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

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

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

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

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

RtkS (SMDB11_2269) is a post-translational activator of T6SS activity.

<p>(A) T6SS-dependent Hcp secretion as measured by immunoblot detection of Hcp in cellular and secreted fractions of wild type (WT) or mutant (Δ<i>tssE</i>, Δ<i>ppkA</i>, Δ<i>pppA</i>, Δ<i>rtkS</i>, Δ<i>ppkA</i>Δ<i>rtkS</i> and Δ<i>pppA</i>Δ<i>rtkS</i>) strains of <i>S</i>. <i>marcescens</i> Db10. (B,D) T6SS-dependent anti-bacterial activity as determined by recovery of target organisms <i>P</i>. <i>fluorescens</i> or <i>S</i>. <i>marcescens</i> ATCC274 following co-culture with strains of <i>S</i>. <i>marcescens</i> Db10 (mutants as above, together with Δ<i>tagF</i>Δ<i>rtkS</i>, Δ<i>ppkA</i>Δ<i>tagF</i> and Δ<i>ppkA</i>Δ<i>tagF</i>Δ<i>rtkS</i>, as indicated). Individual data points are overlaid with mean +/- SEM. (C) Representative images of wild type (WT) and Δ<i>rtkS</i> mutant strains of <i>S</i>. <i>marcescens</i> Db10 expressing TssB-GFP and Fha-mCherry fluorescent fusions. In the lower three panels these strains are further carrying the vector control plasmid (+VC, pSUPROM) or a plasmid directing the expression of RtkS <i>in trans</i> (+ RtkS, pSC590). Panels show individual fluorescence channels (TssB-GFP, Fha-mCherry) and an overlay of the fluorescence channels and the DIC channel (Merge; GFP signal false-coloured green and mCherry red). Scale bar, 5 μm.</p

Phosphorylation of Fha and formation of Fha foci is no longer required for T6SS activity in the absence of the post-translational regulator TagF.

<p>(A) Schematic depiction of the T6SS gene cluster of <i>S</i>. <i>marcescens</i> Db10 showing the genes encoding components of the T6SS post-translational regulatory system in this organism (in red). Core T6SS components are shown in blue, with letters indicating TssA-M, and effector/immunity pairs are shown in purple/pink. (B) T6SS-dependent Hcp secretion as measured by immunoblot detection of Hcp in cellular and secreted fractions of wild type (WT) or mutant (Δ<i>tssE</i>, Δ<i>ppkA</i>, Δ<i>pppA</i>, Δ<i>fha</i>, Δ<i>tagF</i>, Δ<i>ppkA</i>Δ<i>tagF</i>, Δ<i>pppA</i>Δ<i>tagF</i> and Δ<i>fha</i>Δ<i>tagF</i>) strains of <i>S</i>. <i>marcescens</i> Db10. (C) T6SS-dependent anti-bacterial activity as determined by recovery of target organism <i>P</i>. <i>fluorescens</i> following co-culture with wild type or mutant strains of <i>S</i>. <i>marcescens</i> Db10. Individual data points are overlaid with mean +/- SEM (n = 4). (D) Representative images of wild type and mutant strains of <i>S</i>. <i>marcescens</i> Db10 expressing TssB-GFP and Fha-mCherry fluorescent fusions. Panels show individual fluorescence channels (TssB-GFP, Fha-mCherry) and an overlay of the fluorescence channels and the DIC channel (Merge; GFP signal false-coloured green and mCherry red). Additionally, TssB-GFP was monitored in the Δ<i>fha</i>Δ<i>tagF</i> background. The mCherry channel image in this case illustrates the low level but frequently observed background signal unrelated to mCherry expression. Scale bar, 5 μm.</p

RtkS interacts with the periplasmic domain of the kinase PpkA, promoting its stability and consequent phosphorylation of Fha.

<p>(A) Bacterial two-hybrid assay to detect interactions between the periplasmic domain of PpkA (PpkA^P; amino acids 363–482) and mature RtkS (RtkS; amino acids 20–328), each fused with T18 or T25 (PpkA^P-T18, pSC593; T25-PpkA^P, pSC594; RtkS-T18, pSC591; T25-RtkS, pSC592). Negative controls were provided by the empty vectors, pUT18 and pT25 (-), and a positive control by the self-interaction of TssK (+ve; TssK-T18, pSC048, and T25-TssK, pSC053). Shown is the β-galactosidase activity, expressed as Δ405/min/ml/OD₆₀₀, of the reporter strain transformed with the combinations of plasmids indicated. Bars show mean +/- SEM (n = 3 independent transformations). (B) Co-purification of RtkS and PpkA under native conditions. Total membrane fractions of wild type <i>S</i>. <i>marcescens</i> Db10 (no tag) or strains expressing PpkA with a C-terminal HA tag (PpkA-HA), RtkS with a C-terminal His₆ tag (RtkS-His), or both, from the normal chromosomal location were subjected to anti-HA immunoprecipitation (top panels) or His affinity purification using Ni²⁺-NTA (bottom panels). Bound proteins in each case were separated by SDS-PAGE and subjected to anti-HA immunoblot (to detect PpkA-HA) or anti-His₆ immunoblot (to detect RtkS-His), as indicated. (C) Levels of PpkA in the presence or absence of RtkS were determined by immunoblot detection of PpkA-HA in an otherwise wild type background (PpkA-HA) or in the Δ<i>rtkS</i> mutant (PpkA-HA, Δ<i>rtkS</i>), and in strains carrying the vector control plasmid (+VC, pSUPROM) or a plasmid directing the expression of RtkS <i>in trans</i> (+ RtkS, pSC590). PpkA-HA levels were measured in total protein samples, with EFTu (bottom panel) representing a loading control and 0.5x the amount of total protein loaded in the right hand most lane compared with the other samples, as indicated. (D) Quantification of Fha phosphorylation by detemination of the levels of Fha phosphopeptide in otherwise wild type (WT) and mutant (Δ<i>ppkA</i>, Δ<i>pppA</i>, Δ<i>rtkS</i>) strains of <i>S</i>. <i>marcescens</i> Db10 expressing Fha-HA from the normal chromosomal location. Levels of phosphopeptide were determined by label-free mass spectrometry and individual data points are shown, with the mean indicated by a line. Levels of phosphopeptide were quantified in 3/3 replicates for WT and Δ<i>pppA</i>, and in 2/3 replicates for Δ<i>rtkS</i>. The phosphopeptide was not detected in the Δ<i>ppkA</i> mutant.</p

Phosphorylation of Fha promotes formation of Fha and TssB foci in <i>S</i>. <i>marcescens</i> Db10.

<p>(A) T6SS-dependent secretion of Hcp by wild type (WT) <i>S</i>. <i>marcescens</i> Db10 or mutants lacking the kinase PpkA (Δ<i>ppkA</i>) or the phosphatase PppA (Δ<i>pppA</i>), and by derivatives expressing fusions of GFP to the C-terminus of TssB (TssB-GFP) and mCherry to the C-terminus of Fha (Fha-mCherry), in an otherwise wild type, Δ<i>ppkA</i> or Δ<i>pppA</i> mutant background. The T6SS inactive mutant Δ<i>tssE</i> is a negative control and cellular and secreted fractions were subjected to immunoblotting using anti-Hcp antisera. (B) T6SS-dependent anti-bacterial activity of fluorescent reporter strains against a <i>P</i>. <i>fluorescens</i> target. Number of recovered target cells following 4 h co-culture with wild type or mutant strains of <i>S</i>. <i>marcescens</i> Db10 as indicated. Individual data points are overlaid with mean +/- SEM (n = 4). (C) Representative images of cells expressing TssB-GFP and Fha-mCherry fusions, in a wild type, Δ<i>ppkA</i> or Δ<i>pppA</i> mutant background. From left to right, panels show individual fluorescence channels (TssB-GFP, Fha-mCherry), an overlay of the fluorescent channels (Merge; GFP signal false-coloured green and mCherry red), and the corresponding DIC image. Inset panels show a zoomed-in view of the region represented by the white box; for the Δ<i>ppkA</i> mutant, the contrast has been increased in the inset panels to reveal non-focal TssB-GFP fluorescence. Scale bars, 5 μm. (D) Fluorescent signal intensity measurement of the segmented TssB-GFP signal in wild type, Δ<i>ppkA</i>, Δ<i>pppA</i>, Δ<i>tagF</i> and Δ<i>ppkA</i>Δ<i>tagF</i> mutants (au, arbitrary fluorescence units). The boundaries of the boxes represent the 25 and 75 percentiles and the horizontal lines are the median values of the analysed population. The whisker bars represent standard error (10 and 90 percentiles). Regions of interest from at least 32 fields of view were analysed in each case. Relative frequency of TssB-GFP foci (expressed per cell at a single timepoint) were estimated by combining automated focus detection with manual cell counting in a representative 15 of these images.</p

TagF is a negative regulator whose overexpression inhibits T6SS activity.

<p>(A) T6SS-dependent Hcp secretion as measured by immunoblot detection of Hcp in cellular and secreted fractions of wild type (WT) or mutant (Δ<i>tssE</i>, Δ<i>tagF</i>, Δ<i>ppkA</i>Δ<i>tagF</i>, Δ<i>pppA</i>Δ<i>tagF</i> and Δ<i>fha</i>Δ<i>tagF</i>) strains of <i>S</i>. <i>marcescens</i> Db10, carrying either the vector control plasmid (+VC, pSUPROM) or a plasmid directing the expression of <i>tagF in trans</i> (+TagF, pSC701). (B) T6SS-dependent anti-bacterial activity as determined by recovery of target organism <i>P</i>. <i>fluorescens</i> following co-culture with wild type or mutant strains of <i>S</i>. <i>marcescens</i> Db10, carrying vector control or <i>tagF</i> expression plasmid. Individual data points are overlaid with mean +/- SEM (n = 4). (C) Representative images of wild type and Δ<i>pppA</i> mutant strains of <i>S</i>. <i>marcescens</i> Db10 expressing TssB-GFP and Fha-mCherry fluorescent fusions and carrying either vector control or <i>tagF</i> expression plasmid. Panels show individual fluorescence channels (TssB-GFP, Fha-mCherry) and an overlay of the fluorescence channels and the DIC channel (Merge; GFP signal false-coloured green and mCherry red). Scale bar, 5 μm.</p