4,004 research outputs found
A Microinjectable Biological System, the Xenopus Oocyte, as an Approach to Understanding Signal Transduction Protein Function
No full textFibroblast growth factors 1 and 2 differently activate MAP kinase in Xenopus oocytes expressing fibroblast growth factor receptors 1 and 4
No full textAbstractThe mitogen-activated protein kinase (MAP kinase) signalling cascade activated by fibroblast growth factors (FGF1 and FGF2) was analysed in a model system, Xenopus oocytes, expressing fibroblast growth factor receptors (FGFR1 and FGFR4). Stimulation of FGFR1 by FGF1 or FGF2 and FGFR4 by FGF1 induced a sustained phosphorylation of extracellular signal-regulated protein kinase 2 (ERK2) and meiosis reinitiation. In contrast, FGFR4 stimulation by FGF2 induced an early transient activation of ERK2 and no meiosis reinitiation. FGFR4 transduction cascades were differently activated by FGF1 and FGF2. Early phosphorylation of ERK2 was blocked by the dominant negative form of growth factor-bound protein 2 (Grb2) and Ras, for FGF1–FGFR4 and FGF2–FGFR4. The phosphatidylinositol 3-kinase (PI3 kinase) inhibitors wortmannin and LY294002 only prevented the early ERK2 phosphorylation triggered by FGF2–FGFR4 but not by FGF1–FGFR4. ERK2 phosphorylation triggered by FGFR4 depended on the Grb2/Ras pathway and also involved PI3 kinase in a time-dependent manner
RasGAP is involved in signal transduction triggered by FGF1 in Xenopus oocytes expressing FGFR1
No full textAbstractThe role of RasGAP was investigated in the model system of Xenopus oocytes expressing fibroblast growth factor receptor 1 (FGFR1) stimulated by fibroblast growth factor 1 (FGF1). The injection of the SH2-SH3-SH2 domains of RasGAP suppressed Ras activity, extracellular signal-regulated protein kinase 2 (ERK2) phosphorylation and Mos synthesis. The SH2 domain of Src, and PP2, an inhibitor of Src, also abolished Ras activity, ERK2 phosphorylation and Mos synthesis. In addition, Src activity was blocked by the SH2-SH3-SH2 domains of RasGAP. Immunoprecipitation of a chimera composed of the extracellular domain of the platelet-derived growth factor (PDGF) receptor and the intracellular domain of FGFR1 stimulated by PDGF-BB demonstrates the recruitment of phosphorylated RasGAP. This study shows that the transduction cascade induced by the FGFR1–FGF1 interaction in Xenopus oocytes involves RasGAP as a co-activator of Src to stimulate the Ras/mitogen-activated protein kinase cascade and Mos synthesis. It emphasises a new positive regulatory role for RasGAP in FGFR transduction
Fibroblast and epidermal growth factor receptor expression in Xenopus oocytes displays distinct calcium oscillatory patterns
No full textAbstractElectrophysiological study performed with the voltage clamp technique was used to examine the intracellular calcium pathway activated by tyrosine kinase receptor members. Three FGF receptors from Pleurodeles PR1, PR3, PR4, homologs to human receptors, and the human EGF receptor were expressed in Xenopus oocytes. Under FGF1, FGF2 and FGF4 stimulation, PR1 and PR3 display a one phase inward chloride calcium dependent current superimposed by sustained oscillations, whereas PR4 did not show any oscillations. These currents were dependent on intracellular calcium mobilisation, as the responses were reduced by caffeine (10 mM). Solely PR4 responses were affected by an extracellular calcium depleted solution suggesting the involvement of concomitant extracellular and intracellular calcium intervention in the calcium chloride current, whereas PR1 and PR3 did not. Under EGF stimulation, the EGF receptor elicits a two component inward current composed of an undelayed rapid transient dependent on intracellular calcium store recruitment followed by a second slower current dependent on calcium influx. The specific pattern and amplitude of the calcium oscillations induced by the combinatorial action of growth factors on their receptors could be relevant in numerous calcium dependent cell functions
Tissue engineering of a tracheal substitute
No full textLectin histochemistry and scanning electron microscopy (SEM) was used to assess the growth and characterise the differentiation of human respiratory epithelial cells (REC) cultured on two biomaterial scaffolds. The first scaffold, based on a hyaluronic acid derivative, was observed to be non-adhesive for REC. This lack of adhesion was found to be unrelated to the presence of the hyaluronic acid binding domain on the surface of isolated REC. The other scaffold, consisting of equine collagen, was observed to encourage REC spreading and adhesion. Positive Ulex Europaeus agglutinin (UEA) lectin staining of this preparation indicated the presence of ciliated REC on the scaffold surface. However, the marked decrease in peanut agglutinin (PNA) positive staining, relative to that of control cultures and native tissue, indicates a dedifferentiation of the secretory cells in monolayer. SEM analysis of REC cultured on the collagen scaffold confirmed the presence of ciliated cells thereby validating the UEA positive staining. The presence of both established and developing cilia was also verified. This indicates that collagen biomaterials are appropriate for the tissue engineering of REC. Furthermore, that UEA and PNA staining is a useful tool in the characterisation of cells cultured on biomaterials, therefore helpful in identifying biomaterials that are suitable for specific tissue engineering purposes.
The culture of REC at an air liquid interface (ALI) was investigated. Both conventional ALI inserts and the Biofleece scaffold were used. The cells grown the on conventional inserts became multilayered and showed some degree of ciliation after the period of ten days. The cells grown on the Biofleece scaffold became necrotic and died due to nutrient deprivation. The use of ALI culture techniques on scaffold materials needs to be adjusted to allow for sufficient nutrient supply to the cells.
The Biofleece scaffold was found to be suitable for the tissue engineering of cartilage in vitro. Constructs with a cartilage-like morphology were generated with the scaffold after two weeks in culture. The tissue-engineered cartilage was found to contain a higher number of cells and less extracellular matrix (ECM) than the native tissue controls. Suction seeding techniques were used to improve the distribution of cells within the scaffold and thereby increase the overall efficiency of cartilage tissue engineering within the scaffold. Alcian blue (AB) and Papanicolau (PN) stains of the tissue engineered cartilage described two distinct regions within the constructs, namely the developed cartilage-like region and the developing region. The latter is thought to be areas in which the cartilage cells are yet to fully remodel the scaffold material and deposit their own “native” ECM. However, the Biofleece scaffold material was observed to loose 40-50% of its initial volume during the tissue engineering process over a period of two weeks. Thus the degradation of the Biofleece scaffold exceeds the rate of maturation of the cartilage tissue within the scaffold. This rapid biodegradation is most likely a result of matrixmetalloproteinase (MMP), in particular collagenase, production by the maturing chondrocytes. This reduction in size means that the Biofleece scaffold is not an appropriate material for the tissue engineering of a trachea. The optimal biomaterial for the tissue engineering of a trachea would degrade at a rate equal too, or slower than, the time taken for the cells within the scaffold to mature into functional tissue.
The co-culture of REC and chondrocytes was achieved through the use of matrigel as a basement membrane replacement (note that direct growth of REC on cartilage tissue has been observed to be difficult). The co-cultured constructs were not stable because the Biofleece scaffold degrades at a high rate in the presence of both cell types. The constructs were observed to shrink to approximately 35-30% of the original dimensions in a period of 3-7 days. The reason for this accelerated degradation is not known but is most likely the result of severe MMP production by the two cell types when in combination.
It was concluded that the characterisation procedures used in this study (histochemical staining, fluorescent staining and scanning electron microscopy) for both REC and chondrocyte tissue engineered constructs are appropriate for this and further studies. The chondrocyte seeding methodologies in particular are a useful tool for tissue engineering. This study succeeds in many ways to investigate the tissue engineering of a tracheal substitute by detailing how REC and chondrocytes can be cultured on biomaterials and assessed for tissue development. However, the study does not deliver such a viable substitute as an end product. The primary reason for this outcome is the rapid degradation of the Biofleece scaffold materialLectin Histochemie und Elektronenmikroskopie wurden benutzt, um das Wachstum von humanen respiratorischen Epithelzellen (RECs), welche auf zwei Biomaterialien kultiviert wurden, festzusetzen und ihren Differenzierungsgrad zu bestimmen. Das erste Trägermaterial, welches auf einem Hyaluronsäurederivat basiert, ließ keine Anheftung der RECs zu. Diese fehlende Anheftung ließ sich jedoch nicht zurückführen auf das Vorhandensein der Hyaluronsäure bindenden Domaine auf der Oberfläche isolierter RECs. Das andere Trägermaterial, aus Pferdekollagen hergestellt, zeigte dagegen eine verstärkte Teilungsaktivität und Anheftung der REC. Die positive Ulex Europaeus Agglutinin (UEA) Lectin Färbung dieser Proben ließ die Anwesenheit von mit Zilien versehenen RECs auf der Trägerstoffoberfläche vermuten. Darüber hinaus weist das im Vergleich zu Kontrollkulturen und nativem Gewebe deutliche Nachlassen der positiven Peanut Agglutinin–Färbereaktion auf eine Dedifferenzierung der sekretorischen Zellen in der Monolayer-Kultur hin. Die rasterelektronenmikroskopische Untersuchung der auf dem Kollagenbiomaterial kultivierten RECs bestätigte das Auftreten von Zellen mit Zilien und damit auch die Aussagekräftigkeit der positiven UEA–Färbung. Dies zeigt somit, dass Biomaterialien aus Kollagen für das Tissue Engineering von RECs geeignet sind und dass sowohl die UEA–als auch die PNA–Färbung geeignete Methoden zur Charakterisierung von Zellen darstellen, die auf Biomaterialien kultiviert wurden. Somit helfen sie bei der Identifizierung von Biomaterialien für bestimmte Einsatzgebiete im Tissue Engineering.
Des weiteren wurde die Kultivierung von RECs auf einem Air liquid interface (ALI) untersucht, wobei sowohl der konventionelle ALI–Einsatz als auch das Biovliesmaterial zum Einsatz kamen. Dabei wuchsen die Zellen auf dem konventionellen Einsatz in Multilayern und zeigten nach einem Zeitraum von 10 Tagen einen bestimmten Anteil an Ziliierung. Die Zellen auf dem Biovlies dagegen wurden nekrotisch und gingen schließlich an Nahrungsmangel ein. Deshalb muss der Einsatz von ALI–Kulturtechniken bei Trägermaterialien dementsprechend modifiziert werden, dass eine ausreichende Versorgung der Zellen mit Nährstoffen gewährleistet ist.
Für das in vitro–Tissue Engineering von Knorpel erwies sich das Biovlies jedoch als geeignet. Mit ihm konnten nach zwei Wochen Kulturzeit Konstrukte mit einer knorpelähnlichen Morphologie erzeugt werden. Dabei zeigte sich, dass der Tissue Engineering–Knorpel eine höhere Zellzahl bei reduzierter extrazellulärer Matrix (ECM) aufwies als vergleichbares natives Kontrollgewebe. Dabei wurden Saugtechniken benutzt, um die Verteilung der Zellen im Trägerstoff zu verbessern. Die Alzian – Blau – Färbung (AB) und Papanicolau – Färbung (PN) zeigten bei dem Tissue Engineering–Knorpel zwei unterschiedliche Regionen innerhalb des Konstrukts, nämlich eine knorpelähnliche bereits entwickelte Region und eine sich entwickelnde Region. Bei letzterer dürfte es sich wohl um Gebiete handeln, in denen Zellen noch im Begriff sind, den Trägerstoff vollends umzubauen und ihre eigene „native“ ECM abzulagern. Nichtsdestoweniger büßte das Biovlies während des Tissue Engineering Prozesses über einen Zeitraum von zwei Wochen annähernd 40-50 % seines anfänglichen Volumens ein. Somit übersteigt das Ausmaß der Degradation des Biovlieses das des Heranreifens von Knorpelgewebe in dem Trägermaterial. Diese schnelle Biodegradation ist am ehesten das Ergebnis der Aktivität von Matrixmetalloproteinasen (MMP), insbesondere der Kollagenase, welche von reifenden Chondrozyten produziert wird. Diese Schrumpfung bedeutet also, dass das Biovlies kein geeignetes Material für das Tissue Engineering der Trachea darstellt. Denn ein optimales Biomaterial für das Tissue Engineering der Trachea sollte sich innerhalb derselben Zeit bzw. über einen längeren Zeitraum hinweg abbauen, als innerhalb desjenigen, den die sich in dem Trägermaterial befindlichen Zellen benötigen, um zu funktionalem Gewebe heranzureifen.
Durch den Einsatz von Matrigel als Ersatz für die Basalmembran konnte eine Kokultur aus RECs und Chondrozyten etabliert werden (wobei anzumerken ist, dass sich direktes Wachstum von RECs auf Knorpelgewebe als problematisch erweist). Die Konstrukte aus Kokulturen waren nicht stabil, da das Biovlies in Anwesenheit beider Zelltypen hochgradig abgebaut wird. Innerhalb von 3–7 Tagen schrumpften die Konstrukte auf ca. 35–50 % ihrer Ausgangsgröße zusammen. Der Grund für diesen beschleunigten Abbau ist unbekannt, jedoch ist am ehesten eine ausgeprägte Produktion von MMP durch die beiden Zellarten anzunehmen, sobald diese in Kombination vorliegen.
Insgesamt lässt sich sagen, dass die Methoden zur Zell- und Gewebecharakterisierung, welche in dieser Studie benutzt wurden (histochemische Färbungen, Fluoreszenzfärbung und Elektronenmikroskopie) sowohl für mit RECs als auch mit Chondrozyten hergestellte Konstrukte für die vorliegende Arbeit als auch zukünftige Studien als geeignet anzusehen sind. Diese Studie hat in vielerlei Hinsicht erfolgreich das Tissue Engineering einer Luftröhre untersuchen können, indem sie im Detail aufzeigt, wie RECs und Chondrozyten auf Biomaterialien kultiviert und für das Tissue Engineering eingesetzt werden können. Trotzdem kann diese Arbeit kein einsetzbares Ersatzmaterial als Endprodukt liefern. Der Hauptgrund für dieses Ergebnis ist in erster Linie in dem schnellen Abbau des Biovlieses als Trägermaterial zu sehen
Transduction cascades initiated by fibroblast growth factor 1 on Xenopus oocytes expressing MDA-MB-231 mRNAs
No full textERK2 is required for FGF1-induced JNK1 phosphorylation in Xenopus oocyte expressing FGF receptor 1
No full textAbstractA possible connection between the ERK2 and JNK1 MAP kinases transduction cascades was investigated in Xenopus oocytes expressing FGFR1 stimulated by FGF1. Injection of various inhibitors for the Shc/Grb2/Ras/Mos/MEK/ERK2 cascade blocked FGF1-induced germinal vesicle breakdown (GVBD), as well as ERK2 and JNK1 phosphorylation. JNK1 was found to be activated downstream of ERK2, since injection of an active ERK2 triggered JNK1 phosphorylation and inhibition of ERK2 either by a MEK inhibitor or the MKP3 phosphatase blocked JNK1 phosphorylation. These results demonstrated that in FGFR1 signalling JNK1 phosphorylation depends on ERK2
Nanoscale properties of poly(ethylene terephthalate) vascular grafts
No full textThesis (S.B.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2004.Includes bibliographical references (leaves 46-48).Vascular grafts are prosthetic tubes that serve as artificial replacements for damaged blood vessels. Poly(ethylene-terephthalate), PET, has been successfully used in large diameter grafts; however, small caliber grafts are still a major challenge in biomaterials. Due to surface forces, blood plasma proteins adsorb to the graft, resulting in inflammation, infection, thrombus formation, and ultimately, vessel reclosure. The object of this project was to characterize and analyze the nanoscale surface properties of three different commercial vascular grafts, woven collagen-coated, knitted collagen- coated, and knitted heparin-bonded, all PET-based. The study was performed in order to ascertain differences in biocompatibility due to surface coating and morphology. Scanning Electron Microscopy, Atomic Force Microscopy and High Resolution Force Spectroscopy techniques were used to characterize the surface of the samples as well as to measure the forces between these surfaces and blood plasma proteins. The results will serve as a basis for the understanding of the nanoscale interactions between the biomaterial and blood plasma proteins. Such interactions are brought about by the different surface topologies and components, therefore a thorough understanding of surface properties will act as a building block for further changes in small caliber vascular grafts in order to enhance their biocompatibility.by Celia Edith Macias.S.B
Oxygen-alloyed poly-Si passivating contacts for high-thermal budget c-Si heterojunction solar cells
No full textCrystalline silicon solar cells with passivating contacts based on doped poly-Si exhibit high optical parasitic losses. Aiming at minimizing these losses, we developed the oxygen-alloyed poly-Si (poly-SiOx) as suitable material for passivating contacts. From passivation point of view, poly-SiOx layers show excellent passivation quality and carrier selectivity for both n-type (iVOC,flat = 740 mV, contact resistance ρc = 0.7 mΩ/cm2, iVOC,textured = 723 mV) and p-type (iVOC,flat = 709 mV, ρc = 0.5 mΩ/cm2). Optically, due to the incorporation of oxygen, the absorption coefficient of poly-SiOx becomes much lower than that of doped poly-Si at long wavelength. Both n-type and p-type poly-SiOx layers are concurrently deployed in front/back-contacted (FBC) solar cells with a front indium tin oxide (ITO) layer to facilitate the lateral transport of carriers and minimize cell's reflection. A high cell FF of 83.5% obtained in double-side flat FBC solar cell indicates an efficient carrier collection by these passivating contacts. An active-area cell efficiency of 21.0% featuring JSC,EQE = 39.7 mA/cm2 is obtained in front-side textured poly-SiOx FBC cell, with the potential of further improvement in both VOC and FF. The optical advantage of poly-SiOx over poly-Si as passivating contact is also observed with a 19.7% interdigitated back-contacted (IBC) solar cell endowed with poly-SiOx emitter and back surface field. Compared to the reference 23.0% IBC solar cell with poly-Si passivating contacts, the one based on poly-SiOx passivating contacts shows higher IQE at wavelengths above 1100 nm. This indicates that for both FBC and IBC cells, poly-SiOx passivating contacts hold potential in enhancing the cell JSC by maximizing the cell spectral response.Photovoltaic Materials and DevicesElectrical Sustainable Energ
Extrusion foaming of bioplastics for lightweight structure in food packaging
No full textThis thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.This thesis reports the systematic approaches to overcome the key drawbacks of the pure PHBV, namely low crystallisation rate, tensile strength, ductility, melt viscosity, thermal stability and high materials cost. The physical, mechanical, thermal, and rheological properties of the pure PHBV were studied systematically first to lay a solid foundation for formulation development. The influence of blending with other biopolymers, inclusion of filler, and chain extender additives in terms of mechanical properties, rheology, thermal decomposition and crystallization kinetics were then followed. Creating lightweight structures by foaming is considered to be one of the effective ways to reduce material consumption, hence the reduction of density and morphology of PHBV-based foams using extrusion foaming technique were studied comprehensively in terms of extrusion conditions (temperature profiles, screw speed and material feeding rate) and the blowing agent content. The material cost reduction was achieved by adding low-cost filler (e.g. CaCO3) and reduction of density by foaming. The thermal instability was enhanced by incorporation of chain extender (e.g. Joncryl) and blending with a high thermal stability biopolymer (e.g. PBAT). The polymer blend also improved the ductility. Adding nucleation agent enhanced the crystallization rate to reduce stickiness of extruded sheet. The final formulation (PHBV/PBAT/CaCO3 composite) was successfully extruded into high quality sheet and thermoformed to produce prototype trays in an industrial scale trial. The effect of the extrusion conditions (temperature profiles, screw speed and material feeding rate) and the blowing agent content are correlated to the density reduction of the foams. 61 and 47 % density reduction were achieved for the commercial PHBV and the PHBV/PBAT/CaCO3 composite respectively and there exists further scope for more expansion if multiple variable optimisation of the conditions are carried out.This study is funded by the UK Government Department of Trade & Industry (Technology Strategy Board), as part of the project “Biobased Lightweight Sandwich Structures for Packaging Applications”
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