Architecture of low human and environmental impact.

Diante de um cenário de degradação ambiental global, escassez de matéria prima, água, energia, aumento da poluição, crise social e econômica, é feita a proposição de uma Arquitetura de Baixo Impacto Humano e Ambiental - ABIHA.Todas estas variáveis trazem novos elementos à Arquitetura aumentando a sua complexidade e fazendo com que haja necessidade de adaptação.Apesar de todas as incertezas e contradições envolvendo as questões de sustentabilidade, seus conceitos e sua aplicação na Arquitetura são abordados neste contexto.Além das questões conceituais, são apresentadas aplicações práticas da ABIHA, no Jardim Sustentável, na Reciclagem do Galpão da POLI e no MINI labiratório de Conforto e Eficiência Energética.Estas aplicações seriram de base para a proposta de Sistematização que é apresentada no final deste trabalho como parte das conclusões finais. Esta proposta é apenas início de muitas pesquisas que ainda devem ser feiras rumo a uma Arquitetura mais Sustentável.The actual global environmental context is one of fierce degradation : reflecting in the exhaustion of natural sources, increase of air, soil and water pollution, and social and economic crises.Facing such a scenario, it is proposed in this work, principles of architecture of low human environmental impact (arquitetura de baixo impacto humano e ambiental) - ABIHA.All these variables bring new parameters to the design of building, including their entire life cycles.Under these new ciecunstances it is observed an increae of the complexity in this design process, making necessary discussions for change.Despite all the uncertainties and contradictions about issues of sustainability, their concepts, as well as their applications are approached in the context of this work.Besides the conceptual matters, practions applications of ABIHA are carried out, in the example of the sustainable garden, warehouse recycling and the movable environmental laboratory (sensors and data lggers).Such experiences of practical application were fundamental to create the basis for the methodological assessment proposed as part of the final conclusions of this research.However, this procedure of evaluation is understood merely as the beginning of a big range of other researchs, which shlould be developed towards a more sustainable architecture

MOESM7 of Activation peptide of the coagulation factor XIII (AP-F13A1) as a new biomarker for the screening of colorectal cancer

Additional file 7. Table summary of patients selected for the absolute quantification of the two isoforms of tAP-F13A1 by LC-PRM

MOESM3 of Activation peptide of the coagulation factor XIII (AP-F13A1) as a new biomarker for the screening of colorectal cancer

Additional file 3. MS/MS Spectra from the Mascot DAT file imported in Skyline software as MS/MS library

MOESM4 of Activation peptide of the coagulation factor XIII (AP-F13A1) as a new biomarker for the screening of colorectal cancer

Additional file 4. MS/MS Filtering–transition setting tabs in Skyline software

Molecular characterization of Mdm2-dependant ubiquitination of NUB1.

<p>(A) HEK-293T cells were transfected as indicated with Myc-NUB1, Mdm2 together with wild type (WT) or lysine-less mutant (K0) 6HF-Ubiquitin expressing constructs. Twenty-four hours post-transfection, 80% of cells were lysed in Guanidine-HCl containing buffer and 6HF-Ubiquitinated conjugates were isolated on Ni2+-NTA agarose beads. The remaining 20% were lysed in non-denaturing buffer to detect non-modified NUB1. Purified 6HF-Ubiquitinated proteins and cell extracts were resolved by SDS-PAGE and NUB1 was revealed by western blotting using an anti-NUB1 antibody. (B) HEK-293T cells were transfected with Myc-NUB1 and Mdm2, together with wild type (WT), K48R or K11R mutants 6HF-Ubiquitin. Ubiquitination of NUB1 was analyzed as in (A). (C) HEK-293T cells were transfected in 10-cm dishes with Myc-NUB1 alone or together with Mdm2. Twenty-four hours post-transfection, cells were split into six-well plates and allowed to rest overnight. Cells were then treated by supplementation with 100 μg/ml of cycloheximide (CHX) in their culture medium for the indicated time lapses. NUB1 stability was monitored by western blotting using the anti-Myc 9E10 antibody.</p

MOESM5 of Activation peptide of the coagulation factor XIII (AP-F13A1) as a new biomarker for the screening of colorectal cancer

Additional file 5. Representative mass spectra of the AP-F13A1 activation peptide

Mdm2 interacts with NUB1.

<p>(A) Lysates from HEK-293T cells expressing Myc-NUB1 alone or together with Mdm2 were subjected to immunoprecipitation with an anti-Mdm2 antibody. Proteins were separated through SDS-PAGE and NUB1 was detected by western blotting using an anti-Myc antibody (9E10). Amount of precipitated Mdm2 and expression levels of both proteins in cell extracts were controlled. (B) Lysates from HEK-293T cells expressing Mdm2 alone or together with Myc-NUB1 were subjected to immunoprecipitation with the anti-Myc 9E10 antibody. Immunoprecipitates and cell extracts were analyzed by western blotting using an anti-Mdm2 and anti-Myc antibodies. * Shifted NUB1.</p

Mdm2 triggers the ubiquitination of NUB1.

<p>(A) HEK-293T cells were transfected with Myc-NUB1 alone or together with Mdm2, and twenty-four hours later lysates were prepared using an hypotonic / Triton X-100 buffer supplemented or not with N-Ethylmaleimide (NEM). Proteins were separated through SDS-PAGE and NUB1 was detected by western blotting using the anti-Myc antibody. (B) HEK-293T cells were transfected with Myc-NUB1 alone or together with wild type (WT) or catalytically inactive (C462A) Mdm2. Lysates were prepared and proteins were separated through SDS-PAGE and analyzed by western blotting with the appropriate antibodies.</p

Mdm2-mediated ubiquitination controls NUB1 activity and is not a proteolytic signal.

<p>(A) HEK-293T cells were transfected with Myc-NUB1 and 6HF-Nedd8, alone or together. Twenty-four hours post-transfection, cells were treated with 30 μM of MG132 for 4 hours where indicated or with DMSO (vehicle) as a control. 6HF-neddylated proteins were revealed by western blotting using the anti-Flag M2 antibody. (B) HEK-293T cells were transfected with 6HF-Nedd8 and Mdm2, alone or together with WT or K159R Myc-NUB1. Twenty-four hours post-transfection, proteins were separated through SDS-PAGE and 6HF-neddylated proteins were revealed by western blotting using the anti-Flag M2 antibody. (C) HEK-293T cells were transfected with GFP-HHT97 in combination with Myc-NUB1 WT or K159R. 24 hours after transfection cells were dispatched in 6-wells plates and allowed to adhere for 12 more hours. Cells were then treated with 20 μg/ml of Cycloheximide and harvested at indicated time points. Amount of remaining HTT97 was evaluated by western blot of cell lysates (left panel). Densitometry analysis of three independent experiments have been performed and used to establish the half-life of HTT97 and to evaluate the impact of NUB1 WT or K159R expression (right panel).</p

NUB1 is ubiquitinated by Mdm2 on lysine 159.

<p>(A) Workflow of the protocol used to identify NUB1 ubiquitinated lysine. (B) METTRE SPECTRES (C) HEK-293T cells were transfected with wild type (WT), K134R, K159R or K134,159R (2KR) mutants Myc-NUB1 together with 6HF-Ubiquitin and Mdm2 constructs. 6HF-Ubiquitinated proteins were isolated by Ni2+ pull down and NUB1 was revealed by western blotting using an anti-NUB1 antibody. (D) Lysates from HEK-293T cells expressing Mdm2 alone or together with wild type (WT), K134R or K159R mutants Myc-NUB1 were subjected to immunoprecipitation with the anti-Myc 9E10 antibody. Immunoprecipitates and input samples were analyzed by western blotting using an anti-Mdm2 antibody.</p