Fluorescent nanosensors for the monitoring of different intracellular reactive oxygen species (ROS)

Reactive oxygen species (ROS) play important roles for the regulation of normal functions such as proliferation, differentiation, migration and cell death. At low doses they participate in the redox balance, but an excess of these species leads to damage to proteins, lipids or DNA. ROS are involved in the onset and progression of several degenerative diseases (e.g., cancer, neurological disorder, etc). Cancer cells are highly susceptible to ROS-mediated damage and several chemotherapy agents achieve cytotoxicity by inducing oxidative stress. Sensing the variations of different intracellular ROS is crucial for real time assessments of anticancer treatment efficiency. Yet, no sensor currently allows simultaneous and independent monitoring of different ROS live cells. Indeed, existing sensors monitor either the total levels of ROS or the levels of single species (i.e., sensors such as diphenylanthracene, peroxy yellow, anthrafluorescein, etc.). The need to optimise and personalise treatment regimens and for unravelling the mechanisms underpinning ROS-induced cell death requires the introduction of a new set of tools able to provide a real-time report of intracellular ROS levels in response to a given intervention. In this project we developed new fluorescent nanosensors able to respond to different ROS. This was achieved through the synthesis of three conjugatable molecular probes able to respond to individual ROS, namely; an anthrafluorescein-based probe for superoxide anion, a dimethylanthracene-based probe for singlet oxygen and a fluorescein-based probe for hydrogen peroxide. The new probes were grafted onto poly(lactic-co-glycolic acid) (PLGA) and formulated as nanoparticles containing either conjugated or encapsulated sensors. We characterised the fluorescent response of the probes, conjugates and nanospecies in the presence of the target ROS analytes. Lastly, we demonstrated the ability of the nanosensors to enter cells and their potential of to be used as intracellular ROS sensors

Organic optoelectronic devices-flexibility versus performance

International audienceIn this paper, we discuss the effect of flexible substrates on the characteristics of two organic optoelectronic devices, namely P3HT:PCBM-based photovoltaic bulk heterojunctions and pentacene-based phototransistors. In addition, we have developed anode materials deposited by ion beam sputtering, a technique which satisfies the low temperature deposition requirements associated with the use of plastic substrates. The anode materials consisted of indium tin oxide (ITO) and ITO/metal/ITO tri-layers. The use of tri-layer anodes in P3HT:PCBM-based solar cells resulted in an increase in the fill factor and the power conversion efficiency reached a value of 2% with an ITO(70 nm)/Ag(14 nm)/ITO(70 nm) anode deposited on a polyphthalate carbonate substrate. In the case of phototransistors, a photosensitivity of 1.6 × 10 under illumination at 365 nm (with a power intensity of 7 mW/cm) was obtained in the off-state of the transistor. We have fine-tuned the anode structure and deposition/annealing conditions towards flexible organic devices and optimal device characteristics

Determinação do modo de reprodução em Brachiara spp. e Panicum maximum usando microscopia por contraste de interferência em ovários clarificados.

A maioria das plantas superiores se reproduz sexualmente, ou seja, para formação do embrião ocorre a fecundação do óvulo por um gameta masculino. Entretanto, em muitas espécies de gramíneas forrageiras, o embrião se origina por meio de um processo de reprodução assexual via sementes denominado apomixia. Este modo de reprodução possui grande potencial para ser explorado na agricultura, particularmente na produção de sementes híbridas visando à manutenção do vigor híbrido ao longo das gerações. No melhoramento de espécies apomíticas, o cruzamento entre genitores sexuais e apomíticos origina progênies segregando para o modo de reprodução sexual e apomítico. A determinação do modo de reprodução das progênies pode ser realizada por diversas estratégias, uma delas com destaque para as análises citoembriológicas, as quais possibilitam o estudo da morfologia dos ovários das plantas, permitindo separá-las entre sexuais e apomíticas. Apesar de estas análises já serem realizadas em várias espécies, ainda não existe uma publicação que descreva detalhadamente cada etapa e que tenha um acervo fotográfico ilustrando o passo a passo. Assim, neste documento são apresentadas, com detalhes, as análises citoembriológicas por meio de microscopia por contraste por interferência. São relatados todos os procedimentos necessários para a coleta das espiguetas no campo, a fixação destas em fenol ácido acético - FAA, a extração dos ovários e sua clarificação. Posteriormente, é detalhada a contagem das lâminas e a análise dos ovários clarificados, utilizando-se o microscópio com contraste diferencial de interferência (DIC ? differential interference contrast). Por fim, é apresentado como se analisam os resultados do método para se concluir sobre o modo de reprodução de cada planta. Dentro deste contexto, este documento apresenta grande importância para estudantes, professores e pesquisadores que trabalham com sistema reprodutivo de forrageiras.bitstream/item/204549/1/Determinacao-do-modo-de-reproducao-em-Brachiara.pd

Grassroots Agency: Participation and Conflict in Buenos Aires Shantytowns seen through the Pilot Plan for Villa 7 (1971–1975)

open access articleIn 1971, after more than a decade of national and municipal policies aimed at the top-down removal of shantytowns, the Buenos Aires City Council approved the Plan Piloto para la Relocalización de Villa 7 (Pilot Plan for the Relocation of Shantytown 7; 1971–1975, referred to as the Pilot Plan hereinafter). This particular plan, which resulted in the construction of the housing complex, Barrio Justo Suárez, endures in the collective memory of Argentines as a landmark project regarding grassroots participation in state housing initiatives addressed at shantytowns. Emerging from a context of a housing shortage for the growing urban poor and intense popular mobilizations during the transition to democracy, the authors of the Pilot Plan sought to empower shantytown residents in novel ways by: 1) maintaining the shantytown’s location as opposed to eradication schemes that relocated the residents elsewhere, 2) formally employing some of the residents for the stage of construction, as opposed to “self-help” housing projects in which the residents contributed with unpaid labor, and 3) including them in the urban and architectural design of the of the new housing. This paper will examine the context in which the Pilot Plan was conceived of as a way of re-assessing the roles of the state, the user, and housing-related professionals, often seen as antagonistic. The paper argues that residents’ fair participation and state intervention in housing schemes are not necessarily incompatible, and can function in specific social and political contexts through multiactor proposals backed by a political will that prioritizes grassroots agency

Experimental evidence that novel land management interventions inspired by history enhance biodiversity

1. To address biodiversity declines within semi-natural habitats, land-management must cater for diverse taxonomic groups. Integrating our understanding of the ecological requirements of priority (rare, scarce or threatened) species through ‘biodiversity auditing’, with that of the intensity and complexity of historical land-use, encourages novel forms of management. Experimental confirmation is needed to establish whether this enhances biodiversity conservation relative to routine management. 2. Biodiversity auditing and historical land-use of dry-open terrestrial habitats in Breckland (Eastern England) both encourage management incorporating ground-disturbance and spatio-temporal variability. To test biodiversity conservation outcomes, we developed 40 4-ha management complexes over three successive winters, of which 20 were shallow-cultivated (rotovation) and 20 deep-cultivated (ploughing), stratified across 3,850-ha of closed-sward dry grassland and lowland heathland (collectively ‘dry grassland’). Complexes comprised four 1-ha sub-treatments: repeat-cultivation, first-time-cultivation, one-year-old fallow and two-year-old fallow. We examined responses of vascular plants; spiders; true bugs; ground, rove and ‘other’ beetles; bees and wasps; ants; and true flies on treatment complexes and 21 4-ha untreated controls. Sampling gave 132,251 invertebrates from 877 species and 28,846 plant observations from 167 species. 3. Resampling and rarefaction analyses showed shallow- and deep-cultivation both doubled priority species richness (pooling sub-treatments within complexes) compared to controls. Priority spider, ground beetle, other beetle, and true bug richness were greater on both treatments than controls. Responses were strongest for those priority dry-open-habitat associated invertebrates initially predicted (by biodiversity auditing) to benefit from heavy physical-disturbance. 4. Assemblage composition (pooling non-priority and priority species) varied between sub-treatments for plants, ants, true bugs, spiders, ground, rove and other beetles; but only one-year-old fallowed deep-cultivation increased priority richness across multiple taxa. 5. Treatments produced similar biodiversity responses across various dry grassland ‘habitats’ that differed in plant composition, allowing simplified management guidance. 6. Synthesis and applications. Our landscape-scale experiment confirmed the considerable biodiversity value of interventions inspired by history and informed by systematic multi-taxa analysis of ecological requirements across priority biota. Since assemblage composition varied between sub-treatments, providing heterogeneity in management will support the widest suite of species. Crucially, the intended recipients responded most strongly, suggesting biodiversity audits could successfully inform interventions within other systems

Relevance of the Diversity among Members of the Trypanosoma Cruzi Trans-Sialidase Family Analyzed with Camelids Single-Domain Antibodies

The sialic acid present in the protective surface mucin coat of Trypanosoma cruzi is added by a membrane anchored trans-sialidase (TcTS), a modified sialidase that is expressed from a large gene family. In this work, we analyzed single domain camelid antibodies produced against trans-sialidase. Llamas were immunized with a recombinant trans-sialidase and inhibitory single-domain antibody fragments were obtained by phage display selection, taking advantage of a screening strategy using an inhibition test instead of the classic binding assay. Four single domain antibodies displaying strong trans-sialidase inhibition activity against the recombinant enzyme were identified. They share the same complementarity-determining region 3 length (17 residues) and have very similar sequences. This result indicates that they likely derived from a unique clone. Probably there is only one structural solution for tight binding inhibitory antibodies against the TcTS used for immunization. To our surprise, this single domain antibody that inhibits the recombinant TcTS, failed to inhibit the enzymatic activity present in parasite extracts. Analysis of individual recombinant trans-sialidases showed that enzymes expressed from different genes were inhibited to different extents (from 8 to 98%) by the llama antibodies. Amino acid changes at key positions are likely to be responsible for the differences in inhibition found among the recombinant enzymes. These results suggest that the presence of a large and diverse trans-sialidase family might be required to prevent the inhibitory response against this essential enzyme and might thus constitute a novel strategy of T. cruzi to evade the host immune system