476 research outputs found
Characterization and Bioanalysis of Protein-Based Biopharmaceuticals, Peptides and Amino Acids by Liquid Chromatography and Mass Spectrometry
Biopharmazeutika sind zu einer essenziellen Klasse von Therapeutika geworden und werden fĂŒr verschiedene medizinische Indikationen wie Diabetes, Krebs, entzĂŒndliche Erkrankungen und Infektionskrankheiten eingesetzt. Monoklonale Antikörper (mAbs) haben innerhalb der Biopharmazeutika den gröĂten Anteil bezogen auf die Zulassungszahlen. Den Vorteilen bezĂŒglich hoher SpezifitĂ€t und EffektivitĂ€t stehen jedoch Nachteile durch hohe Kosten und erhöhter KomplexitĂ€t gegenĂŒber. Die KomplexitĂ€t ergibt sich einerseits aufgrund des hohen Molekulargewichts und anderseits aufgrund der strukturellen HeterogenitĂ€t, wodurch die analytische Charakterisierung und QualitĂ€tskontrolle von mAbs und anderer Biopharmazeutika zu einer Herausforderung wird. Neben diesen protein-basierten Biopharmazeutika ist auch die AufklĂ€rung der absoluten Konfiguration von therapeutischen und natĂŒrlichen (Lipo)peptiden von besonderem Interesse fĂŒr die Wirkstoffforschung.
Zur BewĂ€ltigung dieser Herausforderungen wurden in der hier prĂ€sentierten Arbeit flĂŒssigchromatographische (LC) und massenspektrometrische (MS) Methoden fĂŒr die umfassende Analyse eingesetzt. Die erste Publikation dieser Dissertation bezog sich auf die Analyse von Ladungsvarianten von mAbs, welche wichtige QualitĂ€tsmerkmale darstellen und die Sicherheit und Wirksamkeit des Arzneimittels beeinflussen können. Zur Charakterisierung der Ladungsvarianten wurden die mAbs auf Ebene des intakten Proteins als auch auf Fragmentebene nach begrenztem Verdau und Reduzierung der DisulfidbrĂŒcken mittels starker KationenaustauschflĂŒssigkeitschromatographie (SCX) analysiert. Die SCX-Methode wurde systematisch mittels statistischer Versuchsplanung (DoE) dahingehend optimiert, die höchstmögliche Anzahl an Ladungsvarianten zu trennen. Die mobile Phase der optimierten SCX-Methode enthielt jedoch eine hohe Konzentration an nicht-flĂŒchtigen Salzen, wodurch sie nicht mit MS Detektion kompatibel ist, welche wiederum entscheidend fĂŒr die Identifikation der Ladungsvarianten ist. Um dieser Herausforderung zu begegnen, wurde erfolgreich eine online zweidimensionale flĂŒssigchromatographische (2D-LC) Methode entwickelt, bei der SCX in der ersten Trenndimension und UmkehrphasenflĂŒssigchromatographie (RP-LC) in der zweiten Trenndimension zur Entsalzung vor der MS Detektion verwendet wurde. Die Entwicklung einer extrem kurzen (†1 min) RP-LC Methode war unabdingbar zur Etablierung einer umfassenden 2D-LC Methode. Dazu wurde eine SĂ€ulenvergleichsstudie mit monolithischen und oberflĂ€chlich porösen PartikelsĂ€ulen (SPP-SĂ€ulen) durchgefĂŒhrt und die Trenneffizienz sowie die Analysengeschwindigkeit untersucht.
Eine noch umfassendere SĂ€ulenvergleichsstudie mit Fokus auf das kinetische Leistungsvermögen wurde in der zweiten Arbeit dieser Dissertation durchgefĂŒhrt. Eine Auswahl von 13 RP-ProteintrennsĂ€ulen inklusive monolithischer, SPP und vollporöser PartikelsĂ€ulen (FPP-SĂ€ulen) wurde hinsichtlich ihrer FĂ€higkeit, Peaks in der kĂŒrzest möglichen Zeit zu trennen, untersucht. Es konnte gezeigt werden, dass SPP-SĂ€ulen mit einer PorengröĂe von etwa 400 Ă
und einer dĂŒnnen, porösen Schicht die beste Performance insbesondere fĂŒr gröĂere Proteinen besitzen.
Proteine selbst können auch potenzielle Ziele fĂŒr Arzneistoffe sein, wie z.B. das Tumorsuppressorprotein p53, welches in der dritten Publikation dieser Arbeit untersucht wurde. Intakte Protein LC-MS wurde erfolgreich verwendet, um die Bindungseffizienz und -spezifitĂ€t des kovalenten Inhibitors an p53 nachzuweisen.
AminosĂ€uren sind die Bausteine von Proteinen und Peptiden und die Mehrheit dieser AminosĂ€uren sind chiral. Die biologische AktivitĂ€t ist in der Regel abhĂ€ngig von der absoluten Konfiguration der AminosĂ€uren, wodurch die enantiomerenselektive Analyse von höchster Wichtigkeit fĂŒr die StrukturaufklĂ€rung und zur QualitĂ€tskontrolle ist. Daher war die Entwicklung schneller und umfassender Trennmethoden zur Analyse von AminosĂ€uren, deren Enantiomeren, Diastereomeren und konstitutionellen Isomeren ein Ziel dieser Arbeit. Dieses konnte durch Derivatisierung mittels 6-Aminochinolyl-N-hydroxysuccinimidylcarbamat (AQC) und anschlieĂender Analyse durch enantioselektiver flĂŒssigchromatographischer IonenmobilitĂ€ts-Massenspektrometrie (LC-IM-MS) erreicht werden. Eine sehr schnelle dreiminĂŒtige Analysenmethode konnte entwickelt und zur StrukturaufklĂ€rung von therapeutischen Peptiden und eines natĂŒrlichen Lipopeptides eingesetzt werden.
Die absolute Konfiguration eines Tetrapeptides als Bestandteil des natĂŒrlichen, antimikrobiellen Peptidpolyensâ Epifadin konnte mittels chiraler LC-MS bestimmt werden, was wiederum entscheidend fĂŒr die StrukturaufklĂ€rung war. In dieser Arbeit konnten alle acht Enantiomerenpaare erfolgreich getrennt werden und die Diastereomerentrennung wurde optimiert.Biopharmaceuticals have become an essential class of therapeutics and are used for different medical indications such as diabetes, cancer, inflammatory diseases, and infectious diseases. Monoclonal antibodies (mAbs) have the biggest share within the biopharmaceuticals regarding the drug approval numbers. However, the benefits in terms of high specificity and efficacy come with the drawback of higher cost and higher complexity. This complexity arises from the high molecular weight on the one hand and high structural heterogeneity on the other hand, making the analytical characterization and quality control of mAbs and other biopharmaceuticals a significant challenge. In addition to these protein-based biopharmaceuticals, the elucidation of the absolute configuration of therapeutic peptides and natural (lipo)peptides is also of particular interest for drug discovery.
To address these challenges, different liquid chromatography (LC) and mass spectrometric (MS) methods were used for the more comprehensive analysis in the presented work. The first publication of this dissertation was dedicated to the analysis of charge variants of mAbs, which is an important quality attribute that might affect safety and efficacy of the drug product. To characterize the charge variants, the mAbs were analysed at the intact protein level and the subunit level after limited digestion and disulphide reduction using strong cation-exchange chromatography (SCX). The SCX method was systematically optimized to enable the separation of the maximum number of charge variants using a design of experiments (DoE) approach. The optimized SCX mobile phase, however, contains high concentrations of non-volatile salt in the mobile phase, which is incompatible with MS detection. On the other hand, MS analysis is essential for the identification of the charge variants. To overcome this limitation, an online two-dimensional liquid chromatographic (2D-LC) method was successfully developed, which uses SCX in the first separation dimension and reversed-phase (RP) LC in the second separation dimension, which can be used for de-salting prior MS analysis. An ultra-short analysis time (†1 min) of the second dimension RP method was essential to establish a full comprehensive 2D-LC analysis. For this purpose, a column comparison study was performed using a set of monolithic and superficially porous particle (SPP) columns, and the separation efficiency and analysis speed were investigated.
An even more comprehensive column comparison study focusing on the kinetic performance was done for the second work presented in this dissertation. A set of 13 RP protein separation columns including monolithic, SPP, and fully porous particle (FPP) columns was investigated regarding their capability to separate peaks in the shortest possible time. It could be demonstrated that SPP columns with a pore size of 400 Ă
and a thin, porous shell provided the best performance especially for large proteins such as mAbs.
Proteins themselves can also be the potential targets of drug products such as the tumour suppressor protein p53 studied in publication III. Intact protein LC-MS was successfully used to investigate the binding efficiency and specificity of covalent inhibitors.
Amino acids are the building blocks of proteins and peptides and most of these amino acids are chiral. As the biological activity is usually dependent on the absolute configuration of the amino acids, the enantioselective analysis is of utmost importance for structural elucidation and quality control. Therefore, one goal of the presented work was to develop a fast and comprehensive method to separate amino acids, their enantiomers, diastereomers, and constitutional isomers. This was achieved by derivatization using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and subsequent analysis by enantioselective liquid chromatography ion mobility-mass spectrometry (LC-IM-MS). A very fast three minutes short analysis method could be developed and was applied for the successful structure elucidation of a therapeutic peptide and a natural lipopeptide.
The absolute configuration of a tetrapeptide originating from the natural antimicrobial peptide-polyene epifadin could be determined using chiral LC-MS, which was crucial for the structure elucidation. In this work, all eight enantiomer peak pairs could be successfully separated and the separation of the diastereomers was optimized
Effects of municipal smoke-free ordinances on secondhand smoke exposure in the Republic of Korea
ObjectiveTo reduce premature deaths due to secondhand smoke (SHS) exposure among non-smokers, the Republic of Korea (ROK) adopted changes to the National Health Promotion Act, which allowed local governments to enact municipal ordinances to strengthen their authority to designate smoke-free areas and levy penalty fines. In this study, we examined national trends in SHS exposure after the introduction of these municipal ordinances at the city level in 2010.MethodsWe used interrupted time series analysis to assess whether the trends of SHS exposure in the workplace and at home, and the primary cigarette smoking rate changed following the policy adjustment in the national legislation in ROK. Population-standardized data for selected variables were retrieved from a nationally representative survey dataset and used to study the policy actionâs effectiveness.ResultsFollowing the change in the legislation, SHS exposure in the workplace reversed course from an increasing (18% per year) trend prior to the introduction of these smoke-free ordinances to a decreasing (â10% per year) trend after adoption and enforcement of these laws (ÎČ2â=â0.18, p-valueâ=â0.07; ÎČ3â=ââ0.10, p-valueâ=â0.02). SHS exposure at home (ÎČ2â=â0.10, p-valueâ=â0.09; ÎČ3â=ââ0.03, p-valueâ=â0.14) and the primary cigarette smoking rate (ÎČ2â=â0.03, p-valueâ=â0.10; ÎČ3â=â0.008, p-valueâ=â0.15) showed no significant changes in the sampled period. Although analyses stratified by sex showed that the allowance of municipal ordinances resulted in reduced SHS exposure in the workplace for both males and females, they did not affect the primary cigarette smoking rate as much, especially among females.ConclusionStrengthening the role of local governments by giving them the authority to enact and enforce penalties on SHS exposure violation helped ROK to reduce SHS exposure in the workplace. However, smoking behaviors and related activities seemed to shift to less restrictive areas such as on the streets and in apartment hallways, negating some of the effects due to these ordinances. Future studies should investigate how smoke-free policies beyond public places can further reduce the SHS exposure in ROK
Bridging Micro- and Macro- Evolution In Tropical Fishes
In marine environments, barriers to dispersal can be challenging to identify because they are often cryptic. Unlike terrestrial environments, where a mountain chain that is visible can physically separate two populations of animals, vast masses of water in the ocean make it challenging to pinpoint these barriers. Therefore, the impact of these barriers on the formation of new species in the ocean is still not well understood. While most marine populations have long been considered to be well connected via long-distance dispersal, molecular ecology studies are increasingly unveiling inconspicuous barriers that promote population divergence and ultimately speciation. The advent of genomic techniques that allow the generation of data for thousands of genes has provided an unprecedented opportunity to uncover marine barriers that were previously invisible using more rudimentary tools. This, in turn, has opened new avenues for understanding of how barriers to dispersal affect population connectivity in the marine environment. The overarching goal of my dissertation is to use genome-wide data to look for genetic patterns that correspond to such barriers, and to test for their effect at short-, intermediate- and long-term evolutionary scales, going through a continuum from micro- to macro-evolution, in a time span from thousands to millions of years.
At the short-scale, I examined two controversial cases of species delimitation. Species delimitation is a major question in biology and is essential for adequate management of organismal diversity. The first challenging case involves the red snappers in the Western Atlantic. Red snappers have been traditionally recognized as two separate species based on morphology: Lutjanus campechanus (northern red snapper) and L. purpureus (southern red snapper). However, recent genetic studies using few molecular markers failed to delineate these nominal species, lumping the northern and southern populations into a single species (L. campechanus). To evaluate if the populations of these fish represent one or two species, my project applied ca. 40,000 genome-wide markers of 178 individuals collected throughout the range of the two species and population and species delimitation analyses. Overall, my results supported the isolation and differentiation of these species, a result that confirmed the morphology-based delimitation scenario, highlighting the benefits of using genome-wide data in complex cases of species delimitation (Chapter I, published in Proc. Roy. Soc. B in 2019).
The second study case involves a species complex of silverside fishes (Chirostoma humboltianum group: Atherinidae) in the Central Mexico plateau. The humboltianum group represents a taxonomically-controversial species complex where previous morphological and molecular studies based on a few genes produced conflicting species delineation scenarios. I applied an integrative approach that considered multiple lines of evidence to investigate the species numbers and boundaries comprising this contentious group. I used ca. 33,000 molecular markers for 77 individuals representing the nine nominal species in the group, spanning their distribution range in the central Mexico plateau, in combination with morphologic and ecologic information. My findings are inconsistent with the morphospecies and ecological delimitation scenarios, identifying three to four species. This study provides an atypical example in which genome-wide analyses delineate fewer species than previously recognized on the basis of morphological data alone. It also highlights the influence of geologic history as a main driver of speciation in the group (Chapter II, published in BMC Eco. Evol. B in 2022).
At the intermediate- scale, I evaluated the influence of historical (e.g., geophysical events) and contemporary barriers (e.g., habitat gaps) hindering genetic flow among populations by studying the spatio-temporal phylogenetic concordance of co-distributed lineages. For this study, I investigated the comparative phylogeography of labrisomid blennies in the genus Malacoctenus. I generated data for ca. 28K genome-wide markers that were sequenced from over 500 individuals collected from 38 locations, representing 23 (out of 25) species of Malacoctenus. With this dataset, I assessed the effect of recognized historical (e.g., the rising of the Isthmus of Panama) and contemporary barriers (e.g., sandy gaps) in the Tropical Eastern Pacific (TEP) and the Tropical Atlantic (TA) biogeographic realms. These blennies represent an ideal system to test the effect of such barriers as they are strongly associated with rocky habitats and coral reefs. Therefore, subtle habitat disruptions may lead to genetic isolation. At the micro-evolutionary scale, the observed population structure patterns identified the Sinaloan and Central American breaks as the major breaks in the TEP; and the Bahamas and Eastern Caribbean breaks as key barriers disrupting connectivity in the TA. All in all, the effect of these breaks varies across species, suggesting that species-specific traits (e.g., habitat preference), also greatly influence their dispersal capabilities. My study identified five instances where marine barriers promoted the diversification of independent evolutionary lineages that could potentially represent species complexes. Some of them supported by evidence of population differentiation from previous morphological analyses as well as by my geometric morphometric analyses. Major environmental variables driving population differentiation in the TEP are depth, temperature, chlorophyll altogether with spatial components, while in the TA suspended particle matter also influences diversification.
At the long-term scale, my results suggest that depth is a primary driver of speciation in the TEP, leading to niche divergence between tide pool- and reef-associated clades. In contrast, in the TA, patterns of environmental association appeared more intricate, where depth, temperature, chlorophyll and physical features significantly contributing to speciation in this region. Finally, our time-calibrated analyses at macroevolutionary scales elucidated an Eastern Atlantic origin of the clade followed by an east-to-west dispersal. Although the historical break attributed to the rise of the Isthmus of Panama had a substantial influence on the evolutionary history of the genus, our analyses demonstrate that it did not triggered synchronous cladogenetic events. In summary, by using a combination of population genomics, comparative phylogeography, phylogenomics, seascape genomics, and geometric morphometric approaches, this study highlights major contemporary and historical barriers hindering population connectivity in the TEP and TA biogeographic regions, enhancing our understanding of the forces and processes generating new species in marine systems (Chapter III, to be submitted for publication).
All in all, my thesis highlights that the use of genome-wide data provides unprecedented resolution to unveil patterns of genetic structure, commonly unraveling cryptic diversity, and the opportunity to address species delimitation problems. By uncovering the spatio-temporal genetic patterns of fishes along the evolutionary continuum, my dissertation provides novel insights into the evolutionary and biogeographic history of marine and freshwater Neotropical fishes. Overall, my dissertation not only helps to understand the evolutionary history of the species under study, but more generally, elucidate factors driving evolutionary process in the marine realm, ranging from population-level scales, to speciation, to higher level relationships among groups
Exoteric effects at nanoscopic interfaces - Uncommon negative compressibility of nanoporous materials and unexpected cavitation at liquid/liquid interfaces
This PhD thesis is devoted to the investigation of some peculiar effects happening at nanoscopic interfaces between immiscible liquids or liquids and solids via molecular dynamics simulations. The study of the properties of interfaces at a nanoscopic scale is driven by the promise of many interesting technological applications, including: a novel technology for developing both eco-friendly energy storage devices in the form of mechanical batteries, as well as energy dissipation systems and, in particular, shock absorbers for the automotive market; biomedical applications related to cavitation, such as High-Intensity Focused Ultrasound (HIFU) ablation of cancer tissues and localised drug delivery, and many more. The kinetics of phenomena taking places at these scales is typically determined by large free-energy barriers separating the initial and final states, and even intermediate metastable states, when they are present. Because of such barriers, the phenomena we are interested in are "rare events", i.e. the system attempts the crossing of the barrier(s) many times before finally succeeding when an energy fluctuation makes it possible. At the same time, the magnitude of the barrier is determined by the energetics and dynamics of atoms, which forces us to model the system by taking into account both the femtosecond atomistic timescale and the timescale of the relevant phenomena, typically exceeding the former by several orders of magnitude. These longer timescales are inaccessible to standard molecular dynamics, so, in order to tackle this issue, advanced MD techniques need to be employed.
The thesis is divided into two parts, corresponding to the main lines of research investigated, which are (I) the interfaces between water and complex nanoporous solids, and (II) planar solid-liquid and liquid-liquid interfaces. Anticipating some results, atomistic simulations helped uncovering the microscopic mechanism behind the (incredibly rare!) giant negative compressibility exhibited by the ZIF-8 metal organic framework (MOF) upon water intrusion. Molecular dynamics simulations also supported experimental results showing how it is possible to change the intermediate intrusion-extrusion performance of ZIF-8 by changing its grain morphology and arrangement, from a fine powder to compact monolith. Free-energy MD calculations allowed to explain the exceptional stability of surface nanobubbles in water, at undersaturated conditions, on a surprisingly wide variety of substrates, characterized by disparate hydrophobicities and gas affinities; and yet, how they catastrophically destabilize in organic solvents. Finally, through simulations, some light was shed upon the working mechanism behind the novelly discovered phenomenon of how the interface between two immiscible liquids can act as a nucleation site for cavitation
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Assembly of polymer colloids at fluid interfaces through external fields and nanoscale surface topography
The superposition of dipolar repulsion and capillary attraction energies between colloidal particles pinned at fluid interfaces dictates their microstructural organization and therefore the macroscopic interfacial material properties of particle-stabilized emulsions and 2D monolayer materials. While isotropic, spherical, particles have been extensively utilized, expanding the possible applications and material property tunability via anisotropic particles has been a challenge due to the propensity of particles to form disordered aggregates at the interface. My thesis presents the synthesis of anisotropic polymer ellipsoids and the development and use of experimental tools to study their interfacial behavior to reveal how dipolar and capillary interactions can be manipulated through external fields and particle topography in order to ultimately create novel 2D monolayer interfacial assemblies.
We first demonstrated a technique to apply an elongation gradient to manufacture multiple batches of lab-scale quantities of colloidal ellipsoids in a single step. A common route to creating anisotropic colloids is the mechanical stretching of spherical polymer colloids into ellipsoids above the glass transition temperature of the polymer. While this general method has been well studied in the production of a single aspect ratio colloidal ellipsoid per batch, we extended the technique to cover the production of multiple monodisperse samples of colloidal ellipsoids. Because these colloidal ellipsoids each underwent the same synthetic procedure, we were able to modulate particle aspect ratio as an independent variable with no sample-to-sample batch variation. Further, we were able to model the elongation of the particles based on the film characteristics to predict their final aspect ratio.
Next, we used these particles to investigate the assembly of ellipsoids under external AC electric fields, both in bulk and at air-water interfaces. We investigated ellipsoidal particle alignment in bulk aqueous solution in order to narrow down the phase space of applied AC electric field strength and frequency, as well as ionic strength to conditions favorable to particle reorientation. We then used these results to inform studies on these same particles at an air-water interface. We developed a first of its kind Mirau interferometer that can measure the relative height profile of the fluid surrounding an interfacially pinned colloid with nanometer precision concurrent with external field application. Under static conditions, increasing the particle aspect ratio decreases the interfacial three-phase contact angle, but increases the relative interfacial deformation and therefore capillary attraction. Applied electric fields change the location of the particle relative to the fluid interface as well as how the fluid interface approaches the three-phase contact line with the particle surface. As the electric field strength increases, the contact angle increases for anisotropic particles. By controlling the contact angle with external fields, the interparticle capillary forces, and thus the final two-dimensional particle assembly, may be controlled in the future.
We also investigated the use of magnetic fields to interfacially assemble spherical polystyrene/iron oxide hybrid colloids. Different combinations of ionic strength and DC magnetic field strengths are applied to monolayers of particles, revealing an intricate state space transitioning between disordered clusters and hexagonal latices. Interfacial assemblies were characterized using 2D finite Fourier transforms to measure the amount of order under different solution-field conditions. The height of the fluid interface surrounding the pinned colloids during magnetic field application was measured using Mirau interferometry, indicating that alteration of capillary interactions via changes to interfacial pinning are occurring simultaneously with induced dipolar forces.
While our previous studies sought to modulate the interparticle potential via induced dipolar and changing capillary forces, we next discovered a way to control interfacial capillary forces through particle engineering. We accomplished this through the introduction of nanoscopic physical heterogeneity to the surface of polymer microellipsoids that alters the interparticle interactions when they are pinned at an aqueous-air interface. Leveraging the experimental tools we developed, we used a combination of Mirau interferometry and video microscopy to show that porous micron-sized ellipsoids at an aqueous-air interface behave in fundamentally different ways than their smooth counterparts. Particles with a nanoscale porous network show no quadrupolar deformation of the fluid interface, a trademark of smooth, homogeneous, colloidal ellipsoids. This causes the capillary interaction energy to be reduced by over an order of magnitude, a result that is confirmed by monitoring the dynamics of pairs of particles as they approach. Our measurements provide direct evidence of a shorter-range attraction with seemingly no orientational specificity between porous ellipsoids, in contrast to homogeneous, smooth ellipsoids. Taken together, these results indicate that incorporating nanoscale surface topography into anisotropic particles is an effective avenue to minimize capillary-driven aggregation and control interparticle interactions. As a result, such particles are promising candidates as building blocks for interfacial assemblies of anisotropic particles with long-range orientational and translational order.
In sum, this thesis presented a combination of novel anisotropic particle synthesis and experimental analysis of interfacial monolayer behavior to inform how the two-dimensional assembly of polymer colloids can be controlled through manipulating dipolar and capillary forces
Ophelia
Ophelia (1851-2) is the title of a Pre-Raphaelite painting by John Everett Millais narrating the final moments of Shakespeareâs heroine in Hamlet (1599-1601): the former is considered the best-known picture in all Victorian art and the latter, the greatest work in English literature. Nonetheless, Ophelia owes its significance and enduring popularity to these monumental artworks, as well as the fantasies of âWomanâ she embodies in successive discourses, and the material, semantic, and social networks she progressively integrates. The eight-hundred years span of such networks, their size and complexity across media and cultures, seem proof enough to consider Ophelia a âhyperobject.â Although Timothy Morton introduced it as a philosophical and ecological concept to deal with âthings that are massively distributed in time and space relative to humans,â Ophelia shows the same characteristic properties (viscosity, nonlocality, temporal undularity, phasing, interobjectivity) and ontological structure, a mesh constituted by a dynamic mixture of strands in which component objects interact, and gaps in which they withdraw remaining unknowable. The reconceptualization constructs Ophelia as a new object of transdisciplinary research, overcoming limitations of previous studies that focused on character analysis, historical period, or discipline. Further, the hyperobject provides an ideal medium in which Ophelia arises, develops, and is resolved or abandoned as problem, and of which the answers to that problem are also part. The chapters that follow will address three questions about Millaisâ Ophelia: What is Millaisâ answer to Ophelia? Where does Ophelia fit in art history and modernity? What did Millais want from Ophelia and what does Ophelia want from the public
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