A needlestick injury is defined by the Center for Disease Control (CDC) as a percutaneous injury due to accidental handling of a sharp. The CDC estimates that approximately 400,000 needlestick incidences occur each year in United States healthcare facilities, and reports from other developed countries, such as the United Kingdom and Spain, share similar frequencies. Further, the World Health Organization (WHO) estimates two million international healthcare workers are exposed annually to infectious disease as a consequence of a needlestick event, resulting in 37.6% and 39% of hepatitis B and hepatitis C cases, respectively. In the United States, federal and state legislation have greatly reduced incidence rates since the late 1980s, providing education, better protocols and effective post-exposure management. Additionally, the introduction of national surveillance databases led to stronger epidemiological support for the causation of needlestick injury and consequently, a stronger national awareness. In an effort to better protect healthcare workers, corporations such as DuPont and BD have further reduced needlestick incidences in the United States by designing products ranging from safety-engineered syringes to adhesive strips surrounded in strong synthetic materials such as Kevlar® and Lycra®. These devices are instrumental in minimizing the needlestick problem in both the clinic and in the operating room. As part of the current United States legislation, healthcare organizations are mandated to implement and utilize these safety-engineered syringes and needles. Despite the rise in protective equipment, national database surveillance and federal/state legislature, the incidence rate remains high as hundreds of thousands of injuries persist each year. We sought to find other solutions for better protecting healthcare workers through the implementation of golden orb weaver spider silk in personal protective equipment. This silk, gathered from the Nephila clavipes, is one of the strongest and toughest biomaterials in known existence. Its characteristically high energy absorption makes it an ideal material for reinforcing gloves and other protective equipment for healthcare workers. We believe that products made from this silk would serve as strong barriers against needlestick injury and bloodborne pathogen exposure. We are in the process of designing and fabricating such a glove and completed preliminary strength testing to ensure the superiority of our material. Tensile testing conducted at Tufts' Department of Biomedical Engineering suggests that our silk possesses the same mechanical profile as N. clavipes silk found in published literature. We plan on utilizing Fourier-transform infrared (DSC-FTIR) microspectroscopy to study the protein structure and possibly conducting enzyme degradation assays to assess the property changes under unique conditions. This information combined with our patented extraction and reinforcing methodology will provide the groundwork for partnering with industry leaders to make this product a reality and help eliminate the incidence of needlestick injury.

Spider silks are biological materials that have inspired the humankind since its beginning. From raising the interest of ancient philosophers to the practical outcomes in the societies, spider silks have always been part of our culture and, thus, of our scientific development. They are protein-based materials with exceptional mechanical and biological properties that from liquid solutions passes to the solid fibres once extruded from the body of the spiders. Spider silks have deeply been investigated in these decades for their possible outcomes in biomedical technology as a supporting material for drugs delivery or tissues regeneration. Furthermore, spiders build webs with the support of different types of silks to create mechanically efficient structures, which are currently under investigation as models for metamaterials and fabrics with superior mechanical properties. This diversity in materials and structures makes spider silks scientific outcomes potentially infinite. In this work, we present some of the outputs of these three years of PhD. We explored the properties of the native material across different aspects (different species and glands) and trying to find possible derived applications (tissue engineering). Then we explored the mechanical behaviour of the natural structures (such as orb webs or attachment discs) coupled with their biological functions. In order to develop to an industrial level this material, we tried to understand and improve the physical properties of artificial spider silk, which helps also in understanding the ones of the native materials.

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abstract: Spider dragline silk is an outstanding biopolymer with a strength that exceeds steel by weight and a toughness greater than high-performance fibers like Kevlar. For this reason, structural and dynamic studies on the spider silk are of great importance for developing future biomaterials. The spider dragline silk comprises two silk proteins, Major ampullate Spidroin 1 and 2 (MaSp1 and 2), which are synthesized and stored in the major ampullate (MA) gland of spiders. The initial state of the silk proteins within Black Widow MA glands was probed with solution-state NMR spectroscopy. The conformation dependent chemical shifts information indicates that the silk proteins are unstructured and in random coil conformation. 15N relaxation parameters, T1, T2 and 15N-{1H} steady-state NOE were measured to probe the backbone dynamics for MA silk proteins. These measurements indicate fast sub-nanosecond timescale backbone dynamics for the repetitive core of spider MA proteins indicating that the silk proteins are unfolded, highly flexible random coils in the MA gland. The translational diffusion coefficients of the spider silk proteins within the MA gland were measured using 1H diffusion NMR at 1H sites from different amino acids. A phenomenon was observed where the measured diffusion coefficients decrease with an increase in the diffusion delay used. The mean displacement along the external magnetic field was found to be 0.35 μm and independent of the diffusion delay. The results indicate that the diffusion of silk protein was restricted due to intermolecular cross-linking with only segmental diffusion observable. To understand how a spider converts the unfolded protein spinning dope into a highly structured and oriented in the super fiber,the effect of acidification on spider silk assembly was investigated on native spidroins from the major ampullate (MA) gland fluid excised from Latrodectus hesperus (Black Widow) spiders. The in vitro spider silk assembly kinetics were monitored as a function of pH with a 13C solid-state Magic Angle Spinning (MAS) NMR approach. The results confirm the importance of acidic pH in the spider silk self-assembly process with observation of a sigmoidal nucleation-elongation kinetic profile. The rates of nucleation and elongation and the percentage of β-sheet structure in the grown fibers depend on pH. The secondary structure of the major ampullate silk from Peucetia viridians (Green Lynx) spiders was characterized by X-ray diffraction (XRD) and solid-state NMR spectroscopy. From XRD measurement, β-sheet nano-crystallites were observed that are highly oriented along the fiber axis with an orientational order of 0.980. Compare to the crystalline region, the amorphous region was found to be partially oriented with an orientational order of 0.887. Further, two dimensional 13C-13C through-space and through-bond solid-state NMR experiments provide structural analysis for the repetitive amino acid motifs in the silk proteins. The nano-crystallites are mainly alanine-rich β-sheet structures. The total percentage of crystalline region is determined to be 40.0±1.2 %. 18±1 % of alanine, 60±2 % glycine and 54±2 % serine are determined to be incorporated into helical conformations while 82±1 % of alanine, 40±3 % glycine and 46±2 % serine are in the β-sheet conformation. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2015

abstract: Spider dragline silk is well known for its outstanding mechanical properties - a combination of strength and extensibility that makes it one of the toughest materials known. Two proteins, major ampullate spidroin 1 (MaSp1) and 2 (MaSp2), comprise dragline silk fibers. There has been considerable focus placed on understanding the source of spider silk's unique mechanical properties by investigating the protein composition, molecular structure and dynamics. Chemical compositional heterogeneity of spider silk fiber is critical to understand as it provides important information for the interactions between MaSp1 and MaSp2. Here, the amino acid composition of dragline silk protein was precisely determined using a solution-state nuclear magnetic resonance (NMR) approach on hydrolyzed silk fibers. In a similar fashion, solution-state NMR was applied to probe the "13"C/"15"N incorporation in silk, which is essential to understand for designing particular solid-state NMR methods for silk structural characterization. Solid-state NMR was used to elucidate silk protein molecular dynamics and the supercontraction mechanism. A "2"H-"13"C heteronuclear correlation (HETCOR) solid-state NMR technique was developed to extract site-specific "2"H quadrupole patterns and spin-lattice relaxation rates for understanding backbone and side-chain dynamics. Using this technique, molecular dynamics were determined for a number of repetitive motifs in silk proteins - Ala residing nanocrystalline &beta-sheet; domains, 3"1"-helical regions, and, Gly-Pro-Gly-XX &beta-turn; motifs. The protein backbone and side-chain dynamics of silk fibers in both dry and wet states reveal the impact of water on motifs with different secondary structures. Spider venom is comprised of a diverse range of molecules including salts, small organics, acylpolyamines, peptides and proteins. Neurotoxins are an important family of peptides in spider venom and have been shown to target and modulate various ion channels. The neurotoxins are Cys-rich and share an inhibitor Cys knot (ICK) fold. Here, the molecular structure of one G. rosea tarantula neurotoxin, GsAF2, was determined by solution-state NMR. In addition, the interaction between neurotoxins and model lipid bilayers was probed with solid-state NMR and negative-staining (NS) transmission electron microscopy (TEM). It is shown that the neurotoxins influence lipid bilayer assembly and morphology with the formation of nanodiscs, worm-like micelles and small vesicles. / Dissertation/Thesis / Ph.D. Chemistry 2014

Cette thèse a visé à comprendre et à recréer artificiellement un mécanisme d'auto-assemblage présent dans la soie d'araignée. Les gouttes de glue microniques qui existent sur la soie d'araignée dîte de capture servent à fournir à la toile ses propriétés adhésives. Ces gouttes jouent pourtant un autre rôle : elles améliorent grandement les propriétés mécaniques de la soie, et permettent de préserver l'intégrité structurelle de la toile. La localisation de l'instabilité de flambage au sein des gouttes de glue, site de surcompression par les ménisques capillaire, implique que ce système de gouttes sur fibre se comporte sous compression comme un liquide, alors que sous tension il possède un régime solide. Les araignées ont donc trouvé un moyen de créer des hybrides mécaniques liquide-solide.La première partie de ma thèse fut dédiée à la caractérisation d'échantillons naturels, qui a permis dans la seconde partie de construire un système entièrement artificiel qui reproduit la soie d'araignée de capture, grâce à des microfibres flexibles longues de plusieurs centimètres. Une simple goutte de liquide mouillant permet la création efficace d'un système semblable aux échantillons naturels. La caractérisation fine de ces systèmes de gouttes sur fibre enroulables a mené à un très bon accord entre les résultats expérimentaux, les simulations numériques et une analogie avec les transitions de phase, notamment pour des propriétés telles que le seuil d'activation, l'existence d'une hystérésis ou encore la morphologie de l'enroulement. Ces résultats ont permis la conception de techniques non conventionnelles dans des domaines tels que les méta-matériaux et la micro-fabrication. / This PhD work aimed to understand and recreate artificially a self-assembling mechanism involving capillarity and elasticity present in spider silk. The primary function of the micronic glue droplets that exist on spider capture silk is to provide the spider web with adhesive properties. These droplets play yet another role: the dramatic enhancement of silk mechanical properties, as well as the preservation of the integrity of the web structure. The localization of the buckling instability within the glue droplets, site of over-compression due to the capillary meniscii implies that under compression this special drop-on-fibre system behaves like a liquid, whereas under tension it has a classical elastic spring regime. Spiders have thus found a way to create liquid-solid mechanical hybrids.The first part of my thesis aimed to the characterization of natural samples, which allowed in the second part to build a completely artificial system that mimics the natural samples, through fabrication of centimeter-long micronic soft fibres. The simple addition of a wetting liquid droplet made for an effective system with mechanical properties quantitatively close to that of spider capture silk.Fine characterization of the created drop-on-coilable-fibre systems yielded very good agreement between experimental results and predictions from numerical simulations and a analogy with phase transition, especially for properties such as the threshold for activation, the existence of an hysteresis and the coiling morphology. All those results added up to the design of unconventional techniques in field such as metamaterials and micro-fabrication.

To improve measurement reliability and repeatability and resolve the orders of magnitude discrepancy between the two different measurements (via reduced model transient electrothermal and lock-in IR thermography), this dissertation details the development of three complementary methods to accurately measure the thermal properties of the natural and synthetic Nephila (N.) clavipes spider dragline fibers. The thermal conductivity and diffusivity of the dragline silk of the N. clavipes spider has been characterized by one research group to be 151-416 W m−1 K −1 and 6.4-12.3 ×10−5 m2 s −1 , respectively, for samples with low to high strains (zero to 19.7%). Thermal diffusivity of the dragline silk of a different spider species, Araneus diadematus, has been determined by another research group as 2 ×10−7 m2 s −1 for un-stretched silk. This dissertation seeks to resolve this discrepancy by three complementary methods. The methods detailed are the transient electrothermal technique (in both reduced and full model versions), the 3ω method (for both current and voltage sources), and the non-contact, photothermal, quantum-dot spectral shape-based fluorescence thermometry method. These methods were also validated with electrically conductive and non-conductive fibers. The resulting thermal conductivity of the dragline silk is 1.2 W m−1 K −1 , the thermal diffusivity is 6 ×10−7 m2 s −1 , and the volumetric heat capacity is 2000 kJ m−3 K −1 , with an uncertainty of about 12% for each property

Spider silk has received significant attention due to its fascinating mechanical properties. Given the solitary and cannibalistic behavior of spiders, spider silk farming is impractical. Unlike spiders, silkworms are capable of producing large quantities of a fibrous product in a manner mimetic to spiders, and there already exists an industry to process cocoons into threads and textiles for many applications. The combination of silk farming (sericulture), a millennia old practice, and modern advancements in genetic engineering has given rise to an innovative biomaterial inspired by nature; transgenic silkworm silk. This project focuses on the creation of chimeric silkworm-spider silk fibers through the genetic modification of silkworms. Advanced genetic engineering techniques were used to introduce the minor ampullate spider silk (MiSp) genes into the silkworm genome. A subset of these transgenic silkworms was cross-bred with other transgenic silkworms containing the same spider silk gene in a different section of the silkworm genome to create hybrid, dual-transgenic silkworms. The transgenic silk samples showed increased mechanical properties compared to native silkworm fibers, with the strongest fibers approaching or surpassing the mechanical properties of native spider silk. The transgenic silk retained the elasticity of the native silkworm silk and gained the strength of the spider silk. Ultimately, genetic engineering opens the door to mass produce synthetic spider silk in an established organism and industry, and the results of this project demonstrate that the properties of silkworm silk can be predictably altered through this technology.