Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 144-161). / Understanding structure-function relationships involved in development of disease is a critical component for the rational design of successful therapeutics, allowing researchers to target precise molecular mechanisms and to anticipate and address future challenges early in development. However, for many diseases and contexts, these precise relationships are unknown or poorly defined. In this thesis, I develop new tools and implement integrated approaches to explore structure-function relationships of disease in two biological contexts: i) the interactions of proteins with glycans that can modify disease susceptibility, and ii) the interactions of viral coat proteins with antibodies that neutralize and prevent viral infection. Section 1: Glycosylation, which is the addition of sugar moieties to proteins and peptides, is one of the most abundant post-translational modifications, modifying a diverse set of biological processes in both health and disease. However, the glycome, or ensemble of glycan structures produced by a cell, is difficult to study, given the low-affinity, high-avidity structural interactions of glycans with host proteins and their non-template-driven biosynthesis. In this section, I investigate how changes in glycomic composition affect disease, and develop and implement tools and approaches that address the aforementioned challenges. In the first part, I leverage a novel in vitro model of lung adenocarcinoma metastasis to study the changes in cell surface glycosylation that correspond with and may influence metastatic progression. Here, I implement and integrate tools, such as glycan biosynthesis gene expression analysis, glycan mass spectrometry analysis frameworks, and lectin binding arrays, to measure the changes in the glycome, identifying several key motifs and glycomic features of metastatic cells. In the second part, I leverage protein-glycan structural insights to understand and predict the susceptibility of a novel seal influenza virus, H3N8, to infect human populations. Here, we used a glycan array, an assay which measures a diversity of glycan binding motifs with biologically relevant avidity and presentation, to demonstrate that unlike human-adapted H3N2, H3N8 lacks the affinity for long a2,6-linked sialylated glycans, which have been shown to determine host specificity and tropism for humans. This result was compared to other experiments, including tissue staining and in vitro replication, to determine that this seal H3N8 virus is unlikely to infect and spread within human populations. These insights further our understanding of how complex ensembles of glycans can influence susceptibility to disease. Section 2: The neutralization of emergent viral pathogens, including Ebola and Zika viruses, by therapeutic antibodies offers the potential to prevent viral infection and to treat patients even after they have been infected. However, given the real-time nature of viral outbreaks, strategies are needed which reduce the development cycle by allowing rational design and selection of potent neutralizing antibodies with minimal susceptibility to antigen escape. Here, I develop a structural, network-based computational and experimental framework which uses information about the viral coat protein and antibodies to identify key features of epitope-paratope interactions. In the context of Ebola virus, I use this analytical framework to identify two novel epitopes on the surface of the trimeric coat glycoprotein which are highly constrained, such that antibodies targeting these regions are likely to be resistant to antigen escape. Furthermore, I use this framework to uncover key differentiators of the overlapping therapeutic antibodies 2G4, 4G7, and KZ52, and describe how these differences may affect their relative susceptibility to epitope escape mutations. In the context of Zika virus, I further develop and expand this analytical framework using a computational Zika E glycoprotein assembly, and use it to understand neutralizing epitopes on the Zika virus surface in the context of their quaternary structure. In doing so, I identify critical interface residues for the 3E31, ZV67, C8, C10, Z23 and Z3L1 antibodies, and use these insights to generate C867, a novel asymmetric IgG bispecific antibody against Zika virus. This antibody has high bispecific purity, maintains in vitro potency, and given its non-overlapping epitopes, has a higher requirement for antigen escape. Taken together, the tools, frameworks, and developments presented here are important additions to the preclinical antibody development toolkit, and enable rational design and faster response to emergent viral pathogens. / by Devin Scott Quinlan. / Ph. D.

Thesis: Sc. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017. / This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Cataloged from student-submitted PDF version of thesis. / Includes bibliographical references (pages 75-83). / Reassortment is a reticulate evolutionary process that results in genome shuffling; the most prominent virus known to reassort is the influenza A virus. Methods to identify reassortant influenza viruses do not scale well beyond hundreds of isolates at a time, because they rely on phylogenetic reconstruction, a computationally expensive method. This thus hampers our ability to test systematically whether reassortment is associated with host switching events. In this thesis, I use phylogenetic heuristics to develop a new reassortment detection algorithm capable of finding reassortant viruses in tens of thousands viral isolates. Together with colleagues, we then use the algorithm to test whether reassortment events are over-represented in host switching events and whether reassortment is an alternative 'transmission strategy' for viral persistence. / by Eric J. Ma. / Sc. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Proteins and polysaccharides are of growing importance as a source for novel therapeutic compounds and target a range of diseases, from cancer to infections from pathogens. However, owing to their large and complex structures, they face a unique set of challenges, compared to small molecules, in their discovery and development as safe, efficacious drugs. Towards addressing these challenges, we describe in this thesis the implementation of structure-function relationship approaches to characterize and engineer polysaccharides and antibodies to improve their therapeutic profiles. The plant polysaccharide pectin, when modified, has demonstrated significant anticancer activity in animal models and small-scale clinical trials. Its development has been hampered, however, due to its complex structure and lack of structure-activity correlates. Using an integrated approach, we engineer a modified pectin that exhibits significant in vivo anticancer activity, which we link to specific structural attributes and cellular functional mechanisms. These results improve our structure-function understanding of anticancer modified pectin, an important step towards the clinical use of this complex polysaccharide. Applying what we learned from pectin, we develop an integrated framework to identify a contaminant in batches of heparin, a polysaccharide anticoagulant drug, associated with an outbreak of allergic-type reactions in 2007-2008. Employing orthogonal analytical approaches to overcome challenges of characterizing structurally complex pharmaceutical heparin, we determine that the structurally related glycan, oversulfated chondroitin sulfate, is the major contaminant. We link its presence to activation of the contact pathway, thereby establishing a structure-function understanding of contaminated heparin and improving the safety profile of this polysaccharide drug. Transitioning knowledge gained from the structure-function characterization of polysaccharides, we engineer, by structure-based design, a broad spectrum neutralizing antibody to dengue virus, which yearly infects more than 200 million people, causing approximately 21,000 deaths. We incorporate complementary approaches of energetics and empirical informatics methods to rationally redesign an existing antibody for greater breadth and potency, resulting in an engineered antibody with binding to all four virus serotypes and good in vitro potency. Overall, this thesis provides important insights into structure-function approaches through the use of complementary methods to characterize and engineer therapeutic polysaccharides and antibodies. / by Luke Robinson. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2010. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 137-146). / Analysis of single cells using flow cytometry techniques has created a wealth of knowledge about cellular phenomena that could not be obtained by population average measurements. As these techniques are integrated with others to increase the number of parameters that can be measured on single cells and these measurements are made more quantitative, their ability to discriminate between sub-populations of cells increases. Microfabricated sensors offer unique advantages in this area because their internal geometries can be engineered at a size scale comparable to the cell's, making them naturally well-suited for single-cell measurements. The suspended microchannel resonator (SMR) is a versatile flow cytometry platform which is capable measuring the mass of single cells with femtogram resolution. The net frequency shift of a resonant cantilever as the cell transits the fluid-filled microchannel running through it is proportional to the buoyant mass of the cell. The resonance frequency of the SMR is also highly sensitive to a cell's position along the cantilever's length. This thesis presents a new method which makes use of this property to accurately quantify the electrophoretic mobility (EPM) of cells transiting the SMR while subjected to oscillatory electric fields. Recorded resonance frequency time courses can be analyzed to extract both the buoyant mass and EPM of individual cells. This instrument has been used to simultaneously measure the EPM and buoyant masses of discrete polystyrene microspheres and Escherichia coli bacteria. As it has been applied to microspheres of known density, the integrated measurement makes it possible to compute the absolute mass and surface charge of individual microspheres. It has been shown that integrated single-microsphere mass and surface charge measurement enables differentiation of complex aqueous suspensions which is not possible by either measurement alone. / by Philip Dextras. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Nucleic acids are subject to extensive chemical modification by all organisms. These modifications display incredible structural diversity, and some are essential for survival. Intriguingly, several of these modifications are unique to bacteria, including many human pathogens. Given the enormous global disease burden due to bacterial infections, and the rapidly increasing rates of antibiotic resistance reported across the world, the need for research to address mechanisms of bacterial survival is more pressing than ever. The goal of this thesis was to determine the function of nucleic acid modifications in pathogenic bacteria, and to evaluate their impact on the three major stages of the infectious disease process: pathogenesis, diagnosis, and therapy. We first used quantitative profiling of tRNA modifications to identify novel stress responses that help mediate host invasion in the world's most common pathogen, Helicobacter pylori. This work uncovered potentially novel targets for the development of new compounds that inhibit pathogenesis. We then developed a new animal model of mycobacterial lung infection that enables drug development and biomarker screening studies in standard laboratories without high-containment facilities. We showed that infection with Mycobacterium bovis bacille Calmette-Guérin produces a granulomatous lung disease in rats that recapitulates many of the important pathological features of human tuberculosis. This model also allowed us to test the utility of nucleic acid modifications as diagnostic biomarkers. Finally, we investigated the effect of the common, transferable bacterial DNA modification phosphorothioation on oxidative and antibiotic stress responses in several pathogens. We showed that phosphorothioation can reduce the effectiveness of antibiotic therapy, which may make it an environmental source of acquired antibiotic resistance. These studies show that nucleic acid modifications play diverse roles in pathogenic bacteria, and that their modulation may be a promising target for developing new tools that can disrupt pathogenesis, improve diagnosis, and strengthen therapy. / by Brandon S. Russell. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2013 / Cataloged from PDF version of thesis. / Includes bibliographical references. / Iron is a cofactor of a number biochemical reactions that are essential for life. In the marine environment, this micronutrient is a scarce resource that limits processes of global importance such as photosynthesis and nitrogen fixation. Given that marine microorganisms play a central role in modulating such biogeochemical cycles, understanding how their distribution, diversity and activity may be affected by changes in iron availability is key. This thesis explores how the availability of iron affects the ecology of marine microbial populations and communities. At the population level, I characterized the prevalence and diversity of iron acquisition strategies in specific populations of marine vibrios with distinct micro-habitat preferences. Using a combination of genomics and functional screens, I showed that siderophore-based iron acquisition is not conserved at the organismlevel but represents a stable trait at the population level. This population-level trait further appears to play a role in driving the diversification of specific vibrio populations, especially of those that are thought to prefer particles as a micro-habitat. At the community level, I measured whole microbial community responses to iron addition in microcosm experiments in different regions of the Pacific Ocean. Using metagenomics, I characterized the impact of iron availability on the microbial community structure of the Central Equatorial Pacific Ocean. This study showed that addition of iron to an iron-limited ecosystem triggers a phytoplankton bloom dominated by Pseudo-nitZschia-like diatoms, which in turn stimulate a Bacteroidetes population functionally distinct from the ambient free-living population. In the North Pacific Subtropical Gyre, I explored how iron availability impacts microbial community gene expression dynamics. Using a metatranscriptomic approach I showed that in that environment, the impact of iron was tightly connected to the supply of other limiting macronutrients, and seems to mostly affect photosynthetic organisms. This initial study paves the way for more in depth and longer-term studies to further investigate the effects of iron on the dynamics of the microbial community in the North Pacific Subtropical Gyre. Taken together data and analyses presented in this thesis demonstrate how iron availability can shape the ecology of marine microorganisms at population, community and functional levels. / by Laure-Anne Ventouras. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2013. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 151-165). / Immunization is a powerful approach for the prevention and control of infectious disease, however despite the successes of modem vaccine development, there remain several notable obstacles for the advancement of vaccine-mediated improvements in global healthcare. Many of the current limitations in vaccine availability and administration are the result of obligate needle-based delivery, which in addition to contributing to reduced speed, ease, and compliance in administration, has been shown to contribute to reduced overall safety due to needle re-use and needle-based injuries. Needle-based vaccine delivery to immunologically passive tissues such as muscle may limit efficacy, thus motivating the targeting of more inherently potent immune-competent sites. These inherent limitations of needle-based vaccination on global health have led to a strong impetus to develop needle-free vaccination strategies which have the potential to improve vaccine efficacy and availability, enhance the ease, speed, and safety of vaccine administration, and reduce vaccination associated costs world-wide. Here we present the design and preclinical testing of several parallel materials strategies for the noninvasive delivery of subunit vaccines to the skin. We have utilized laser ablative micro-molding of poly(dimethylsiloxane) to generate bio resorbable poly(lactide-co-glycolide) micro-structured skin patches bearing -100 micron-scale needles arrayed across their surface. Upon topical application, these 'microneedle arrays' are able to safely, and painlessly insert into the immune-competent epidermal skin layers to generate microscopic conduits through which otherwise impermeant vaccines and therapeutics are able to passage into the body. We have leveraged this approach in combination with layer-by-layer (LbL) directed assembly to generate vaccine-loaded conformal coatings on the surface of these microneedle arrays, which are then delivered into the skin through topical patch application. The construction of coatings containing antigen-expressing plasmid DNA (pDNA), together with immune-stimulatory RNA, and degradable cationic polymers provided tunable control over vaccine dosage, rapid and effective vaccine delivery in murine and primate skin models, and potent immunogenicity against a model HIV antigen in mice. In this case, DNA vaccine delivery was able to elicit strong functional CD8' T cell and humoral responses matching or exceeding the potency of in vivo electroporation, currently the most promising approach for clinical DNA delivery in humans. Further efforts have explored the use of LbL for encapsulation and delivery of soluble and particulate protein subunit vaccines, giving enhanced generation of diverse and potent humoral responses in mice. In other work, we have developed an approach enabling rapid delivery of micron-scale degradable polymer matrices or hydrogel depots using dissolvable composite microneedle structures for the delivery of vaccines with programmable kinetics. These efforts have demonstrated the potential of persistent vaccine release on tuning immune potency following non-invasive microneedle delivery, including induction of potent effector and memory CD8* T cell responses and more powerful and diverse antigen-specific humoral responses. Finally, we have developed an approach for simple loading and delivery of clinically advanced recombinant adenoviral vaccine vectors from sugar-glass coatings on bioresorbable microneedles. Formulation in microneedle coatings improved vaccine stability at room temperature and preclinical testing of these vaccine patches in mice and nonhuman primates demonstrated equivalent immunogenicity compared to parenteral injection, eliciting strong systemic and disseminated mucosal CD8' and CD4* T cell responses to a model HIV antigen. These cellular responses were correlated with a similarly potent systemic and mucosal humoral response, together suggesting the utility of this approach for non-invasive adenoviral immunization in a model close to humans. Together these results strongly demonstrate the potential of materials engineering strategies for the effective formulation, delivery, and release of recombinant vaccines by microneedle patches targeting the skin. In addition to the significant practical advantages enabled by microneedle delivery including improved safety, convenience, and storage, we have shown that advanced formulation strategies paired with controlled release are able to initiate humoral and cellular adaptive immunity more potently than possible through parenteral injection. Comprehensive tests in both mice and primates have suggested that these principles may be broadly applied to enhance various recombinant vaccination strategies potentially targeting numerous disease targets. Finally, initial tests performed in nonhuman primates have indicated the promise of engineered microneedle approaches for successful translation to humans. Overall, these findings provide a strong basis for the continued development of similar vaccination strategies for the comprehensive transformation of conventional vaccination enabling significant vaccine-mediated improvements in global health. / by Peter C. DeMuth. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 135-148). / Polymeric microparticles have a wide variety of uses, ranging from traditional applications in paints and coatings, to specialized applications in medical therapeutics and diagnostics. For biological applications - including drug delivery, analytical assays, and tissue engineering - it is important to tailor the interactions between the microparticles and their external environment. To do this, it is necessary to precisely control the physical and chemical properties of the engineered microparticles. Recently, it has become apparent that in addition to particle chemistry, the physical properties of a particle - for example, size, shape, internal structure, and mechanical deformability - play an important role in determining particle behaviour in biological environments. However, it remains largely unknown exactly how these various physical properties influence particle behaviour and function, and how these properties should be exploited for different applications. This thesis focuses on the development and characterization of polymeric hydrogel microparticles with well-controlled physical and chemical properties, and shows several applications of these custom microparticles. In particular, we explore particle motifs inspired by biological entities, designing particles with different shapes, internal structure, and mechanical deformability, functionalized with proteins and nucleic acids. We employ microfluidic tools for synthesis and characterization of these hydrogel microparticles, and also investigate the interaction of functionalized particles with nucleic acids and cells, in the context of biomolecule detection and specific cell capture, respectively. Based on the microfluidic particle synthesis technique, stop flow lithography, we fabricate custom particles - including non-spherical 3D capsules and 2D extruded cylindrical rings with systematically varied internal architecture. We design microfluidic channels to study the flow and deformation of these particles, investigating the effects of internal structure, size, and stiffness on passage through microfluidic constrictions. We expand on this work, designing a microfluidic platform to specifically position particles in hydrodynamic traps, based on particle physical properties. This platform enables subsequent encapsulation of immobilized particles in monodisperse, isolated aqueous droplets. We demonstrate the platform's utility with chemically functionalized microparticles enabling sensitive, multiplexed microRNA detection. To further explore the interactions of functionalized microparticles with biological systems, we study how antibody-functionalized microparticles of varying shape can capture specific cells for future diagnostic applications. / by Lynna Chen. / Ph. D.

Thesis (Sc. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, February 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references. / With constantly increasing demand for lightweight power sources, electrode architectures that eliminate the need for conductive and organic additives will increase mass specific energy and power densities. The increased demand for lightweight power is coupled with increasing device miniaturization. As the scale of devices decreases, current battery technologies add mass on the same scale as the device itself. A dual functional electro-mechanical material that serves as both the device structural material and the power source would dramatically improve device integration and range for powered movement. To address the demand for lightweight power with the objective of a dual functional electro-mechanical material, the M 13 bacteriophage was used to create novel 3-dimensional nano-architectures. To synthesize 3-dimensional nanowire scaffolds, the M13 virus is covalently linked into a hydrogel that serves as a 3-dimensional bio-template for the mineralization of copper and nickel nanowires. Control of nanowire diameter, scaffold porosity, and film thickness is demonstrated. The nanowire scaffolds are found to be highly conductive and can be synthesized as free-standing films. To demonstrate the viability of the 3-dimensional nanowire networks for electrical energy storage, copper nanowires were galvanically displaced to a mixed phase copper-tin system. These tin based anodes were used for lithium rechargeable batteries and demonstrated a high storage capacity per square area and stable cycling approaching 100 cycles. To determine the viability of the 3-dimensional nanowire networks as dual functional electro-mechanical materials and the mechanical stability of processing intermediates, phage hydrogels, aerogels, and metal nanowire networks were examined with nano-indentation. The elastic moduli of the metal networks are in the range of open cell metal foams The demonstration of 3-dimensional virus-templated metal nanowire networks as electrically conductive and mechanically robust should facilitate their implementation across a broad array of device applications to include photovoltaics, catalysis, electrochromics, and fuel cells. / by F. John Burpo. / Sc.D.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 103-123). / Objectives: 1) To perform a quantitative comparison of proteins released to media on combination with cytokine (IL-1[beta[ or TNF-[alpha]) and Injury as compared to either treatment alone, and to thus identify proteins which may be responsible for the synergism seen between cytokine and injury in causing catabolism of cartilage in vitro. 2) To identify proteins which contribute most to some commonly observed phenotypes on treatment of cartilage with cytokine or injury or both. Methods : Cartilage explants from calves were treated with (i)IL-1 (10 ng/ml), (ii)TNF[alpha] (100 ng/ml), (iii)Injurious compression (50% strain at 100%/sec) and IL-1[beta] (10 ng/ml) or (iv)Injurious compression (50% strain at 100%/sec) and TNF-[alpha](10 ng/ml), cultured for 5 days post treatment, and the pooled media collected, labeled with one of four iTRAQ labels and subjected to nano-2D-LC/MS/MS on a quadrupole time of flight instrument. Peptides were identified and quantified using Protein PilotTM, and MATLAB scripts used to obtain protein ratios. These results were analyzed using different statistical techniques. Data from two iTRAQ experiments were combined to generate data for all possible injury and cytokine treatment conditions, and proteins on which injury and cytokines acted synergistically identified. PLSR analysis was performed using Unscrambler®X software with the combined data set to determine which proteins are most relevant to some observed phenotypes. The phenotypes chosen were sGAG released to media in 5 days post treatment, proline and sulfate incorporation rates on day 6 post treatment, and nitrite accumulation in media in 5 days post treatment Results and Discussion: TNF-[alpha]+injury and IL-[beta] +injury treatment conditions show a very high correlation with each other. Most cytosolic, ER lumen and nuclear protein levels are highly elevated with both cytokine+injury conditions, while ECM proteins are either highly down regulated or marginally elevated. Many collagen telopeptides are down regulated, possibly indicating reduced anabolism. However, attempts at repair exist, as shown by increased levels of TGF-[beta] and activin A, and reduced levels of LTBP1. Also, biglycan and lumican, SLRPs known to be involved in early development are significantly increased, possibly indicating repair attempts. Other SLRPs such as PRELP and chondroadherin are also highly elevated, with one or both injury+cytokine treatments. While MMPs are mildly down regulated or remain the same, ADAMTS1 increases with TNF-a+injury, indicating increased catabolism. Among ECM structural proteins, COMP shows high down regulation with TNF-[alpha]+injury, possibly due to reduced synthesis. Proenkephalin, a signaling molecule possibly involved in tissue/repair and apoptosis, AIMPI, a multifunctional proapoptotic, inflammatory and pro-repair cytokine and Annexin A5, a protein indicating mineralization and apoptosis are all highly elevated with cytokine+injury indicating heightened apoptosis and/or repair. When results of two 4-plex iTRAQ experiments are combined to obtain data for all possible combinations of injury and cytokine, we again find a very high correlation between TNF-a+injury and IL-1 +injury (-95%), slightly higher than the correlation between TNF-[alpha] alone and IL-[beta] alone (-90%), and much higher than the correlation of either cytokine+injury condition with cytokine alone (-70%) or injury alone (-75%). / (cont.) This shows that IL-1[beta] and TNF-[alpha] in combination with injury act through very similar pathways in chondrocytes to produce their effect on cartilage tissue. TNF-a and injury were seen to act synergistically in a positive fashion on aggrecan, CILP-2, COL6A3 and histone H4, and in a negative fashion on SPARC and IGFBP7, suggesting that these proteins may be involved in causing synergism between injury and cytokine in releasing sGAG to the media. A PLSR analysis shows that SPARC and IGFBP7 project close to proline and sulfate incorporation, and far away from sGAG, indicating that SPARC and IGFBP7 may be proteins involved in anabolism. The highest phenotype-protein positive correlations obtained using PLSR are sGAG with Perlecan, SAA3, Complement factor B, CILP-2 and pleiotropin, indicating that all these 5 proteins are associated strongly with catabolism and can serve as markers of catabolism. The correlation of inflammatory proteins SAA3 and complement factor B with sGAG indicates the role of inflammation with catabolism. Conclusion: The combination of injury and cytokine affects tissue differently at a molecular level as compared to either chemical or mechanical stresses alone. Increased catabolism and increased attempts at tissue repair are observed due to a combination of injury and cytokine, and a combination of injury and cytokine may thus serve as a useful model to study OA in vitro. / by Krishnakumar Swaminathan. / S.M.