Stimulation of vascular cells by extracellular signals - A biophysical analysis

Stimulation of vascular cells by extracellullar signals Treatment of vascular diseases often requires the selective addressing of endothelial (ECs) and smooth muscle cells (SMCs). The two vascular cell types are important for the wound healing after stent implantation. Recent research designs new materials and coatings for stents to improve the complex healing process. The aim of my work was to find and investigate different reactions in the two vascular cell types (ECs and SMCs) through surface chemistry, topography and other stimulating factors like electrical fields or applied external stretching forces. On various iridium-oxide stent coatings ECs seem to be more sensitive to chemical differences than SMCs. On PDMS micro-nano-grooves ECs and SMCs align not significant differently to the structure. Cell assays on nano-structured and bio-functionalized surfaces reveal a universal ligand distance dependency for both cell types. Upon application of uniaxial mechanical stretch or an directed electrical field, ECs and SMCs show significant different responses. General characteristics of the two cell types can be quantitatively described by an automatic controller model. These findings are promising for further studies to improve wound healing after implantation and even more to allow the artificial generation of new blood vessels (angiogenesis)

Disposable MMP-9 sensor based on the degradation of peptide cross-linked hydrogel films using electrochemical impedance spectroscopy

Barts and The London Charity and Queen Mary Innovation Lt

Biomimetic sensors for HbA1c

Diabetes mellitus is a growing health problem worldwide. Suitable long-term control and management of this disease are enabled by determination of glycated haemoglobin (HbA1c) in blood. The results are given as %HbA1c of total haemoglobin. Presently available tests vary in cost and convenience and there is an identified need to introduce improved equipment for self-monitoring. This dissertation focuses on fast and straightforward detection of glycated haemoglobin (HbA1c) using cyclic voltammetry and chronoamperometry. Haemoglobin was determined by monitoring its reaction with potassium ferricyanide on screen printed electrodes at an oxidative potential +500 mV. A working electrode was modified with carbon nanotubes to enhance electron transfer. A calibration curve was linear in a range from 0.83 to 83 mg/mL. Another innovative approach to detecting haemoglobin using its enzymatic activity was also developed. Detection of haemoglobin was performed with hydroquinone and hydrogen peroxide on screen printed electrodes at a potential -400 mV in a Flow Injection Analysis system (FIA). Cont/d.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Long‐lived Snell dwarf mice display increased proteostatic mechanisms that are not dependent on decreased mTORC1 activity

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/111144/1/acel12329.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/111144/2/acel12329-sup-0001-SuppInfo.pd

A Specific Activity-Based Probe to Monitor Family GH59 Galactosylceramidase, the Enzyme Deficient in Krabbe Disease

Galactosylceramidase (GALC) is the lysosomal β-galactosidase responsible for the hydrolysis of galactosylceramide. Inherited deficiency in GALC causes Krabbe disease, a devastating neurological disorder characterized by accumulation of galactosylceramide and its deacylated counterpart, the toxic sphingoid base galactosylsphingosine (psychosine). We report the design and application of a fluorescently tagged activity-based probe (ABP) for the sensitive and specific labeling of active GALC molecules from various species. The probe consists of a β-galactopyranose-configured cyclophellitol-epoxide core, conferring specificity for GALC, equipped with a BODIPY fluorophore at C6 that allows visualization of active enzyme in cells and tissues. Detection of residual GALC in patient fibroblasts holds great promise for laboratory diagnosis of Krabbe disease. We further describe a procedure for in situ imaging of active GALC in murine brain by intra-cerebroventricular infusion of the ABP. In conclusion, this GALC-specific ABP should find broad applications in diagnosis, drug development, and evaluation of therapy for Krabbe disease

Structure and Stability of the Spinach Aquaporin SoPIP2;1 in Detergent Micelles and Lipid Membranes

Background: SoPIP2;1 constitutes one of the major integral proteins in spinach leaf plasma membranes and belongs to the aquaporin family. SoPIP2;1 is a highly permeable and selective water channel that has been successfully overexpressed and purified with high yields. In order to optimize reconstitution of the purified protein into biomimetic systems, we have here for the first time characterized the structural stability of SoPIP2;1. Methodology/Principal Finding: We have characterized the protein structural stability after purification and after reconstitution into detergent micelles and proteoliposomes using circular dichroism and fluorescence spectroscopy techniques. The structure of SoPIP2;1 was analyzed either with the protein solubilized with octyl-beta-D-glucopyranoside (OG) or reconstituted into lipid membranes formed by E. coli lipids, diphytanoylphosphatidylcholine (DPhPC), or reconstituted into lipid membranes formed from mixtures of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPE), 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE), 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS), and ergosterol. Generally, SoPIP2;1 secondary structure was found to be predominantly a-helical in accordance with crystallographic data. The protein has a high thermal structural stability in detergent solutions, with an irreversible thermal unfolding occurring at a melting temperature of 58 degrees C. Incorporation of the protein into lipid membranes increases the structural stability as evidenced by an increased melting temperature of up to 70 degrees C. Conclusion/Significance: The results of this study provide insights into SoPIP2;1 stability in various host membranes and suggest suitable choices of detergent and lipid composition for reconstitution of SoPIP2;1 into biomimetic membranes for biotechnological applications

FIB patterning of stainless steel for the development of nano-structured stent surfaces for cardiovascular applications

Stent implantation is a percutaneous interventional procedure that mitigates vessel stenosis, providing mechanical support within the artery and as such a very valuable tool in the fight against coronary artery disease. However, stenting causes physical damage to the arterial wall. It is well accepted that a valuable route to reduce in-stent re-stenosis can be based on promoting cell response to nano-structured stainless steel (SS) surfaces such as by patterning nano-pits in SS. In this regard patterning by focused ion beam (FIB) milling offers several advantages for flexible prototyping. On the other hand FIB patterning of polycrystalline metals is greatly influenced by channelling effects and redeposition. Correlative microscopy methods present an opportunity to study such effects comprehensively and derive structure–property understanding that is important for developing improved patterning. In this chapter we present a FIB patterning protocol for nano-structuring features (concaves) ordered in rectangular arrays on pre-polished 316L stainless steel surfaces. An investigation based on correlative microscopy approach of the size, shape and depth of the developed arrays in relation to the crystal orientation of the underlying SS domains is presented. The correlative microscopy protocol is based on cross-correlation of top-view scanning electron microscopy, electron backscattering diffraction, atomic force microscopy and cross-sectional (serial) sectioning. Various FIB tests were performed, aiming at improved productivity by preserving nano-size accuracy of the patterned process. The optimal FIB patterning conditions for achieving reasonably high throughput (patterned rate of about 0.03 mm2/h) and nano-size accuracy in dimensions and shapes of the features are discussed as well

The Relative Importance of Topography and RGD Ligand Density for Endothelial Cell Adhesion

The morphology and function of endothelial cells depends on the physical and chemical characteristics of the extracellular environment. Here, we designed silicon surfaces on which topographical features and surface densities of the integrin binding peptide arginine-glycine-aspartic acid (RGD) could be independently controlled. We used these surfaces to investigate the relative importance of the surface chemistry of ligand presentation versus surface topography in endothelial cell adhesion. We compared cell adhesion, spreading and migration on surfaces with nano- to micro-scaled pyramids and average densities of 6×102–6×1011 RGD/mm2. We found that fewer cells adhered onto rough than flat surfaces and that the optimal average RGD density for cell adhesion was 6×105 RGD/mm2 on flat surfaces and substrata with nano-scaled roughness. Only on surfaces with micro-scaled pyramids did the topography hinder cell migration and a lower average RGD density was optimal for adhesion. In contrast, cell spreading was greatest on surfaces with 6×108 RGD/mm2 irrespectively of presence of feature and their size. In summary, our data suggest that the size of pyramids predominately control the number of endothelial cells that adhere to the substratum but the average RGD density governs the degree of cell spreading and length of focal adhesion within adherent cells. The data points towards a two-step model of cell adhesion: the initial contact of cells with a substratum may be guided by the topography while the engagement of cell surface receptors is predominately controlled by the surface chemistry

Gating of aquaporins by heavy metals in Allium cepa L. epidermal cells

Changes in the water permeability, aquaporin (AQP) activity, of leaf cells were investigated in response to different heavy metals (Zn2+, Pb2+, Cd2+, Hg2+). The cell pressure probe experiments were performed on onion epidermal cells as a model system. Heavy metal solutions at different concentrations (0.05 μM–2 mM) were used in our experiments. We showed that the investigated metal ions can be arranged in order of decreasing toxicity (expressed as a decrease in water permeability) as follows: Hg>Cd>Pb>Zn. Our results showed that β-mercaptoethanol treatment (10 mM solution) partially reverses the effect of AQP gating. The magnitude of this reverse differed depending on the metal and its concentration. The time course studies of the process showed that the gating of AQPs occurred within the first 10 min after the application of a metal. We also showed that after 20–40 min from the onset of metal treatment, the water flow through AQPs stabilized and remained constant. We observed that irrespective of the metal applied, the effect of AQP gating can be recorded within the first 10 min after the administration of metal ions. More generally, our results indicate that the toxic effects of investigated metal ions on the cellular level may involve AQP gating