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Toward Photo-control of Peptide Structure in VivoChi, Lei 15 February 2011 (has links)
An ability to manipulate the activity of a specific protein inside living cells offers exciting prospects for the study of protein function in vivo. Azobenzene derivatives introduced as intramolecular bridges have been demonstrated to reversibly photoregulate secondary structures and functions of peptides and proteins in vitro. My overall goal is to create a generally-applicable process for the reversible photocontrol of protein-protein interactions within the complex environment of a living cell. Results of studies toward this aim are presented.
A blue-green absorbing (~480 nm) azobenzene derivative cross-linker was designed that reversibly controlled the helical content of attached peptides with a half-life of the cis state of ~50 ms. This rapid photoswitch may prove useful as a tool for probing dynamic processes in biochemical systems using light.
The effect of cross-linker position (N-terminus, middle, C-terminus) on a photo-switchable 32-residue helical peptide was studied. Although the activation energies for thermal cis – trans relaxations were not the same, linker position did not affect the change in helix content. This work provides useful information for the effective photoregulation of much longer helices such as occur in coiled-coils.
Fluorescently labeled, cross-linked, modified Fos/Jun peptides with and without cell-penetrating peptide (CPP) tags were prepared for the purpose of photocontrolling peptide-peptide interactions in vivo. One of the peptides showed a degree of photocontrol of helicity. Cell uptake of CPP-tagged peptides was demonstrated. However, overall peptide behavior was dominated by undesired aggregation.
A simple reporter, a cross-linked peptide bearing an environmentally sensitive fluorophore at a key site, was designed for detecting photoswitching in vivo. Photoisomerization of the cross-linker caused changes in the local chemical environment and changes in fluorescence intensity of the environmentally sensitive dyes in vitro. However, no change in fluorescence was observed in the living systems we investigated.
Conclusions and suggestions for further work aimed at achieving the overall goal stated above are discussed.
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Toward Photo-control of Peptide Structure in VivoChi, Lei 15 February 2011 (has links)
An ability to manipulate the activity of a specific protein inside living cells offers exciting prospects for the study of protein function in vivo. Azobenzene derivatives introduced as intramolecular bridges have been demonstrated to reversibly photoregulate secondary structures and functions of peptides and proteins in vitro. My overall goal is to create a generally-applicable process for the reversible photocontrol of protein-protein interactions within the complex environment of a living cell. Results of studies toward this aim are presented.
A blue-green absorbing (~480 nm) azobenzene derivative cross-linker was designed that reversibly controlled the helical content of attached peptides with a half-life of the cis state of ~50 ms. This rapid photoswitch may prove useful as a tool for probing dynamic processes in biochemical systems using light.
The effect of cross-linker position (N-terminus, middle, C-terminus) on a photo-switchable 32-residue helical peptide was studied. Although the activation energies for thermal cis – trans relaxations were not the same, linker position did not affect the change in helix content. This work provides useful information for the effective photoregulation of much longer helices such as occur in coiled-coils.
Fluorescently labeled, cross-linked, modified Fos/Jun peptides with and without cell-penetrating peptide (CPP) tags were prepared for the purpose of photocontrolling peptide-peptide interactions in vivo. One of the peptides showed a degree of photocontrol of helicity. Cell uptake of CPP-tagged peptides was demonstrated. However, overall peptide behavior was dominated by undesired aggregation.
A simple reporter, a cross-linked peptide bearing an environmentally sensitive fluorophore at a key site, was designed for detecting photoswitching in vivo. Photoisomerization of the cross-linker caused changes in the local chemical environment and changes in fluorescence intensity of the environmentally sensitive dyes in vitro. However, no change in fluorescence was observed in the living systems we investigated.
Conclusions and suggestions for further work aimed at achieving the overall goal stated above are discussed.
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Nucleic acid detection using oligonucleotide cross-linked polymer compositesFerrier, David Christopher January 2017 (has links)
There has been much interest in recent years about the potential of microRNA as a new source of biomarkers for the diagnosis of disease. The delivery of new diagnostic tools based on this potential has been limited by shortcomings in current microRNA detection techniques. This thesis explores the development of a new method of microRNA detection through the incorporation of conductive particles into oligonucleotide-functionalised polymers to form oligonucleotide cross-linked polymer composites. Such composites could provide a simple, rapid, and low-cost means of microRNA detection that could be easily multiplexed, providing a valuable tool for point-of-care medical diagnostics. This work presents oligonucleotide-functionalised carbon/polyacrylamide composites which demonstrate a selective swelling response in the presence of analyte oligonucleotide sequences and for which the electrical conductivity decreases with swelling. The composites were synthesised via UV-initiated free-radical polymerisation of carbon/- monomer mixtures upon custom electrode devices, consisting of interdigitated platinum electrodes fabricated upon a silicon substrate. The optimal cross-linker density and carbon loading concentration were determined as well as the best means of dispersing the carbon particles within the polymer. Various types of carbon particles, with differing sizes and aspect ratios, were compared and their performances as conductive additives for polymer swelling transduction evaluated. The swelling behaviour of these composites was evaluated by analysing images of composite microdroplets as they swell. The electrical characteristics of the composites were determined by measuring either the two-terminal resistance or the complex impedance of composite microdroplets on the electrode devices. Alternating and direct current measurement techniques were compared to determine the best approach for the transduction of composite swelling. The volumetric and electrical responses of oligonucleotide-functionalised carbon/polyacrylamide composites were analysed in solutions of analyte oligonucleotide and non-complementary controls. It has been demonstrated that, using carbon nanopowder composites and a direct current two-terminal resistance measurement, it is possible to differentiate between analyte and control solutions to concentrations as low as 10 nM, with single-base precision, in less than three minutes. However, the inability to detect at concentrations below this value, difficulties in differentiating between different analyte concentrations and thermal instability mean that, in their current form, oligonucleotide cross-linked polymer composites are unsuitable for the detection of circulating microRNA at clinically relevant concentrations. Potential avenues of work to address these challenges are discussed. Also presented are collaborative results for oligonucleotide-responsive polymers functionalised with morpholino nucleic acid analogues, in what is believed to be the first example of such a material. These morpholino-functionalised polymers offer significant advantages, in terms of stability and sensitivity, over their nucleic acid equivalents for bio-responsive polymer applications.
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Microtensile bond strength of resin-dentin bonds following application of a chemical collagen cross-linker using different dentin bonding systemsZidane, Bassam Naoraldean 01 July 2015 (has links)
Introduction: The stabilization of dentinal collagen fibers against enzymatic degradation by the use of biocompatible cross-linker agents is of clinical importance for effective dentin bonding to surpass the test of time.
Objective: The present study aims to evaluate and compare the effect of the application of two versions of a desensitizer solution to sound coronal dentin, on the microtensile bond strength (μTBS) of the resin-sound coronal dentin using 4th and 6th generation dentin bonding systems.
Materials and Methods: Extracted human third molars were collected from an unidentified bank of teeth followed by IRB approval. A flat surface of all 12 teeth was prepared utilizing a water-cooled high-speed diamond disc, leaving an entire hard sound dentinal area for testing. Subsequently, according to the assigned group, specimens followed specific manufacturer’s instructions for application of dentin bonding systems: specimens were subdivided into 6 groups (n=20). Group 1 (G1) First positive control group. Specimens received an application of a 4th generation dentin bonding system (DBS). Group 2 (G2) Second positive control group. Specimens received an application of a 6th generation DBS. Group 3 (G3) Specimens were exposed to Gluma Desensitizer agent, blot-dried and followed by application of a 4th generation DBS. Group 4 (G4) Specimens were exposed to Gluma Desensitizer agent, blot-dried and followed by application of a 6th generation DBS. Group 5 (G5) Specimens were exposed to Gluma Desensitizer PowerGel agent, blot-dried and followed by application of a 4th generation DBS. Group 6 (G6) Specimens were be exposed to Gluma Desensitizer PowerGel agent, blot-dried and received an application of a 6th generation DBS. After application of the adhesive systems, all specimens were restored using a microhybrid resin composite. The root portion was sectioned 1mm below the CEJ, and discarded. All specimens were thermocycled at 5-55 Cº for 7000 cycles on distilled water. Then each restored tooth was sectioned perpendicular to the bonding interface into 1mm x 1mm x 8mm beams with a slow speed diamond wafering blade under thorough irrigation. Then specimens were subjected to μTBS testing at a crosshead speed of 1mm/min. Subsequently; specimens were subjected to fracture analysis and SEM evaluation of the different failure’s mode of the involved surfaces. Statistical analysis was performed by usingone- way ANOVA, two-way ANOVA and Fisher’s PLSD test (p<0.05).
Results: For the first aim of the study and after obtaining the μTBS in MPa: Group G1: 15.50 ± 6.28, Group G2: 13.06 ± 11.53, Group G3: 19.20 ± 9.43, Group G4: 12.76 ± 4.61, Group G5: 14.38 ± 5.95, Group G6: 18.54 ± 9.49. Statistical analysis showed that there is no significant influence of variables on the μTBS (Welch ANOVA [F (5,114) =2.21, p=0.057]). Treatment with Gluma desensitizing agent and Gluma desensitizing PowerGel has no significant influence on the bond strength. For the second aim of the study and to analyze group differences for type of fracture data was first recoded into two groups: (1) Adhesive failure and (2) Cohesive failure. Group differences were analyzed by type of fracture using a Fisher’s exact test. No difference was found between the groups by type offracture (5, N = 120) = 8.62, p = 0.090
Conclusion: Within the limitations of this in vitro study it can be concluded that Gluma desensitizing agent and Gluma desensitizing PowerGel did not significantly affect the μTBS of both 4th and 6th generation bonding system using extracted human teeth.
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THE DEVELOPMENT OF INTRACELLULAR NANOSENSORS: ACID-DEGRADABLE POLYMERIZED PHOSPHOLIPID VESICLES AND FLUORESCENT LABELSRoberts, David January 2010 (has links)
Phospholipid vesicles are biocompatible, and have potential for intracellular applications, but minimal membrane integrity limits their use in membrane-rich environments. Stabilized membranes overcome this limitation while maintaining biocompatible surface structures. Additionally, the modularity of phospholipid bilayer makes them ideal components when designing biologically inspired sensors. Membrane composition can be tailored to specific applications, transmembrane proteins can provide added functionalities, and the isolated interior can prevent cytotoxic and interfering detection chemistries from altering the cellular environment. This work has focused on expanding the capabilities of stabilized phospholipid membranes, and determining which formulations hold promise in developing stabilized phospholipid vesicle nanosensors.Current membrane stabilization methods suffer from either incomplete stabilization, or irreversible stabilization limiting the applications of vesicle nanosensors. Therefore, a facile method to prepare robust phospholipid vesicles using commonly available phospholipids stabilized via the formation of an interpenetrating, acid-labile, cross-linked polymer network that imparts controlled polymer destabilization and subsequent vesicle degradation was developed. Upon exposure to acidic conditions, the highly cross-linked polymer network was converted to linear polymers, substantially reducing vesicle stability upon exposure to chemical and physical insults. The resultant transiently stabilized vesicles have potential for enhanced drug delivery and chemical sensing applications requiring minimal membrane defects, and allow for improved physiological clearance.Some vesicle nanosensor schemes may require the passive diffusion of low molecular weight species across the membrane in addition to controllable degradation. Therefore, the acid-degradable, polymer-stabilized, phospholipid vesicle production method was extended to bis-SorbPC membranes by simultaneously polymerizing the vesicle with an acetal-containing cross-linker. The vesicles display prolonged stability under physiological conditions, and significant additional stability compared to vesicles composed of naturally occurring phospholipids. The vesicles demonstrated potential utility for sensing and therapeutic applications.Phospholipid vesicles can also serve as labels to observe movement in macromolecular biological assemblies, but a dearth of caged fluorescent labels limits design and function. Therefore, the first caged fluorescent thiol was synthesized, shown to label amines rapidly, and demonstrated the required photolytic properties. The caged fluorescent thiol has potential as a label in observing the movement of macromolecular biological assemblies and as a fluorescent probe for observing endosomal trafficking and release.
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Oligomer cross-linked gelatin hydrogels for peripheral nerve regenerationKohn-Polster, Caroline 08 May 2020 (has links)
The use of autografts is the gold standard for peripheral nerve regeneration (PNR) while
biomedical engineering made some contributions to improve PNR. A next generation of nerve guidance conduits (NGC) is required to transmit topographical and biochemical signals towards severed nerves. In this thesis, the gelatin hydrolyzate Collagel® (COL) and anhydride-containing cross-linkers (oPNMA, oPDMA) were used to fabricate crosslinked hydrogels (cGEL) for PNR. At first, established cGEL formulations were adjusted towards an injection-molding tool with static mixer. Therefore, the gelation kinetic was modified by variation of the gelation base. Hence, high reactive oPNMA was available for fabrication of robust cGEL based NGC. Secondly, novel cGEL and molding technique were adapted towards the fabrication of
cGEL-based filler for polymer-derived braided NGC. Shear-thinning filler was developed that allowed direct application inside the conduit lumen with minimal mechanical stiffness but sufficient scaffolding properties. Besides pristine filler, chemically modified filler was designed with a small mimetic of the nerve growth factor, LM11A-31, that was grafted to oPNMA. In a rat sciatic nerve model, the performance of this derivatized filler was comparable to the control and underlined the potential of chemical cues in PNR. A number of small diamines were further integrated into oPNMA and oPDMA to modify cGEL bulk. In addition to chemical feasibility, the cytocompatibility and cellular response were tested on L929 mouse fibroblasts and human adipose-derived stem cells. The functionalization showed an impact on the cell behavior with differences in cell proliferation, migration and spreading. Finally, modified oPNMA-derived hydrogels were tested on neonatale Schwann cells. The cell viability and extension was maintained in all hydrogels while the impact of LM11A-31 was not as pronounced. This thesis emphasizes the potential of cGEL hydrogels in nerve implants as fillers or conduits and, thus, is a promising building block for a new generation of NGC.
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Enhancing the durability of fluorocarbon-free Durable Water Repellant (DWR) formulation / Förbättring av hållbarheten för fluorkarbonfria vattenrepellerande formuleringarSolomon, Meron January 2017 (has links)
The focus of the project was to alter and optimize the water repellant textile coating formulations to reach enhanced durability. For this purpose, the project was approached with three methods. Firstly, bio-based components were implemented in the mother emulsion to act as surfactant and crosslinking agent and to provide hydrophobic properties. Secondly different binders were added to crosslink and increase the coating resistance towards washes. Lastly additives at nano-scale were added to increase surface roughness in order to obtain higher hydrophobicity and improved of crosslinking capacity due to the presence of more functional groups. The stability of all emulsions was controlled using different techniques such as optical microscopy to determine particle size, distribution and any observable instability (flocculation etc.), normal aging at room temperature and accelerated aging using higher temperature. All coatings were applied using a laboratory padder on standard PA and PES pieces of textiles and hydrophobic performance was evaluated through ISO 4920 spray test. By standard washing and repeating spray test, durability could be assessed. Further structure and property studies have been run using other tests such as: contact angle measurement, breathability of the coating and SEM observations. Based on the obtained results the incorporation of low HLB, bio-based surfactants in low amount (~0,25%) resulted in an increase in the hydrophobic performance of the tested textiles. However, a decrease in shelf life could be observed with these surfactants at room temperature. Sonication was successfully used to increase both stability and shelf life significantly. Some binders and nanoparticles proved to be successful in increasing the coating quality and thus the durability. Overall many of the developed formulations could enhance performance on PA compared to the already present commercial product. On PES textile, however, the developed strategies yielded hydrophobic effect close to the commercial product.
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Fabrication and characterizations of hydrogels for cartilage repairKaur, Payal, Khaghani, Seyed A., Oluwadamilola, Agbabiaka, Khurshid, Z., Zafar, M.S., Mozafari, M., Youseffi, Mansour, Sefat, Farshid 26 September 2017 (has links)
Yes / Articular cartilage is a vascular tissue with limited repair capabilities, leaving an afflicted person in extreme pain. The tissue experiences numerous forces throughout its lifetime. This study focuses on development of a novel hydrogel composed of chitosan and β-glycerophosphate for articular cartilage repair. The aim of this study was to investigate the mechanical properties and swelling behaviour of a novel hydrogel composed of chitosan and β-glycerophosphate for cartilage repair. The mechanical properties were measured for compression forces. Mach-1 mechanical testing system was used to obtain storage and loss modulus for each hydrogel sample to achieve viscoelastic properties of fabricated hydrogels. Two swelling tests were carried out to compare water retaining capabilities of the samples. The hydrogel samples were made of five different concentrations of β-glycerophosphate cross-linked with chitosan. Each sample with different β-glycerophosphate concentration underwent sinusoidal compression forces at three different frequencies -0.1Hz, 0.316Hz and 1Hz. The result of mechanical testing was obtained as storage and loss modulus. Storage modulus represents the elastic component and loss modulus represents the viscosity of the samples. The results obtained for 1Hz were of interest because the knee experiences frequency of 1Hz during walking.
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Self-assembly effects of filamentous actin bundlesSchnauß, Jörg 30 September 2015 (has links) (PDF)
Das Zytoskelett einer eukaryotischen Zelle besteht aus drei Hauptbestandteilen: Aktin, Intermediärfilamenten und Mikrotubuli. Die vorliegende Arbeit beschäftigt sich mit dem Protein Aktin, welches unter physiologischen Bedingungen dynamische Filamente durch Polymerisation ausbildet. Diese Filamente können sowohl in Netzwerken als auch Bündeln angeordnet werden. Diese Anordnungen bilden die Grundlage für eine Vielfalt von Strukturen zur Realisierung diverser zellulärer Funktionen. Konventionell wurde die Ausprägung solcher Strukturen durch zusätzliche Proteine erklärt, welche Aktin beispielsweise vernetzen oder sogar aktive, dissipative Prozesse durch ATP Hydrolyse ermöglichen. Durch diese Erklärungen prägte sich ein sehr komplexes Bild zellulärer Funktionen heraus. Die dissipative Natur der meisten Prozesse führte dazu, dass meist auf grundlegende physikalische Beschreibungen, welche auf nicht-dissipativen Gleichgewichtszuständen beruhen, verzichtet wurde. Diese Arbeit widmet sich solchen nicht-dissipativen Prozessen und beschreibt deren inhärente Bedeutung auch in aktiven, dissipativen Systemen.
Ein erstes Beispiel beschreibt die Generierung von kontraktilen Kräften in Aktinbündeln durch eine hohe makromolekulare Dichte der Umgebung. Diese hohe Dichte führt zu einem entropischen Effekt, welcher durch Volumenausschluss hochkonzentrierter inerter Polymere Aktinfilamente in Bündel ordnet. Werden diese Strukturen aus ihrem energetischen Minimum ausgelenkt, so entsteht eine rücktreibende Kraft, welche nach Ausschaltung der auslenkenden Kraft zu einer Kontraktion des gesamten Bündels führt. Dieses Bespiel zeigt klar, dass selbst in sehr einfachen Systemen äußerst komplexe Prozesse ablaufen können, welche konventionell mittels dissipativer Umwandlung von chemischer Energie in mechanische Arbeit beschrieben wurden.
Die Komplexität der Eigenschaften von Aktinbündeln nimmt zudem drastisch zu sobald zusätzliche Proteine mit eigenen mechanischen Eigenschaften das System beeinflussen. Zur Untersuchung eines solchen Mehrkomponentensystems wurden Aktinfilamente mittels transienter Vernetzungsproteine gebündelt. Versuche auf unterschiedlichen Zeitskalen zeigten klar differenzierbare mechanische Antworten auf induzierte, aktive Biegedeformationen. Im Falle kurzer Deformationen verhielt sich das System völlig elastisch, während für lange Deformationszeiten deutliche plastische Effekte auftraten. Als Ursprung dieser Plastizität wurde die dynamische Umordnung der Vernetzungsproteine identifiziert.
Jedoch führen nicht nur zusätzliche Proteine zu einer erhöhten Komplexität. Bereits die Anordnung von reinen Aktinbündeln in Netzwerke mittels entropischer Kräfte führt zu einer überraschenden Variabilität von entstehenden Mustern. Im besonderen Fokus dieser Untersuchung stehen Aster ähnliche Muster, welche regelmäßige Netzwerkstrukturen ausbilden und nur in Verbindung mit Aktin assoziierten Proteinen bekannt waren. Störungen der isotropen Ausgangssituation führen zu veränderter Musterbildung, welche die initiale Störung direkt widerspiegeln.
Mit den präsentierten Resultaten leistet die Arbeit einen wichtigen Beitrag zum Verständnis der Dynamik von Aktinbündeln sowie deren Interaktionen.
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A Novel Microspheres Composite Hydrogels Cross-linked by Methacrylated Gelatin Nanoparticles: Enhanced Mechanical Property and BiocompatibilityWang, Chunhua, Mu, C., Lin, W. 25 June 2019 (has links)
Content: Nowadays, protein-based nanoparticle as a biodegradable, biocompatible product attracts considerable interest for new uses in specialized technical areas. Gelatin is a denatured, biodegradable,
and nonimmunogenic protein obtained by controlled hydrolysis of the triple-helix structure of collagen into single-strain molecules. As an amphiphilic biopolymer, gelatin can easily assemble into different kinds of aggregates under the defined pH and temperature and the resulting gelatin nanoparticles have been developed to be applied in the food industry and biomedical fields. Herein we report a novel
macromolecular microsphere composites (MMC) hydrogels with the use of prepared methacrylated gelatin nanoparticles (MA-GNP) as the cross-linker. MA-GNP have the ability of chemical crosslinking by the
polymerization of C=C bonds, such that the composite hydrogels can be formed by radical polymerization of acrylamide (AAm) on the surface of MA-GNP. The smooth spherical particles with an average size of
~100 nm have been synthesized through a modified two-step desolvation method as proved by atomic force microscopy (AFM). The results of nuclear magnetic resonance and dynamic light scattering further
confirm the presence of reactive groups (C=C bonds) in the particles and its narrow sizes distribution. The resulting composite hydrogels (MA-GNP/PAAm) are porous materials with tunable pore sizes and exhibit enhanced compressive resistance and elasticity as well. Increasing appropriately the dosage of MA-GNP reduces the equilibrium swelling ratio and improves thermal stability of the gels. Moreover, all the hydrogels exhibit prolonged blood-clotting time, nonhemolytic nature and strong suitability for cell proliferation, indicating the improved antithrombogenicity and excellent cyto-compatibility. It suggests
that the novel MA-GNP/PAAm hydrogels have potential application as tissue engineer scaffold materials, and the MA-GNP can be a promising macromolecular microsphere cross-linker for application in biomedical materials. The present work not only exploits new strategies to fabricate MMC hydrogels but also advance the potential application of biodegradable gelatin-based nanoparticles in biomedical fields.
Take-Away:
1. A well-dispersed methacrylated gelatin nanoparticle (MA-GNP) with an average size of ~100 nm is presented by a modified two-step desolvation method.
2. MA-GNP is readily introduced into the polyacrylamide (PAAm) system as a cross-linker to prepare macromolecular microsphere composites (MMC) hydrogels via a free radical polymerization reaction.
3. MA-GNP is an effective cross-linker, improving both the compressive resistance and elasticity of MMC hydrogels as well as the biocompatibility.
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