160 research outputs found
University of Windsor Graduate Calendar 2023 Spring
https://scholar.uwindsor.ca/universitywindsorgraduatecalendars/1027/thumbnail.jp
Multi-Scale Fluctuations in Non-Equilibrium Systems: Statistical Physics and Biological Application
Understanding how fluctuations continuously propagate across spatial scales is fundamental for our understanding of inanimate matter. This is exemplified by self-similar fluctuations in critical phenomena and the propagation of energy fluctuations described by the Kolmogorov-Law in turbulence. Our understanding is based on powerful theoretical frameworks that integrate fluctuations on intermediary scales, as in renormalisation group or coupled mode theory. In striking contrast to typical inanimate systems, living matter is typically organised into a hierarchy of processes on a discrete set of spatial scales: from biochemical processes embedded in dynamic subcellular compartments to cells giving rise to tissues. Therefore, the understanding of living matter requires novel theories that predict the interplay of fluctuations on multiple scales of biological organisation and the ensuing emergent degrees of freedom.
In this thesis, we derive a general theory of the multi-scale propagation of fluctuations in non-equilibrium systems and show that such processes underlie the regulation of cellular behaviour. Specifically, we draw on paradigmatic systems comprising stochastic many-particle systems undergoing dynamic compartmentalisation.
We first derive a theory for emergent degrees of freedom in open systems, where the total mass is not conserved. We show that the compartment dynamics give rise to the localisation of probability densities in phase space resembling quasi-particle behaviour. This emergent quasi-particle exhibits fundamentally different response kinetics and steady states compared to systems lacking compartment dynamics. In order to investigate a potential biological function of such quasi-particle dynamics, we then apply this theory to the regulation of cell death. We derive a model describing the subcellular processes that regulate cell death and show that the quasi-particle dynamics gives rise to a kinetic low-pass filter which suppresses the response of the cell to fast fluituations in cellular stress signals. We test our predictions experimentally by quantifying cell death in cell cultures subject to stress stimuli varying in strength and duration.
In closed systems, where the total mass is conserved, the effect of dynamic compartmentalisation depends on details of the kinetics on the scale of the stochastic many-particle dynamics. Using a second quantisation approach, we derive a commutator relation between the kinetic operators and the change in total entropy. Drawing on this, we show that the compartment dynamics alters the total entropy if the kinetics of the stochastic many-particle dynamics violate detailed balance. We apply this mechanism to the activation of cellular immune responses to RNA-virus infections. We show that dynamic compartmentalisation in closed systems gives rise to giant density fluctuations. This facilitates the emergence of gelation under conditions that violate theoretical gelation criteria in the absence of compartment dynamics. We show that such multi-scale gelation of protein complexes on the membranes of dynamic mitochondria governs the innate immune response.
Taken together, we provide a general theory describing the multi-scale propagation of fluctuations in biological systems. Our work pioneers the development of a statistical physics of such systems and highlights emergent degrees of freedom spanning different scales of biological organisation. By demonstrating that cells manipulate how fluctuations propagate across these scales, our work motivates a rethinking of how the behaviour of cells is regulated
University of Windsor Graduate Calendar 2023 Winter
https://scholar.uwindsor.ca/universitywindsorgraduatecalendars/1026/thumbnail.jp
Rationales Design von polyfluorierten und enzymatisch abbaubaren Biomaterialien auf Peptidbasis
Amphiphilic peptide-based biomaterials are of great interest for pharmaceutical and biomedical applications and mainly associated with pronounced biocompatibility and biodegradability. In fact, introducing fluorine-containing amino acids into peptides & proteins offers an unique opportunity to enhance their biophysical properties such as membrane permeability. Through its influence on hydrophobicity and polarity, the degree of fluorination dictates the extent of fluorine-specific interactions on peptide folding and stability, intermolecular interactions, and biological activity.
The first study of this doctoral thesis describes the folding, self-assembly, and hydrogelation of single-strand amphipathic peptides with different degrees of fluorination on the amino acid side chains by the iterative incorporation of monofluoroethylglycine (MfeGly), difluoroethylglycine (DfeGly), and trifluoroethylglycine (TfeGly). A combination of experimental and theoretical approaches proved a higher degree of side chain fluorination to promote β-sheet formation and the rheological stability of peptide-based hydrogels in physiological conditions, whereas secondary structure formation was inhibited at a low fluorine content due to fluorine-induced polarity.
In a follow-up study, the selective modification of antimicrobial peptides (AMPs) by fluorinated amino acids was investigated. A β-hairpin-forming peptide motif, whose amphipathic structure enables the targeted disruption of bacterial cell membranes, was therefore examined. Extensive MIC screening with Gram-negative and Gram-positive bacteria confirmed highly fluorinated amino acids such as trifluoroethylglycine (TfeGly) or pentafluoropropylglycine (PfpGly) to strengthen the bioactivity of the AMPs through enhanced intrinsic hydrophobicity without causing a simultaneous increase in toxic & hemolytic properties.
Numerous studies on the singular incorporation of fluorinated amino acids have been published to date, whereas synthetic peptides with larger or exclusive amounts of these building blocks remained unexplored. That drove the motivation for the herein-described development and characterization of so-called "fluoropeptides". In brief, β-sheet to α-helix or fluorine-induced PPII-helix transitions were observed in SDS-supplemented buffer (pH 7.4). In situ SEIRAS experiments with POPC:POPG-based membrane models functioned to investigate the fluoropeptide’s lipid insertion and (re)folding. Thus, the highest α-helical secondary structure content was found for the nonfluorinated homooligopeptide and decreased in the order of tri-, di-, and mono-fluorination of the side chains. An important focus of this doctoral thesis was the evaluation of biodegradability for especially higher polyfluorinated sequences. In fact, all peptides prepared in this work could be hydrolyzed by various proteases regardless of the fluorine content. In cooperation with the University College Dublin, first data on the microbial digestion of fluorinated peptides and individual amino acids could be generated. The enzyme-catalyzed cleavage of the C-F bond on the side chain for both kind of substrates was, for instance, proven by detection of released fluoride ions in solution.
The results of this work will contribute to the rational design and potential application of polyfluorinated peptides, whose enzymatic degradability is going to be of great interest for the future development of fluorinated biomaterials.Amphiphile peptidbasierte Biomaterialien sind vom großen Interesse für pharmazeutische und biomedizinische Anwendungen und überzeugen zumeist durch ihre Biokompatibilität und Bioabbaubarkeit. Die Einführung von fluorhaltigen Aminosäuren in Peptide & Proteine bietet hierbei die einzigartige Möglichkeit, ihre biophysikalischen Eigenschaften wie etwa die Membranpermeabilität zu verstärken. Insbesondere der Fluorierungsgrad spielt eine entscheidende Rolle, da er durch seinen Einfluss auf die Hydrophobie und Polarität die Gesamtheit fluor-spezifischer Wechselwirkungen auf die Peptidfaltung und -stabilität, intermolekularen Wechselwirkungen und biologische Aktivität steuern kann.
Die erste Studie dieser Doktorarbeit beschreibt die Faltung, Selbstassemblierung und Hydrogelierung von einzelsträngigen amphipathischen Peptiden mit unterschiedlichen Fluorierungsgraden der Aminosäureseitenketten durch den iterativen Einbau von Monofluorethylglycin (MfeGly), Difluorethylglycin (DfeGly) und Trifluorethylglycin (TfeGly). Mittels einer Kombination aus experimentellen und theoretischen Ansätzen konnte gezeigt werden, dass bei physiologischen Bedingungen ein höherer Fluorierungsgrad die Bildung von β-Faltblattstrukturen und die rheologische Stabilität der peptid-basierten Hydrogele fördert, jedoch diese Sekundärstruktur von Peptiden mit niedrigem Fluorgehalt durch die fluor-induzierte Polarität inhibiert wird.
In einer weiteren Studie wurde die gezielte Modifizierung der biologischen Eigenschaften antimikrobieller Peptide (AMP) durch den Einbau fluorierter Aminosäuren untersucht. Hierzu wurde ein β-Hairpin bildendes Peptidmotiv ausgewählt, dessen amphipathische Struktur die zielgerichtete Disruption bakterieller Zellmembrane ermöglicht. Die ermittelten minimalen Hemmkonzentrationen (MHK) gegen verschiedene Gram-negative und Gram-positive Bakterien zeigen, dass hochfluorierte Aminosäuren wie Trifluorethylglycin (TfeGly) und Pentafluorpropylglycin (PfpGly) die Bioaktivität antimikrobieller Peptide durch Erhöhung der intrinsischen Hydrophobie selektiv verstärken können, ohne eine gleichzeitige Zunahme toxischer & hämolytischer Eigenschaften zu verursachen.
Zahlreiche Studien zum singulären Einbau fluorierter Aminosäuren wurden bis dato veröffentlicht, während synthetische Peptide mit größeren bzw. ausschließlichen Mengen dieser Bausteine unerforscht blieben. Dies war die Motivation zur Entwicklung und Charakterisierung sogenannter "Fluoropeptide". In SDS-beinhaltenden Puffer (pH 7.4) wurden, unter anderem, Übergänge von β-Faltblatt Strukturen zu α-Helices oder Fluor-induzierte PPII-Helices beobachtet. In-situ SEIRAS-Studien mit POPC:POPG-basierten Membranmodellen dienten zum Studium der Lipidinsertion und (Rück-)-Faltung der Fluoropeptide in Abhängigkeit zum gesamten Fluoranteil. Hierbei wurde der höchste Gehalt an α-helikaler Sekundärstruktur für das nichtfluorierte Homooligopeptid bestimmt, welcher in der Reihenfolge der Tri-, Di- und Monofluorierung der Seitenkette abnahm.
Ein wichtiger Schwerpunkt dieser Doktorarbeit war die Bewertung der biologischen Abbaubarkeit für insbesondere höher polyfluorierte Sequenzen. Tatsächlich konnten alle in dieser Arbeit hergestellten Peptide unabhängig vom Fluorgehalt durch verschiedene Proteasen hydrolysiert werden. In Zusammenarbeit mit dem University College Dublin konnten zudem erste Daten zum mikrobiellen Verdau fluorierter Peptide und Aminosäuren generiert werden. Die enzymkatalysierte Spaltung der C-F-Bindung an der Seitenkette für beide Substratarten wurde beispielsweise durch den Nachweis von freigesetzten Fluorid-Ionen in Lösung nachgewiesen.
Die Ergebnisse dieser Arbeit werden zum rationalen Design und potenzieller Anwendbarkeit neuartiger polyfluorierter Peptide beitragen, deren enzymatische Abbaubarkeit von groĂźem Interesse fĂĽr die kĂĽnftige Entwicklung fluorhaltiger Biomaterialien sein wird
Roadmap for optical tweezers
ArtĂculo escrito por un elevado nĂşmero de autores, solo se referencian el que aparece en primer lugar, el nombre del grupo de colaboraciĂłn, si le hubiere, y los autores pertenecientes a la UAMOptical tweezers are tools made of light that enable contactless pushing, trapping, and manipulation of objects, ranging from atoms to space light sails. Since the pioneering work by Arthur Ashkin in the 1970s, optical tweezers have evolved into sophisticated instruments and have been employed in a broad range of applications in the life sciences, physics, and engineering. These include accurate force and torque measurement at the femtonewton level, microrheology of complex fluids, single micro- and nano-particle spectroscopy, single-cell analysis, and statistical-physics experiments. This roadmap provides insights into current investigations involving optical forces and optical tweezers from their theoretical foundations to designs and setups. It also offers perspectives for applications to a wide range of research fields, from biophysics to space explorationEuropean Commission (Horizon 2020, Project No. 812780
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Membrane Rupture, Membrane Fusion and the Regulation of Exocytosis
Biological membranes form the structural boundaries and compartments of cells, owing to their robustness and impermeability facilitated by phospholipid bilayers. The strength of biological membranes is intricately linked to the behavior of membrane pores, whose formation and expansion can lead to membrane rupture. However, processes essential for drug delivery, gene editing via genetic material transfer, and antimicrobial peptide action necessitate controlled membrane disruption for efficient cellular entry. Likewise, fundamental phenomena such as exocytosis, including neurotransmitter release between neurons and hormone secretion for physiological responses, rely on membrane breach to release cargo beyond cell confines. Exocytosis involves the fusion of cargo-contained vesicle membranes with the cell's plasma membrane, resulting in the release of cargo into the extracellular milieu. Post-release, these fused vesicles may either integrate with the plasma membrane, remain stationary, enlarge, or depart the release site through fusion pore closure, which, in turn, can modulate exocytosis rate through site availability. However, the precise mechanism of membrane rupture remains elusive. Similarly, the pathway of membrane fusion facilitated by SNARE proteins, pivotal in cellular fusion machinery, remains a subject of debate. Additionally, the mechanisms governing exocytosis remain incompletely understood.
To address these inquiries, we employ ultra-coarse-grained molecular dynamics simulations which can explore these phenomena in physiological timescale. These simulations explore membrane rupture mechanisms via pore formation and expansion under varying membrane tension. Furthermore, the research addresses how SNARE proteins drive membrane fusion. In addition, we also rigorously analyze confocal microscopy data from Ling-Gang Wu's research group and develop a quantitative model to elucidate exocytosis rate regulation. Furthermore, the research verifies the robustness of a mathematical model outlining Ca2+-mediated membrane fusion and establishes that hemifusion diaphragms (HDs), where only the outer leaflets of membranes fuse, act as hubs in the Ca2+-mediated fusion network. This finding casts new light on the role of membranes in SNARE-mediated fusion. In the extra study, we analyzed fission yeast contractile ring behavior based on z-stack confocal microscopy data from Mohan Balasubramanian's research group, offering insights into the mechanism behind a critical step in cytokinesis.
Chapter one examines membrane pore energetics and bilayer rupture times through highly coarse-grained simulations operating at submillisecond time scales. No metastable states are detected during pore formation. At lower tensions, small hydrophobic pores mature into large hydrophilic pores that ultimately rupture from reversible hydrophilic pores, aligning with classical tension-dependent rupture times. At higher tensions, membranes rupture directly from small hydrophobic pores, with rupture times exhibiting exponential tension dependence. Upon reaching a minimum hydrophobic pore size, a critical tension threshold prompts immediate rupture. This analysis corroborates established experimental findings but reveals that the high-tension exponential regime is not related to long-lived pre-pore defects but rather to the instability of hydrophilic pores beyond a critical tension, leading to significant changes in pore dynamics and rupture kinetics.
Chapter two describes utilizing ultra-coarse-grained simulations to dissect the core requirements of membrane fusion and unravel the intricacies of SNARE-mediated fusion. Remarkably, simulations conducted on a millisecond timescale expose the inefficiency of fusion through simple body forces pushing vesicles together. Successful inter-vesicle fusion hinges on the rod-like structure of fusogens, ensuring their sufficient length for effective fusion and subsequent clearance from the fusion site via entropic forces. Simulations featuring rod-shaped fusogens and SNARE proteins demonstrate the fusion of 50-nanometer vesicles in submilliseconds, propelled by entropic forces that direct a predictable fusion pathway. The entropic force hypothesis of SNARE-mediated membrane fusion garners strong support from these findings, emphasizing the necessity of the rod-like configuration of the SNARE complexes for entropic force generation and fusion.
Chapter three focuses on the spatiotemporal dynamics of dense-core vesicle exocytosis events in chromaffin cells, deducing a novel mechanism for exocytosis regulation based on the availability of release sites. Repeated fusion supports membrane reservoir comprising incompletely merged or closed vesicles, occupying release sites and dampening exocytosis frequency. Mathematical modeling suggests reservoir formation relies on locally reduced membrane tension, eliminating the driving force for vesicle merging. Endocytosis facilitates the clearance of unmerged vesicles from the reservoir, ultimately restoring release site availability for subsequent exocytosis events.
Chapter four introduces a mathematical model pinpointing the hemifusion diaphragm (HD) as the decision nexus dictating the outcomes of pathways and the fate of final products during multivalent cation-mediated membrane interactions. Transient formation of a high-tension hemifusion interface between membrane-enclosed compartments underscores the model's prediction of fusion, dead-end hemifusion, or vesicle lysis. This comprehensive framework offers predictive insights into interactions mediated by cationic fusogens within membrane-enclosed compartments.
Chapter five offers a unique exploration of writhing contractile rings in fission yeast cell ghosts, resulting from controlled digestion of the cell wall and subsequent membrane permeabilization. This innovative approach unveils the intricate dynamics of contractile rings under exceptional circumstances. Writhing of rings is attributed to the detachment of sections from the weakened membrane, followed by their coiling due to apparent twisting torques at anchoring points. Iterative rotations give rise to multiple coils within the rings
Mesoscale Modeling of Controlled Degradation in Polymer Networks and Melts
Controlled degradation of polymers finds various applications in fields ranging from the design of functional soft materials to recycling of polymers. In several of these applications, the characteristic length scale at which relevant processes occur ranges from nanometers to microns, typically referred to as the mesoscale. Although analytical models and continuum approaches inform our current understanding, analysis of degradation at the mesoscale is exceptionally limited. For modeling degradation at the mesoscale, we use the Dissipative Particle Dynamics (DPD) technique and the LAMMPS simulation software. Within the DPD framework, we model controlled degradation or the breaking of covalent bonds within a polymer as a stochastic process that reproduces first order degradation reaction kinetics. A known limitation of the DPD approach is polymer chains crossing through each other. Previous researchers had developed a modified segmental repulsive potential (mSRP) framework which prevents such crossing of polymers by introducing extra repulsion between the bonds of polymer chains. We modified the existing model in LAMMPS to enable switching off the extra repulsion when a bond is broken. We implemented this feature within the LAMMPS framework, and it is now available for the general scientific community as a part of the online open-source project. Later, we extended this feature to introduce the extra repulsion when a bond is formed to simulate the hydrosilylation reaction used in the synthesis of polymer derived ceramics. As a model polymer network for studying degradation, we use the tetra-arm polyethylene glycol (tetra-PEG) based hydrogel films. Tetra-PEG networks have a uniform network structure and hence superior mechanical properties. We tracked the degradation iii of these networks by measuring the evolution of the weight average molecular weight and dispersity during degradation. By tracking the fraction of degradable bonds broken, we identified the “reverse gel point”, the point where the polymer network dissolves into the surrounding solvent. Additionally, we tracked the erosion or mass loss from the degrading network by accounting for polymer fragments which dissociate and diffuse away from the network. We identified that the mass loss from the network depends on the initial thickness of the hydrogel films. As a second system, we modeled the controlled degradation of nanogels that are either suspended in a single solvent or adsorbed onto a liquid-liquid interface. Controlled degradation of nanogels at an interface provides a dynamic approach to control interface topography at the nanoscale. We tracked the degradation of these particles by analyzing the evolution of their shape and size along with the molecular weights and dispersity in the system. In bulk, the particles swell almost homogenously while at the interface, the particles spread and cover the interface as degradation occurs. We found that the reverse gel point for these particles varies with the total initial number of precursors. The evolution of particle shape and size is significantly affected by the surrounding solvent and the surface tension between the two liquid phases. The final part of this dissertation focuses on developing an initial framework to extend the above approach to model degradation of polyolefin melts under a local temperature gradient. The long term goal of this project is to study thermal degradation of polyolefins caused by introducing microwave absorbing nanosheets and subjecting the polymer to microwave irradiation
Roadmap for optical tweezers
Optical tweezers are tools made of light that enable contactless pushing, trapping, and manipulation of objects, ranging from atoms to space light sails. Since the pioneering work by Arthur Ashkin in the 1970s, optical tweezers have evolved into sophisticated instruments and have been employed in a broad range of applications in the life sciences, physics, and engineering. These include accurate force and torque measurement at the femtonewton level, microrheology of complex fluids, single micro- and nano-particle spectroscopy, single-cell analysis, and statistical-physics experiments. This roadmap provides insights into current investigations involving optical forces and optical tweezers from their theoretical foundations to designs and setups. It also offers perspectives for applications to a wide range of research fields, from biophysics to space exploration.journal articl
Roadmap for Optical Tweezers 2023
Optical tweezers are tools made of light that enable contactless pushing, trapping, and manipulation of objects ranging from atoms to space light sails. Since the pioneering work by Arthur Ashkin in the 1970s, optical tweezers have evolved into sophisticated instruments and have been employed in a broad range of applications in life sciences, physics, and engineering. These include accurate force and torque measurement at the femtonewton level, microrheology of complex fluids, single micro- and nanoparticle spectroscopy, single-cell analysis, and statistical-physics experiments. This roadmap provides insights into current investigations involving optical forces and optical tweezers from their theoretical foundations to designs and setups. It also offers perspectives for applications to a wide range of research fields, from biophysics to space exploration
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