, pero el pronóstico en estos pacientes es muy pobre (2),(3). Solamente nuevas terapias dirigidas (2) como las terapias anti-angiogénicas (4); o terapias génicas muestran un beneficio real en grupos limitados de pacientes con defectos moleculares específicos conocidos (4). De este modo, se hace necesario el desarrollo de nuevas terapias farmacológicas para atacar los tumores cerebrales. Frente a las terapias los gliomas malignos son con frecuencia quimioresistentes, y esta resistencia parece depender de al menos dos mecanismos: en primer lugar, la pobre penetración de muchas drogas anticáncer a través de la barrera hematoencefálica (BBB: Blood Brain Barrier), la barrera del fluido sangre-cerebroespinal (BCSFB: Blood-cerebrospinal fluid barrier) y la barrera sangre-tumor (BTB: blood-tumor barrier). Dicha resistencia se debe a la interacción de la droga con varios transportadores o bombas de eflujo de droga ABC (ABC: ATP-binding cassette) que se sobre expresan en las células endoteliales o epiteliales de estas barreras. En segundo lugar, estos transportadores de eflujo de drogas ABC propios de las células tumorales confieren un fenotipo conocido como resistencia a multidrogas (MDR: multidrug resistance), el cual es característico de varios tumores sólidos. Este fenotipo también está presente en los tumores del SNC y su papel en gliomas es objeto de investigación (5). Por consiguiente el suministro de medicamentos a través de la BBB es uno de los problemas vitales en los tratamientos de terapia dirigida. Estudios recientes han demostrado que algunas moléculas pequeñas utilizadas en estas terapias son sustratos de la glicoproteína P (Pgp: P-gycoprotein), así como también de otras bombas de eflujo como las proteínas relacionadas con la resistencia a multidrogas (MRPs: multidrug resistance-related proteins (MRPs) o la proteína relacionada con cáncer de seno (BCRP: breast-cancer resistance related protein)) que no permiten que las drogas de este tipo alcancen el tumor (1). Un sustrato de Pgp y BCRP es la DOXOrubicina (DOXO), un fármaco utilizado en la terapia anti cáncer, el cual es muy eficaz para atacar las células del tumor cerebral in vitro, pero con un uso clínico limitado por la poca entrega a través de la barrera hematoencefálica (BBB) y por la resistencia propia de los tumores. Por otra parte las células de BBB y las células del tumor cerebral tienen también proteínas superficiales, como el receptor de la lipoproteína de baja densidad (LDLR), que podría utilizarse como blanco terapéutico en BBB y tumores cerebrales. Es asi como la importancia de este estudio se basa en la generación de estrategias terapéuticas que promuevan el paso de las drogas a través de la barrera hematoencefalica y tumoral, y a su vez, se reconozcan mecanismos celulares que induzcan el incremento en la expresión de los transportadores ABC, de manera que puedan ser utilizados como blancos terapéuticos.Este estudio demostró que el uso de una nueva estrategia basada en el “Caballo de Troya”, donde se combina la droga DOXOrubicina, la cual es introducida dentro de un liposoma, salvaguarda la droga de manera que se evita su reconocimiento por parte de los transportadores ABC tanto de la BBB como de las células del tumor. La construcción del liposoma permitió utilizar el receptor LDLR de las células asegurando la entrada a través de la BBB y hacia las células tumorales a través de un proceso de endocitosis. Este mecanismo fue asociado al uso de estatinas o drogas anticolesterol las cuales favorecieron la expresión de LDLR y disminuyeron la actividad de los transportadores ABC por nitración de los mismos, incrementando la eficiencia de nuestro Caballo de Troya. Por consiguiente demostramos que el uso de una nueva estrategia o formulación denominada ApolipoDOXO más el uso de estatinas favorece la administración de fármacos a través de la BBB, venciendo la resistencia del tumor y reduciendo los efectos colaterales dosis dependiente de la DOXOrubicina. Además esta estrategia del "Caballo de Troya", es un nuevo enfoque terapéutico que puede ser considerado como una nueva estrategia para aumentar la eficacia de diferentes fármacos en varios tumores cerebrales y garantiza una alta eficiencia incluso en un medio hipóxico,característico de las células cancerosas, donde la expresión del transportador Pgp se vió aumentada. Teniendo en cuenta la relación entre algunas vías de señalización reconocidas como moduladores de la actividad de Pgp, este estudio presenta no solo la estrategia del Caballo de Troya, sino también otra propuesta terapéutica relacionada con el uso de Temozolomide más DOXOrubicina. Esta estrategia demostró que el temozolomide logra penetrar la BBB por que interviene en la via de señalización de la Wnt/GSK3/β-catenina, la cual modula la expresión del transportador Pgp. Se demostró que el TMZ disminuye la proteína y el mRNA de Wnt3 permitiendo plantear la hipótesis de que la droga al disminuir la transcripción del gen Wnt3 en células de BBB, incrementa la activación de la vía fosforilando la β-catenina y conduciendo a disminuir la β-catenina nuclear y por tanto su unión al promotor del gen mdr1. Con base en los resultados este estudio permitió el reconocimiento de tres mecanismos básicos relacionados con la expresión de los transportadores ABC y asociados a las estrategias empleadas: el primero fue el uso de las estatinas, el cual condujo a la nitración de los transportadores disminuyendo su actividad por la via del factor de transcripción NFκB; el segundo a partir del uso del temozolomide, el cual metila el gen de Wnt3 reduciendo la actividad de la via de señalización de la la β-catenina, disminuyendo la expresión del transportador Pgp. El tercero consistió en la determinación de la relación entre el eje RhoA/RhoA quinasa como un modulador de la via (no canónica) GSK3/β-catenina. Se demostró que la proteína quinasa RhoA promovió la activación de la proteína PTB1, la cual al fosforilar a GSK3 indujo la fosforilación de la β-catenina, lo cual dio lugar a su destrucción por el proteosoma, evitando su unión al promotor del gen mdr1 y por tanto reduciendo su expresión. En conclusión las estrategias propuestas en este trabajo incrementaron la citotoxicidad de las células tumorales al aumentar la permeabilidad no solo de la barrera hematoencefálica, sino también de la propia barrera tumoral. Igualmente, la estrategia del “Caballo de Troya” podría ser útil para la terapia de otras enfermedades asociadas al sistema nervioso central. Por otra parte estos estudios indican que el reconocimiento de mecanismos asociados a la expresión de los transportadores ABC podría constituir una herramienta clave en el desarrollo de nuevas terapias anticáncer.

Análise in silico de um novo “cluster” de PKS II Metagenômico

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

"Mutações no gene OCT4 em células eritroleucêmicas humanas: implicações sobre o fenótipo de resistência a múltiplas drogas (MDR)”

O fator de transcrição OCT4 é um importante marcador de células tronco e tem sido relacionado com o conceito de células tronco tumorais (CTTs). Recentemente, ele tem sido também relacionado ao fenótipo de resistência a múltiplas drogas (MDR). O objetivo deste trabalho foi testar se o OCT4 está ligado ao fenótipo MDR em células eritroleucêmicas derivadas da linhagem LMC-K562. Para isso, foi realizada a análise de expressão de genes associados à superfamília de transportadores ABC (ATP Binding Cassette) e, também, de fatores de transcrição relacionados a células tronco. Os primeiros resultados apontaram uma relação direta entre OCT4 e transportadores ABC na linhagem MDR derivada de K562 (Lucena). O sequenciamento de promotores ABC não revelou qualquer mutação que pudesse explicar a expressão diferenciada do OCT4 na linhagem MDR. Posteriormente, o sequenciamento da região contendo o domínio homeobox do gene OCT4 de ambas as linhagens celulares evidenciou, pela primeira vez, que este fator de transcrição é alvo de mutações que podem estar relacionados com o fenótipo MDR. As mutações encontradas implicam substituições de vários aminoácidos em ambas as linhagens celulares. K562 teve sete substituições (três exclusivas), enquanto Lucena teve 13 (nove exclusivas). Além disso, um busca in silico por motivos de fosforilação dentro da sequência de aminoácidos comparada, revelou que o OCT4 humano normal possui sete motivos potenciais de fosforilação. Entretanto, K562 perdeu um motivo e Lucena dois deles. Moléculas com diferentes padrões de fosforilação podem ter sua função modificada. Estes resultados indicam o OCT4 com uma alvo potencial para o tratamento do câncer, especialmente aqueles resistentes à quimioterapia.

The key lessons learnt from producing the ABC programme Talking Heads a talk show/documentary hybrid in a fast turnaround environment

The following exegesis will detail the key advantages and disadvantages of combining a traditional talk show genre with a linear documentary format using a small production team and a limited budget in a fast turnaround weekly environment. It will deal with the Australian Broadcasting Corporation series Talking Heads, broadcast weekly in the early evening schedule for the network at 18.30 with the presenter Peter Thompson. As Executive Producer for the programme at its inception I was responsible for setting it up for the ABC in Brisbane, a role that included selecting most of the team to work on the series and commissioning the music, titles and all other aspects required to bring the show to the screen. What emerged when producing this generic hybrid will be examined at length, including: „h The talk show/documentary hybrid format needs longer than 26¡¦30¡¨ to be entirely successful. „h The type of presenter ideally suited to the talk show/documentary format requires someone who is genuinely interested in their guests and flexible enough to maintain the format against tangential odds. „h The use of illustrative footage shot in a documentary style narrative improves the talk show format. iv „h The fast turnaround of the talk show/documentary hybrid puts tremendous pressure on the time frames for archive research and copyright clearance and therefore needs to be well-resourced. „h In a fast turnaround talk show/documentary format the field components are advantageous but require very low shooting ratios to be sustainable. „h An intimate set works best for a talk show hybrid like this. Also submitted are two DVDs of recordings of programmes I produced and directed from the first and third series. These are for consideration in the practical component of this project and reflect the changes that I made to the series.

Measuring the success of ABC training in South Africa: A case study in the production of Western Liberal broadcast news values

In 1993 the Australian Broadcasting Corporation was contracted by the Australian Government to assist in the reshaping of the South African Broadcasting Corporation from a state-run broadcaster to a respected and trusted national broadcaster for all people in the newly democratic South Africa. Broadcast journalism training was identified by ABC consultant Bob Wurth as possibly the greatest need for SABC Radio. This thesis examines the ABC's role in South Africa and the effectiveness of its radio journalism training project considering the organisational, structural, cultural and political constraints of the SABC. This thesis will show through interviews and participant observation the difficulties in achieving the production of Western Liberal journalism values at the SABC within the time constraints set by the project funded by the Australian Government and the particular South African morays.

Public service broadcasting, creative industries and innovation infrastructure : the case of ABC's Pool

The proliferation of media services enabled by digital technologies poses a serious challenge to public service broadcasting rationales based on media scarcity. Looking to the past and future, we articulate an important role that the Australian Broadcasting Corporation (ABC) might play in the digital age. We argue that historically the ABC has acted beyond its institutional broadcasting remit to facilitate cultural development and, drawing on the example of Pool (an online community of creative practitioners established and maintained by the ABC), point to a key role it might play in fostering network innovation in what are now conceptualised as the creative industries.

Simple as ABC? An examination of Activity Based Costing (ABC) Implementation in Fiji

We review the level of Activity Based Costing (ABC) in Fiji in this article. This exercise is important in furthering our understanding of the use of ABC in Fiji. ABC is a popular costing system and strategic tool for organizations in developed countries (Chenhall and Langfield-Smith 1998). However, little is known about the benefits and challenges of implementing ABC in developing countries. Such an understanding could stimulate discussion on the development or modification of ABC to suit developing country circumstances. The article also has a practical objective in informing and education organizations in Fiji regarding the benefits and challenges of ABC implementation in Fiji and also provides practical suggestions for improving and easing the implementation of ABC.

Extending the ABC's public service remit through ABC Pool

Historically, the public service broadcaster (PSB) acted beyond its institutional broadcasting remit by initiating and facilitating activities to support cultural infrastructure and national identity (Wilson, Hutchinson and Shea 2010). The recent focus to develop new content delivery platforms and services (Debrett 2010) signifies a semantic shift from the PSB to the public service media (PSM) organisation. The Australian PSM organisation, the Australian Broadcasting Corporation (ABC) has moved beyond the era of ‘online’ publishing to incorporate Web 2.0 technologies to foster new relationships with the audience (Walker 2009) and engage in production activities with participatory cultures (Jenkins 2006). This shift presents opportunities and challenges to traditional media production, the existing editorial policies and governance models, and raises questions around the value of PSM experimental and innovative activities. Further, the incorporation of information communication technologies and participatory cultures challenge the core values of ‘public service’ within PSM. This paper examines ABC Pool (abc.net.au/pool) as a means of extending the ABC’s public service remit by incorporating participatory cultures into the production and governance models of the corporation and critically analyses the public value of such innovative experiments.

Importing Wikipedia principles into the cultural institution of a public service broadcaster : the case of ABC Pool

Web 2.0 technologies have mobilised collaborative peer production and participatory cultures for online content creation. However, not all online communities engaging in these activities are independently facilitated and often operate within the auspices of the cultural institutions that develop and resource them. Borrowing from the principles of Wikipedia that supports collaborative online content creation and online community, ABC Pool (abc.net.au/pool) is one such institutional online community operating with the support of the Australian Public Service Broadcaster (PSB), the Australian Broadcasting Corporation (ABC). This paper explores the collaborative, creative, and governance activities of an institutional online community and how the role of the community manager is an intermediary within these arrangements.

The cultural impact of institutional remix : the formalisation of textual reappropriation within the ABC

In 2009, Mark Deuze proposed an updated approach to media studies to incorporate ‘media life’, a concept he suggests addresses the invisibleness of ubiquitous media. Media life provides a useful lens for researchers to understand the human condition in media and not with media. At a similar time, public service media (PSM) strategies have aligned audience participation with the so‐called Reithian trinity which suggest the PSB should inform, educate and entertain while performing its core values of public service broadcasting (Enli 2008). Remix within the PSM institution relies on audience participation, employing ‘the people formerly known as the audience’ (Rosen 2006) as cultural artifact producers, and draws on their experience from within the media. Remix as a practice then enables us to examine the shift of the core PSM values by understanding how audience participation, informed by a human condition mobilised from our existence of being in media and not merely with media. However, remix within PSM challenges the once elitist construction of meaning models with an egalitarian approach towards socially reappropriated texts, questioning its affect on the cultural landscape. This paper draws on three years of ethnographic data from within the Australian Broadcasting Corporation (ABC), exploring the remix culture of ABC Pool. ABC Pool operates under a Creative Commons licensing regime to enable remix practice under the auspices of the ABC. ABC Pool users provide a useful group of remix practitioners to examine as they had access to a vast ABC archival collection and were invited to remix those cultural artefacts, often adding cultural and fiscal value. This paper maintains a focus on the audience participation within PSM through remix culture by applying media dependency theory to remix as cultural practice and calls to expand and update the societal representation within the ABC.

Communication models of institutional online communities : the role of the ABC cultural intermediary

The co-creation of cultural artefacts has been democratised given the recent technological affordances of information and communication technologies. Web 2.0 technologies have enabled greater possibilities of citizen inclusion within the media conversations of their nations. For example, the Australian audience has more opportunities to collaboratively produce and tell their story to a broader audience via the public service media (PSM) facilitated platforms of the Australian Broadcasting Corporation (ABC). However, providing open collaborative production for the audience gives rise to the problem, how might the PSM manage the interests of all the stakeholders and align those interests with its legislated Charter? This paper considers this problem through the ABC’s user-created content participatory platform, ABC Pool and highlights the cultural intermediary as the role responsible for managing these tensions. This paper also suggests cultural intermediation is a useful framework for other media organisations engaging in co-creative activities with their audiences.

The role of the cultural intermediary within professional participatory communication : lessons from ABC Pool

As media institutions are encouraged to explore new production methodologies in the current economic crisis, they align with Schumpeter’s creative destruction provocation by exhibiting user-led political, organisation and socio-technical innovations. This paper highlights the significance of the cultural intermediary within the innovative, co-creative production arrangements for cultural artefacts by media professionals in institutional online communities. An institutional online community is defined as one that is housed, resourced and governed by commercial or non- commercial institutions and is not independently facilitated. Web 2.0 technologies have mobilised collaborative peer production activities for online content creation and professional media institutions face challenges in engaging participatory audiences in practices that are beneficial for all concerned stakeholders. The interests of those stakeholders often do not align, highlighting the need for an intermediary role that understands and translates the norms, rhetoric tropes and day-to-day activities between the individuals engaging in participatory communication activities for successful negotiation within the production process. This paper specifically explores the participatory relationship between the public service broadcaster (PSB), the Australian Broadcasting Corporation (ABC) and one of its online communities, ABC Pool (www.abc.net.au/pool). ABC Pool is an online platform developed and resourced by the ABC to encourage co-creation between audience members engaging in the production of user-generated content (UGC) and the professional producers housed within the ABC Radio Division. This empirical research emerges from a three-year research project where I employed an ethnographic action research methodology and was embedded at the ABC as the community manager of ABC Pool. In participatory communication environments, users favour meritocratic heterarchical governance over traditional institutional hierarchical systems (Malaby 2009). A reputation environment based on meritocracy requires an intermediary to identify the stakeholders, understand their interests and communicate effectively between them to negotiate successful production outcomes (Bruns 2008; Banks 2009). The community manager generally occupies this role, however it has emerged that other institutional production environments also employ an intermediary role under alternative monikers(Hutchinson 2012). A useful umbrella term to encompass the myriad of roles within this space is the cultural intermediary. The ABC has experimented with three institutional online community governance models that engage in cultural intermediation in differing decentralised capacities. The first and most closed is a single point of contact model where one cultural intermediary controls all of the communication of the participatory project. The second is a model of multiple cultural intermediaries engaging in communication between the institutional online community stakeholders simultaneously. The third is most open yet problematic as it promotes and empowers community participants to the level of cultural intermediaries. This paper uses the ABC Pool case study to highlight the differing levels of openness within cultural intermediation during the co-creative production process of a cultural artifact.

Communication models of institutional online communities : the role of the ABC cultural intermediary

The co-creation of cultural artefacts has been democratised given the recent technological affordances of information and communication technologies. Web 2.0 technologies have enabled greater possibilities of citizen inclusion within the media conversations of their nations. For example, the Australian audience has more opportunities to collaboratively produce and tell their story to a broader audience via the public service media (PSM) facilitated platforms of the Australian Broadcasting Corporation (ABC). However, providing open collaborative production for the audience gives rise to the problem, how might the PSM manage the interests of all the stakeholders and align those interests with its legislated Charter? This paper considers this problem through the ABC’s user-created content participatory platform, ABC Pool and highlights the cultural intermediary as the role responsible for managing these tensions. This paper also suggests cultural intermediation is a useful framework for other media organisations engaging in co-creative activities with their audiences.

Collaboration, connections and consequences : a study of cultural intermediation within the ABC Pool institutional online community

This thesis addresses the question of what it means to be a public broadcaster in the context of a rapidly changing media landscape, in which audiences no longer only watch and consume but now also make and share media content. Through a close investigation of the ABC Pool community, this thesis documents how the different interests of the stakeholders within an institutional online community intersect and how those interests are negotiated within the Australian Broadcasting Corporation. It demonstrates a new approach towards the cultural intermediation of user-created content within institutional online communities. The research moves beyond the exploration of the community manager role as one type of intermediary to demonstrate the activities of multiple cultural intermediaries that engage in collaborative peer production. Cultural intermediation provides the basis for institutional online community governance.