11 research outputs found

    Machining of Aluminum Alloy

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    Import 23/07/2015Diplomová práce se zabývá kvalitou opracované hliníkové slitiny EN AW-6082, speciálně drsností povrchu a měřením a vyhodnocením složek řezných sil. Teoretická část objasňuje základní pojmy věnované čelnímu frézování, obrobitelnosti hliníku, obráběným materiálům, řezným podmínkám a geometrii obrábění. V návrhu experimentální části práce je popsáno použití stroje, nástroje a vyměnitelných břitových destiček, přístrojů na měření drsnosti, velikosti řezných sil a navržené řezné podmínky. V experimentální části práce jsou změřeny drsnosti povrchu a presentovány výsledky naměřených hodnot drsnosti Ra a Rz. Řezné síly byly měřeny na piezoelektrickém dynamometru.This master thesis is concerned with the quality of machined aluminium alloy EN AW-6082, especially surface roughness and the measurement and evaluation components of the cutting forces. The theoretical part explains the basic concepts of frontal milling, machinability aluminium, machined material, cutting conditions and geometry processing. In the proposal of the experimental part is described the using of machine, tool and indexable inserts, devices for measuring roughness, cutting forces and proposed cutting conditions. In the experimental part of the work are measured surface roughnesses and presented the results of the measured values of roughness Ra and Rz. Cutting forces were measured on the piezoelectric dynamometer.346 - Katedra obrábění, montáže a strojírenské metrologievelmi dobř

    A Flower-like Brain Targeted Selenium Nanocluster Lowers the Chlorogenic Acid Dose for Ameliorating Cognitive Impairment in APP/PS1 Mice

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    Aβ aggregation-related neuroinflammation and imbalance of brain glucose homeostasis play important roles in the pathological process of Alzheimer’s disease (AD). Chlorogenic acid (CGA) is one of the most common dietary polyphenols with neuroprotective effects. However, due to the low bioavailability of CGA, its application dose is usually high in vivo. In our previous study, the spherical selenium nanoparticles act as drug carriers to improve the bioactivity of resveratrol. Here, the brain-targeting peptide (TGN peptide) and CGA were used to prepare a new flowerlike selenium nanocluster (TGN-CGA@SeNCs) for enhancing the bioavailability of CGA. After decoration on selenium nanoclusters, the solubility and stability of CGA was obviously increased. Oral administration of a low dose of CGA (80 mg/kg/body weight) only slightly inhibited Aβ aggregate-related neuroinflammation and glucose homeostasis disorder in the brain. Moreover, CGA showed less effect on increasing the diversity and richness of gut microbiota. At the same concentration, the CGA-modified selenium nanocluster (CGA@SeNCs) and TGN-CGA@SeNCs showed better function in ameliorating the gut microbiota disorder. Especially, TGN-CGA@SeNCs significantly increased the relative abundance of Turicibacter, Colidextribacter, Ruminococcus, Alloprevotella, and Alistipes against oxidative stress, inflammation, and glucose homeostasis imbalance. Notably, only TGN-CGA@SeNCs can transport through the blood–brain barrier (BBB), and TGN-CGA@SeNCs showed better effects than CGA@SeNCs in regulating Aβ aggregation and improving brain glucose homeostasis. These results broadened the application of TGN-CGA@SeNCs, effectively improving the bioactivity of CGA, which also lowers the CGA dose for preventing AD progression

    Adipose Stromal Cells Amplify Angiogenic Signaling <em>via</em> the VEGF/mTOR/Akt Pathway in a Murine Hindlimb Ischemia Model: A <em>3D</em> Multimodality Imaging Study

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    <div><p>Although adipose-derived stromal cell (ADSC) transplantation has been demonstrated as a promising therapeutic strategy for peripheral arterial disease (PAD), the mechanism of action behind the observed therapeutic efficacy of ADSCs remains unclear. This study was designed to investigate the long-term outcome and therapeutic behavior of engrafted ADSCs in a murine hindlimb ischemia model using multimodality molecular imaging approaches. ADSCs (1.0×10<sup>7</sup>) were isolated from Tg(<em>Fluc</em>-<em>egfp</em>) mice which constitutively express dual-reporter firefly luciferase and enhanced green fluorescent protein (Fluc<sup>+</sup>-eGFP<sup>+</sup>, mADSCs<sup>Fluc+GFP+</sup>), then intramuscularly injected into the hindlimb of BALB/c-nu mice after unilateral femoral artery ligation and excision. Abbreviated survival (∼5 weeks) of post-transplant mADSCs within the ischemic hindlimb was longitudinally monitored using noninvasive bioluminescence imaging (BLI), fluorescence imaging (FRI), and bioluminescence tomography with micro-computed tomography (BLT/micro-CT). Use of the BLT/micro-CT system enabled quantitative 3-dimensional (<em>3D</em>) imaging of the cells’ distribution and kinetics <em>in vivo</em>. Engrafted mADSCs improved blood perfusion recovery, ambulatory performance and prognosis of the ischemic hindlimb, probably by inducing angiogenesis and formation of collateral vessels, which could be visualized using laser Doppler perfusion imaging (LDPI), micro-CT angiography, vascular-cast imaging, and immunofluorescence. mADSCs augmented activation of the pro-angiogenic VEGF/mTOR/Akt pathway <em>in vivo</em>, even though the cells failed to incorporate into the host microvasculature as functional components. Downregulation of VEGF/mTOR/Akt signaling using small molecule inhibitors counteracted mADSC-induced angiogenesis and perfusion restoration. This study demonstrates for the first time the spatiotemporal kinetics and functional survival of transplanted mADSCs in a PAD model using <em>in vivo 3D</em> multimodality imaging. Our study indicates that mADSCs potentiate pro-angiogenic signal amplification <em>via</em> a VEGF/mTOR/Akt-dependent pathway, and thereby promote recovery from hindlimb ischemia.</p> </div

    Immunofluorescence assessment of mADSC engraftment and angiogenesis.

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    <p>Confocal microscopy of ischemic gastrocnemius muscle tissue sections triply stained with GFP (green), endothelial marker CD31 (PECAM-1, red) and 4′,6-diamidino-2-phenylindole (DAPI, blue) visualized the para-vascular survival of mADSCs<sup>Fluc+GFP+</sup> (<b>a</b> and <b>b</b>). Quantitative analysis of CD31<sup>+</sup> blood vessels (<b>c</b>) and GFP<sup>+</sup> cells (<b>d</b>) within the same-sized regions. n = 20 random fields. Scale bars represent 50 µm. Error bars: mean±SD. *<i>P</i><0.001 <i>vs.</i> Control, #<i>P</i><0.001 <i>vs.</i> Day 1 within ADSC group.</p

    Blood perfusion imaging and function assessment of ischemic hindlimb.

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    <p>Laser Doppler perfusion imaging (LDPI) visualized dynamic changes in hindlimb perfusion (<b>a</b>). Colored scale bar represents blood flow velocity in LDPI index. Blood perfusion was quantified using perfusion ratio (PR), i.e., the ratio of average LDPI index of ischemic (red arrows) to nonischemic hindlimb (<b>b</b>). Representative photos show hindlimb autoamputation or salvage (yellow arrows) among groups on day 21 (<b>c</b>). Significantly lower autoamputation percentage (<b>d</b>), scores of ischemic damage (<b>e</b>) and ambulatory impairment (<b>f</b>) were observed in the ADSC group compared to the control group. n = 20 for each. Error bars: mean±SD. *<i>P</i><0.001 <i>vs.</i> Control, #<i>P</i><0.05 <i>vs.</i> Control, †<i>P</i><0.001 <i>vs.</i> Control.</p

    mADSCs<sup>Fluc+GFP+</sup> quantification by reporter gene imaging.

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    <p>Representative bioluminescence/fluorescence images of mADSCs<sup>Fluc+GFP+</sup><i>in vitro</i> (<b>a</b>). Linear correlation of cell quantities with optical signal of firefly luciferase (Fluc) and eGFP is displayed, with correlation coefficient <i>r</i><sup>2</sup> values and linear functions (<b>b</b>). The Fluc enzymatic activity of mADSC<sup>Fluc+GFP+</sup> was stable in 8 passages (<b>c</b>). BLI (<b>d</b>) and quantification (<b>e</b>) of mADSCs<sup>Fluc+GFP+</sup> over osteogenesis induction or normal culture (control) for 14 days. Colored scale bars represent Fluc bioluminescence intensity in photons/second/cm<sup>2</sup>/steridian (P·s<sup>−1</sup>·cm<sup>−2</sup>·sr<sup>−1</sup>), and eGFP fluorescence intensity in efficiency. n = 20 for each. Error bars: mean±SD.</p

    mADSCs<sup>Fluc+GFP+</sup> characterization.

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    <p>Fibroblastoid morphology (<b>a</b>) and enhanced green fluorescent protein (eGFP) expression (<b>b</b>) of Fluc<sup>+</sup>−GFP<sup>+</sup> murine adipose-derived stromal cells (mADSCs<sup>Fluc+GFP+</sup>, 3<sup>rd</sup> passage). Multipotent differentiation of mADSC<sup>Fluc+GFP+</sup> is shown in <b>c–f</b>. Osteogenesis was demonstrated using alizarin red-S (ARS, <b>c</b>) or alkaline phosphatase (ALP, <b>d</b>) staining. Adipogenesis and chondrogenesis were detected using oil red-O staining (<b>e</b>) and collagen-II immunohistochemistry staining (<b>f</b>) respectively. Flow cytometry of mADSCs labeled with phycoerythrin(PE)/allophycocyanin(APC)-conjugated cell markers or isotype IgG controls for immunophenotype identification (<b>g</b>). Scale bar represents 100 µm (a,b,c,d), 15 µm (e,f).</p

    Longitudinal tracking of transplanted mADSCs<sup>Fluc+GFP+</sup> using multimodality imaging <i>in vivo</i>.

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    <p><i>In vivo</i> BLI/FRI/BLT/micro-CT tracked mADSC<sup>Fluc+GFP+</sup> survival in the ischemic hindlimb of the same representative animal. Progressive decay of bioluminescence (<b>a</b> upper and <b>b</b>) and fluorescence signal intensity (<b>a</b> lower and <b>c</b>) over time is indicative of donor mADSCs<sup>Fluc+GFP+</sup> death. Tomography and reconstruction of bioluminescence signals were performed by our BLT/micro-CT platform to show the <i>in vivo</i> kinetics of mADSCs<sup>Fluc+GFP+</sup> (red mass) in detailed <i>3D</i> resolution (<b>d</b>). Reconstruction of bioluminescence total power (nanoWatts) also displayed a downward trend (<b>e</b>), confirmed by <i>ex vivo</i> Fluc assays (<b>f</b>). n = 20 for each. Error bars: mean±SD. *<i>P</i><0.01 <i>vs.</i> day 0, #<i>P</i><0.001 <i>vs.</i> day 0, †<i>P</i><0.05 <i>vs.</i> day 0, **<i>P</i><0.001 <i>vs.</i> day 0 (within ADSC group).</p

    Transplanted mADSCs activated VEGF/mTOR/Akt pathway <i>in vivo</i>.

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    <p>Levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF) and stromal cell derived factor-1α (SDF-1α) in PBS or ADSC-treated ischemic hindlimbs on day 0 (baseline), 3 and 7 using ELISA (<b>a</b>). Western blot analysis (<b>b</b>) of VEGFR2(Tyr951)/VEGFR2 (<b>c</b>), mTOR(Ser2448)/mTOR (<b>d</b>), Akt(Ser473)/Akt (<b>e</b>) and β-actin expression within ischemic hindlimbs on day 7. Protein expression was quantified by the integrated optical density (IOD) ratio of each pair. n = 20 for each. LDPI follow-up showed that combined treatment of anti-VEGF monoclonal antibody (mAb) (<b>f</b>), PP242 (<b>g</b>) or triciribine (<b>h</b>) with mADSCs attenuated mADSC-induced blood perfusion restoration, compared with combined treatment of nonspecific IgG or vehicle controls. Error bars: mean±SD. *<i>P</i><0.05 <i>vs.</i> day 0 within Control group, **<i>P</i><0.05 <i>vs.</i> Control, #<i>P</i><0.05 <i>vs.</i> Sham, ##<i>P</i><0.001 <i>vs.</i> Control, §<i>P</i><0.001 <i>vs.</i> ADSC, ‖<i>P</i><0.01 <i>vs.</i> ADSC, ¶<i>P</i><0.01 <i>vs.</i> ADSC, †<i>P</i><0.01 <i>vs.</i> ADSC+anti-VEGF mAb, ††<i>P</i><0.001 <i>vs.</i> ADSC+PP242, ‡<i>P</i><0.05 <i>vs.</i> ADSC+triciribine.</p
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