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Novel Immunogens Of Cellular Immunity Revealed Using In Vitro Human Cell-based ApproachSchanen, Brian 01 January 2012 (has links)
In the last 150 years, tremendous headway has been made in our understanding of the human immune system. Pioneers in the field such as Paul Ehrlich, Elie Metchnikoff, Louis Pasteur, Robert Koch and Walter Reed carried out seminal studies that established the groundwork for our understanding of humoral and cellular immunity in humans. However, this direct line of evidence into human immunology was diverted in the mid-20th century with the adoption of a model which allowed for investigators to use a reductionist-approach with the promise to resolve immunity at a molecular level. This revolutionary model was the scientific commercialization of various inbred strains of mice. It seems inconceivable how a four-legged nocturnal rodent managed to become the focus of billions of dollars of research to improve our understanding of human immunity. Nevertheless, this strange surrogate for human immunity did provide us with major conceptual advances in areas, such as identification of dendritic cell population heterogeneity, T cell help for B cell antibody production, MHC-restricted recognition of virusinfected cells, and even the discovery of cell types like NKT cells. However, these prior advances have now been prefaced with decades-worth of disappointing, non-translational findings. The best examples of such disappointments are in murine models of autoimmunity, cancer immunotherapy, and vaccinology where numerous studies have revealed promising outcomes in mice but were met with failure or limited success upon translation into humans. We do not look at this as a failure of the murine model; rather we consider it a call to arms to innovate in vitro surrogates to examine human immunity when otherwise bound by ethical limitation from working directly in humans. To overcome these challenges, we developed a system to interrogate novel immunogens that begins by generating human dendritic cells (DCs), a cell type necessary to mounting a protective immune response. DCs for research and clinical applications are typically derived from purified blood monocytes that are cultured in a cocktail of cytokines for a week or more. Because it has been iv suggested that these cytokine-derived DCs may be deficient in some important immunological functions and might not accurately represent antigen presenting cell (APC) populations found under normal conditions in vivo, there is an interest in developing methods that permit the derivation of DCs in a more physiologically relevant manner in vitro. Here, we describe a simple and reliable technique for generating large numbers of highly purified DCs that is based on a one-way migration of blood monocytes through a layer of human umbilical vein endothelial cells (HUVECs) that are cultured to confluency in the upper chamber of a Transwell device. The resultant APCs, harvested from the lower Transwell chamber, resemble other cultured DC populations in their expression of major histocompatibility (MHC) and costimulatory molecules, ability to phagocytose protein antigens and capacity to trigger primary antigen-specific T cell responses. This technique offers several advantages over the standard method of in vitro cytokine-driven DC development, including: (1) the rapidity of this approach, as DC differentiation occurs in only 2 days, (2) the differentiation process itself, which is more akin to the development of DCs under physiologic conditions and (3) the cost effectiveness of the system, since no monocyte pre-selection is required and DC development occurs in the absence of expensive recombinant cytokines. Taken together, this approach allows for the exploration of novel immunogens utilizing a physiologically representative population of APCs enriched from circulating blood. The outbreak of the swine-origin H1N1 influenza in the spring of 2009 took epidemiologists, immunologists, and vaccinologists by surprise and galvanized a massive worldwide effort to produce millions of vaccine doses to protect against this single virus strain. Of particular concern was the apparent lack of pre-existing antibody capable of eliciting cross-protective immunity against this novel virus, which fueled fears this strain would trigger a particularly far-reaching and lethal pandemic. Given that disease caused by the swine-origin virus was far less severe than expected, we hypothesized v cellular immunity to cross-conserved T cell epitopes might have played a significant role in protecting against the pandemic H1N1 in the absence of cross-reactive humoral immunity. We collaborated with bioinformaticians to develop an immunoinformatics approach to predict CD4+ T cell epitopes conserved between the 2008-2009 seasonal H1N1 vaccine strain and pandemic H1N1 (A/California/04/2009) hemagglutinin proteins that could act as novel immunogens and function as potential vaccine candidates or compliments to current vaccine formulations. We examined these peptides using T cells from human donors not exposed to the pandemic virus demonstrating that pre-existing CD4+ T cells can elicit cross-reactive effector responses against the pandemic H1N1 virus. As well, we showed the computational tools created by our collaborators were 80-90% accurate in predicting CD4+ T cell epitopes and their HLA-DRB1-dependent response profiles in donors that were chosen at random for HLA haplotype. Combined, these results confirm the power of coupling immunoinformatics to define broadly reactive CD4+ T cell epitopes with a highly sensitive in vitro model to verify these in silico predictions as a means to understand human cellular immunity, including cross-protective responses, and to define CD4+ T cell epitopes for potential vaccination efforts against future influenza viruses and other pathogens. It is thought that utilizing highly conserved peptides as novel immunogens of cellular immunity for future vaccination strategies may require an adjuvant for efficacy. However, the FDA has approved the use of only two adjuvant compounds (Alum or MPL®) which may not be compatible or offer effective immune enhancement in novel vaccine preparations, thereby soliciting the need for novel adjuvants. Nanoparticles have since been a topic of adjuvant potential. Nanoparticles harbor great potential because they possess unique physicochemical properties compared to their larger counter parts as a result of quantum-size effects and their inherent large surface area to volume ratio. These physicochemical properties govern how a nanoparticle will behave in its environment. However, vi researchers have only just begun to catalogue the biological effect these properties illicit. Moreover, little is known about the interaction between the immune system and NPs. However, in light of the recent development in new adjuvants that involves composites and coatings of polymers, lipids, ligands, TLR agonist, the ability of a simple metal oxide nanopowder to effectively induce or couple immunomodulation would provide researchers a basic alternative to costly and complex adjuvant development. Considering the evidence suggesting NPs can act as immunopotentiators, we questioned whether these materials can act not only as innate adjuvants, but as novel immunogens to cellular immunity. To accomplish this, we under took a set of studies to investigate any nanoparticle size-induced effects using TiO2, one of the most widely manufactured nanoparticles, as a model. We explored titanium dioxide synthesized into its three most commonly nanoarchitectures: anatase (7-10 nm), rutile (15-20 nm), and nanotube (10-15 nm diameters, 70-150 nm length) in comparison to a micron-sized formulation. We used the fully human autologous MIMIC® immunological construct has been utilized as a predictive, nonanimal alternative to diagnose nanoparticle immunogenicity. Cumulatively, treatment with titanium dioxide nanoparticles in the MIMIC® system led to elevated levels of proinflammatory cytokines and increased maturation and expression of costimulatory molecules on dendritic cells. Additionally, these treatments effectively primed activation and proliferation of naïve CD4+ T cells in comparison to dendritic cells treated with titanium dioxide microspheres, characteristic of an in vivo inflammatory response, providing evidence of a size induced difference between the nano-sized and micron-sized material, revealing novel immune cell recognition and activation by a crystalline nanomaterial in a size-dependent manner. Having identified nanomaterial size as a contributing feature of nanoparticle induced immunopotentiation, we became interested if additional physicochemical properties such as surface vii reactivity or catalytic behavior could also be immunostimulatory. Moreover, because we witnessed a stimulatory effect to dendritic cells following nanoparticle treatment, we were curious how these nanoparticle-touched dendritic cells would impact adaptive immunity. Since TiO2 acts as an oxidant we chose an antioxidant nanoparticle, CeO2, as a counterpart to explore how divergent nanoparticle surface reactivity impacts innate and adaptive immunity. We focused on the effect these nanoparticles had on human dendritic cells and TH cells as a strategy towards defining their impact to cellular immunity. Combined, we report that TiO2 nanoparticles potentiate DC maturation inducing the secretion of IL- 12p70 and IL-1B, while treatment with CeO2 nanoparticles induced IL-10, a hallmark of suppression. When delivered to T cells alone TiO2 nanoparticles induced stronger proliferation in comparison to CeO2 which also stimulated TReg differentiation. When co-cultured in allogeneic T cell assays, the materials directed alternate TH polarization whereby TiO2 drives largely a TH1 dominate response, whereas CeO2 drove a TH2 bias. Combined, we report a novel immunomodulatory capacity of nanomaterials with catalytic activity. While unintentional exposure to these nanomaterials could pose a serious health risk, development and targeted use of such immunomodulatory nanoparticles could provide researchers with new tools for novel adjuvant strategies or therapeutics.
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A Linear Multiplexed Electrospray Thin Film Deposition SystemLojewski, Brandon 01 January 2013 (has links)
Liquid spray is essential to industries requiring processes such as spray coating, spray drying, spray pyrolysis, or spray cooling. This thesis reports the design, fabrication, and characterization of a thin film deposition system which utilizes a linear multiplexed electrospray (LINES) atomizer. First, a thorough review of the advantages and limitations of prior multiplexed electrospray systems leads to discussion of the design rationale for this work. Next, the line of charge model was extended to prescribe the operating conditions for the experiments and to estimate the spray profile. The spray profile was then simulated using a Lagrangian model and solved using a desktop supercomputer based on Graphics Processing Units (GPUs). The simulation was extended to estimate the droplet number density flux during deposition. Pure ethanol was electrosprayed in the cone-jet mode from a 51-nozzle aluminum LINES atomizer with less than 3% relative standard deviation in the D10 average droplet diameter as characterized using Phase Doppler Interferometry (PDI). Finally a 25-nozzle LINES was integrated into a thin film deposition system with a heated, motion controlled stage, to deposit TiO2 thin films onto silicon wafers from an ethanol based nanoparticle suspension. The resulting deposition pattern was analyzed using SEM, optical profilometry, and macro photography and compared with the numerical simulation results. The LINES tool developed here is a step forward to enabling the power of electrospray for industrial manufacturing applications in clean energy, health care, and electronics
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Novel Nanostructures And Processes For Enhanced Catalysis Of Composite Solid PropellantsDraper, Robert 01 January 2013 (has links)
The purpose of this study is to examine the burning behaviour of composite solid propellants (CSP) in the presence of nanoscale, heterogenous catalysts. The study targets the decomposition of ammonium perchlorate (AP) as a key component in the burning profile of these propellants, and seeks to identify parameters of AP decomposition reaction that can be affected by catalytic additives. The decomposition behavior of AP was studied in the presence of titanium dioxide nanoparticles in varying configurations, surface conditions, dopants, morphology, and synthesis parameters with the AP crystals. The catalytic nanoparticles were found to enhance the decomposition rate of the ammonium perchlorate, and promote an accelerated burning rate of CSP propellants containing the additives. Furthermore, different configurations were shown to have varying degrees of effectiveness in promoting the decomposition behaviour. To study the effect of the catalyst’s configuration in the bulk propellant, controlled dispersion conditions of the nanoparticle catalysts were created and studied using differential scanning calorimetry, as well as model propellant strand burning. The catalysts were shown to promote the greatest enthalpy of reaction, as well as the highest burn rate, when the AP crystals were recrystalized around the nanoparticle additives. This is in contrast to the lowest enthalpy condition, which corresponded to catalysts being dispersed upon the AP crystal surface using bio-molecule templates. Additionally, a method of facile, visible light nanoparticle tracking was developed to study the effect of mixing and settling parameters on the nano-catalysts. To accomplish this, the titania nanoparticles were doped with fluorescent europium molecules to track the dispersion of the catalysts in the propellant binder. This method was shown to succesfully allow for dispersion and agglomeration monitoring without affecting the catalytic effect of the TiO2 nanoparticles.
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Fabrication Of Functional Nanostructures Using Polyelectrolyte Nanocomposites And Reduced Graphene Oxide AssembliesChunder, Anindarupa 01 January 2010 (has links)
A wide variety of nanomaterials ranging from polymer assemblies to organic and inorganic nanostructures (particles, wires, rods etc) have been actively pursued in recent years for various applications. The synthesis route of these nanomaterials had been driven through two fundamental approaches - 'Top down' and 'Bottom up'. The key aspect of their application remained in the ability to make the nanomaterials suitable for targeted location by manipulating their structure and functionalizing with active target groups. Functional nanomaterials like polyelectrolyte based multilayered thin films, nanofibres and graphene based composite materials are highlighted in the current research. Multilayer thin films were fabricated by conventional dip coating and newly developed spray coating techniques. Spray coating technique has an advantage of being applied for large scale production as compared to the dip coating technique. Conformal hydrophobic/hydrophilic and superhydrophobic/hydrophilic thermal switchable surfaces were fabricated with multilayer films of poly(allylaminehydrochloride) (PAH) and silica nanoparticles by the dip coating technique, followed by the functionalization with thermosensitive polymer-poly(N-isopropylacrylamide)(PNIPAAM) and perfluorosilane. The thermally switchable superhydrophobic/ hydrophilic polymer patch was integrated in a microfluidic channel to act as a stop valve. At 70 degree centigrade, the valve was superhydrophobic and stopped the water flow (close status) while at room temperature, the patch became hydrophilic, and allowed the flow (open status). Spray-coated multilayered film of poly(allylaminehydrochloride) (PAH) and silica nanoparticles was fabricated on polycarbonate substrate as an anti-reflection (AR) coating. The adhesion between the substrate and the coating was enhanced by treating the polycarbonate surface with aminopropyltrimethoxylsilane (APTS) and sol-gel. The coating was finally made abrasion-resistant with a further sol-gel treatment on top of AR coating, which formed a hard thin scratch-resistant film on the coating. The resultant AR coating could reduce the reflection from 5 to 0.3% on plastic. Besides multilayered films, the fabrication of polyelectrolyte based electrospun nanofibers was also explored. Ultrathin nanofibers comprising 2-weak polyelectrolytes, poly(acrylic acid) (PAA) and poly(allylaminehydrochloride) (PAH) were fabricated using the electrospinning technique and methylene blue (MB) was used as a model drug to evaluate the potential application of the fibers for drug delivery. The release of MB was controlled in a nonbuffered medium by changing the pH of the solution. Temperature controlled release of MB was obtained by depositing temperature sensitive PAA/poly(N-isopropylacrylamide) (PNIPAAM) multilayers onto the fiber surfaces. The sustained release of MB in a phosphate buffered saline (PBS) solution was achieved by constructing perfluorosilane networks on the fiber surfaces as capping layers. The fiber was also loaded with a real life anti-depressant drug (2,3-tertbutyl-4-methoxyphenol) and fiber surface was made superhydrophobic. The drug loaded superhydrophobic nanofiber mat was immersed under water, phosphate buffer saline and surfactant solutions in three separated experiments. The rate of release of durg was monitored from the fiber surface as a result of wetting with different solutions. Time dependent wetting of the superhydrophobic surface and consequently the release of drug was studied with different concentrations of surfactant solutions. The results provided important information about the underwater superhydrophobicity and retention time of drug in the nanofibers. The nanostructured polymers like nanowires, nanoribbons and nanorods had several other applications too, based on their structure. Different self-assembled structures of semiconducting polymers showed improved properties based on their architectures. Poly(3-hexylthiophene) (P3HT) supramolecular structures were fabricated on P3HT-dispersed reduced graphene oxide (RGO) nanosheets. P3HT was used to disperse RGO in hot anisole/N, N-dimethylformamide solvents, and the polymer formed nanowires on RGO surfaces through a RGO induced crystallization process. The Raman spectroscopy confirmed the interaction between P3HT and RGO, which allowed the manipulation of the composite's electrical properties. Such a bottom-up approach provided interesting information about graphene-based composites and inspired to study the interaction between RGO and the molecular semiconductor-tetrasulphonate salt of copper phthalocyanine (TSCuPc) for nanometer-scale electronics. The reduction of graphene oxide in presence of TSCuPc produced a highly stabilized aqueous composite ink with monodispersed graphene sheets. To demonstrate the potential application of the donor (TSCuPc)'acceptor (graphene) composite, the RGO/TSCuPc suspension was successfully incorporated in a thin film device and the optoelectronic property was measured. The conductivity (dark current) of the composite film decreased compared to that of pure graphene due to the donor molecule incorporation, but the photoconductivity and photoresponsivity increased to an appreciable extent. The property of the composite film overall improved with thermal annealing and optimum loading of TSCuPc molecules.
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Aluminium surface impregnated with nano constituents for enhanced mechanical performanceCooke, Kavian O., Chudasama, P. 04 August 2022 (has links)
Yes / Aluminium alloys are widely used structural materials in automotive, aerospace, and transportation, among several other notable industries. However, aluminium alloys' low hardness and poor tribological performance prevent potential use in applications requiring high contact pressures and wear resistance. This paper presents a novel two-step technique for enhancing the mechanical properties of the aluminium alloy by impregnating the surface with Ni-coating containing hard TiO2 nanoparticles using a high-intensity electric arc generated during tungsten inert gas welding. The results show that the process significantly changes the Microstructure and mechanical properties. The surface hardness increased from 0.48 GPa to 0.65 GPa with a corresponding change of Young's modulus from 15 GPa to 24 GPa of the treated surface.
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SYNTHESIS AND OPTICAL PROPERTIES OF ULTRAFINE METAL NANOPARTICLES ON DIELECTRIC ANTENNA PARTICLESWei, Qilin, 0000-0003-1729-1951 January 2022 (has links)
Effective light energy conversion into other forms of energy in metal and metal compound nanoparticles has been of great interest in past decades. Being illuminated by incident light, electrons in the nanoparticles can be excited to higher energy states followed by deposition of energy into other molecules around their surface and the lattices in the following relaxation process. Ultrafine nanoparticles are thus preferred in these processes due to their high specific surface areas. Moreover, the portion of excited electrons with higher energies is higher in smaller nanoparticles than in larger ones. However, the overall light power absorbed by nanoparticles is proportional to the square of particle size, which causes the ultrafine nanoparticles not to efficiently absorb the incident light, or to drive further chemical or physical processes.Light antennae materials are usually employed to enhance the light absorption of these ultrafine nanoparticles. Plasmonic nanoparticles, e.g., Ag, Au, Cu, and Al nanoparticles, enhance the light absorption of loaded nanoparticles mainly through strong electromagnetic fields generated near their surfaces and have been proven to be effective light antennae to benefit the light energy conversion of ultrafine nanoparticles. On the other hand, spherical dielectric particles, e.g., silicon dioxide nanospheres, represent a different type of light antennae with the advantages of low cost, simple synthesis, and negligible Ohmic loss when being illuminated. When the sizes of high geometric symmetry dielectric nanospheres are comparable with the wavelength of the incident light, Mie scattering can happen based on the difference in refractive index between the sphere and the surrounding medium, generating size-dependent scattering resonances at various wavelengths. At these wavelengths, strong electric fields can be created on the surface of dielectric spheres to enhance the light absorption of the nanoparticles loaded on the surface. Previous works have shown that silica nanospheres with a diameter of several hundreds of nanometers can effectively enhance the light absorption of ultrafine Pt nanoparticles and benefit photocatalytic reactions, e.g., selective oxidation of benzyl alcohol. Over the past few years, this concept has been broadened to other ultrafine nanoparticles to study their novel photo-to-chemical/physical properties. However, the availability and comprehensive understanding of the optical properties of this class of composite particles still need to be improved. These challenges limit the further development of these composite materials in new light energy conversion processes. This dissertation aims at studying this class of novel ultrafine nanoparticles/dielectric sphere composite particles synthesis and optical properties.
In Chapter 2, a synthesis protocol of ultrafine ruthenium oxyhydroxide nanoparticles on the surface of silica nanospheres’ surfaces is introduced. Unlike the traditional synthesis of nanoparticles in solution followed by a loading process, the method developed in this chapter only requires the injection of aqueous ruthenium salt solution into a silica nanosphere dispersion. The obtained ultrafine nanoparticles with sizes of 2-3 nm are characterized to be ruthenium oxyhydroxide (RuOOH) nanoparticles. The silica nanospheres are crucial in stabilizing these ultrafine RuOOH nanoparticles and enhancing their light absorption. Due to the presence of ruthenium-oxygen bonds in the nanoparticles, the absorbed photons are converted to heat and transferred to the surrounding media with a photo-to-thermal conversion efficiency close to the unity. Experimental results have shown that heat can be effectively used in accelerating the reaction rate of selective oxidation of benzyl alcohol by molecular oxygen. Kinetics data also have shown that these ultrafine RuOOH nanoparticles are able to activate molecular oxygen adsorbed on their surfaces, which represents a novel property of these ultrafine RuOOH nanoparticles that is not observed in other traditional ruthenium catalysts.
In Chapter 3, a more general synthesis method of ultrafine metal and metal oxyhydroxide nanoparticles on silica nanospheres is developed, inspired by the synthetic route in Chapter 2. Instead of functionalizing silica surfaces with silane agents with amino groups, the silica surfaces are selectively etched by an aqueous base to create a high density of surface hydroxyl groups. These hydroxyl groups can provide basic sites to stabilize metal ions in aqueous dispersion, which are nuclei for the further growth of larger metal oxyhydroxide nanoparticles. In this chapter, more than ten kinds of metal ions are loaded onto silica spheres, forming oxyhydroxide nanoparticles with average sizes below 5 nm. Some oxyhydroxide nanoparticles can be reduced by 5% H2/N2 to form metal nanoparticles with their ultrafine sizes maintained. The synthesis protocol is promising in preparation of bimetallic samples. The composition and optical absorption of all obtained composite particles are analyzed, demonstrating the practicability of utilizing the reported method to prepare high-quality light-absorbing composite particles.
In Chapter 4, the optical absorption property of the composite particle is systematically studied. Using ultrafine Pt nanoparticles as the light absorbing material, the light absorptions of composite particles consisting of silica spheres with diameters from 100 to 1100 nm loaded with these Pt nanoparticles are studied. Through the combination of theoretical calculation based on Mie theory and the measured optical absorption spectra, the scattering resonance peaks are successfully located in each sample. It is also found that the photonic crystal effect and the general absorption of Pt nanoparticles can contribute to the light absorption spectra, especially at higher wavelengths. The relationship between the general absorption of Pt nanoparticles and the packing density of the powder is further studied. The successful deconvolution of several components in the absorption spectra can guide the further rational design of composite particles in optical-related applications.
In Chapter 5, the composite particle system is further broadened to using high refractive index zinc sulfide nanospheres as a light antenna. The use of a higher refractive index light antenna is promising for obtaining higher light absorption enhancement in loaded ultrafine nanoparticles, even though the sample is dispersed in organic media with a high refractive index as well. After the successful loading of Pt nanoparticles to the surface of silica-coated zinc sulfide nanospheres, a protocol for analyzing their light absorption spectra in organic media is proposed. Size-dependent scattering resonance peaks are observed in bare zinc sulfide nanospheres and can be utilized to enhance the light absorption of Pt nanoparticles, even when the sample is sealed in high refractive index polymeric matrices. The composite particles are further employed in photothermal tests, the results prove that the better light absorption enhancement using zinc sulfide than silica nanospheres.
The results introduced in this dissertation represent the first systematic and comprehensive study of ultrafine metal and metal oxyhydroxide nanoparticles loaded on the surface of dielectric light antenna particles. The conclusions open an avenue to further rational design of high-performance light-absorbing composite particles to be used in photo-to-thermal/chemical processes. / Chemistry
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Lipid-Based Delivery Systems for Therapeutic Small Molecules and RNAZhang, Chi January 2022 (has links)
No description available.
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Silver Doped Nanoceria (AgCNP) Integrated Silk Scaffold For Chronic Wound HealingVenkatesan, Architha K 01 January 2023 (has links) (PDF)
Chronic wound healing can be seriously impeded by the formation of biofilms, infections, peri-wound edema, hematoma, osteomyelitis, and the formation of reactive oxidative species (ROS). We hypothesize that a scaffold created from Silver-Doped Nanoceria (AgCNP) embedding silk can be beneficial to aid the wound healing process, inhibit inflammation and prevent microorganisms from forming a biofilm over the wound. Current wound healing methods such as intradermal injections are not advantageous to use since they can cause unwanted responses elsewhere in the body other than the wound site. Silk, however, has a positive impact on the wound healing effect and can be used as an alternative delivery method to deliver the drugs to the target site rather than intradermal injections since its degradability is controllable and it is bioresorbable, therefore it can get absorbed by the body and degrade safely without causing bodily harm. AgCNPs are used as they have antimicrobial/antioxidant properties to scavenge harmful ROS species at the wound site and can also modify silk for UV protection. As silk's degradability can be controlled, our experiment will involve collecting data on release studies conducted in vitro to see how long it takes for the silk patch to release the drugs. Our goal is to ensure the drug is not released immediately but rather over a longer controlled time manner to protect the wound while healing.
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The structural basis for lipid interactions of serum amyloid AFrame, Nicholas 07 October 2019 (has links)
Serum amyloid A (SAA) is a small, evolutionarily well-conserved, acute-phase protein best known as the protein precursor for amyloid A amyloidosis. During acute injury, infection, or inflammation, SAA plasma concentration rapidly rises 1000-fold, but the benefit of this dramatic increase is unclear. SAA functions in the innate immune response, cell signaling, and lipid homeostasis. Most SAA circulates on plasma high-density lipoproteins (HDL), where it reroutes HDL for lipid recycling. The aim of this dissertation is to provide a structural basis for understanding SAA-lipid interactions and to elucidate the structure-function relationship in this ancient protein.
SAA is an intrinsically disordered protein that acquires ~50% helical structure when bound to lipids, and is ~80% helical in three available atomic-resolution x-ray crystal structures. We took advantage of these crystal structures of lipid-free SAA to propose the binding site for various lipids, including lipids in HDL. We postulated that SAA, as a monomer, binds lipids via two amphipathic helices, h1 and h3, that form a concave hydrophobic surface, and that the curvature of this surface defines the binding preference of SAA for HDL versus larger lipoproteins.
Next, we used murine SAA1.1 and a membrane-mimicking model phospholipid, palmitoyl-oleoyl phosphocholine (POPC), to reconstitute SAA-lipid complexes and characterize their overall structure, stability and stoichiometry using an array of spectroscopic, electron microscopic, and biochemical methods. We observed preferential formation of ~10 nm particles that mimic HDL size, accompanied by the α-helical folding.
To probe the local protein conformation and dynamics in these SAA-POPC particles, we used hydrogen-deuterium exchange mass spectrometry. Analysis of the amount and the kinetics of deuterium uptake clearly established h1 and h3 as the lipid-binding site. Moreover, we determined that SAA binding to lipid follows a mixed model that combines induced fit, promoting α-folding in h3, with conformational selection, stabilizing pre-existing conformations in h1 and around the h2-h3 linker. Taken together, our results provided the structural basis necessary for understanding SAA-lipid interactions, which are central to beneficial functions of SAA as a housekeeping molecule, and to its misfolding in amyloid. This research sets the stage for understanding SAA interactions with its numerous other functional ligands.
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Functionalization of 1D and 2D Nanostructures and Their ApplicationsLi Sip, Yuen Yee 01 January 2023 (has links) (PDF)
Material discovery and development has been playing a significant role in shaping human civilizations, by studying and improving materials for appealing observations to aid in our survival as well as to satisfy our curiosity. From the common earthly materials that give us strong building structures and hunting weapons to the Silicon Age that contributes to the creation of modern electronics and computers, the development of novel and enhanced materials continues to grow. Recently, a new field has emerged that is rapidly expanding the engineering circle; these are called nanomaterials. By shrinking bulk materials into structures with nanoscale dimensions, there is a deviation from classical physics, and quantum effects begin to dominate the properties of these materials. The nanometer range brings a high surface area-volume ratio which enhances the reactivity of the material, and thus size-dependent properties are materialized. Such behaviors can be applicable in several areas such as biomedical, catalysis, optics, processing, sensing and more. Nanomaterials can be further functionalized to grant new and enhanced functions, features and capabilities needed for a specific application. This dissertation aids to explore the functionalization of 1D and 2D nanomaterials for various applications. The proposed 1D and 2D nanostructures for testing will be polymer hydrogel nanofibers and silica nanoparticulate thin films, respectively. Nanofibers are unique by acting like swollen nanoreactors to enable functionalization via aqueous absorption and reaction. Silica nanoparticulate films have high nano-porosity, which can wet the thin coating intrinsically with aqueous and organic solvents or with non-organic solvents upon additional surface chemistry modification. In this dissertation, the functionalization of 1D and 2D nanostructures with chemical compounds and metal colloids will be tested, and the performance of the nanomaterials and nanocomposites for various applications will be evaluated.
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