Measurements and implications of forced molecular unbinding

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2009.Cataloged from PDF version of thesis.Includes bibliographical references.The main goal of this thesis is to study the coupled interactions between chemically and mechanically characterized materials and cells that are relevant to microvascular physiology and pathology. In particular, the mechanical characterization of cell surface structure and force generation are realized via various atomic force microscopy (AFM) imaging techniques including AFM cell force spectroscopy and functionalized force imaging. In these approaches, the recognition of mechanical responses of cells or mapping of cell surface receptors is mediated by chemomechanically characterized AFM cantilevers. The high spatial and force resolution of AFM imaging techniques and force spectroscopy enabled investigation of mechanical interaction at the cell-cell or cell-material interfaces. This interaction was studied via the mapping of specific receptors on endothelial cell surfaces and the detection of pN-scale force transmission through ligand-receptor pairs on the plasma membrane with biophysical interpretation of cellular force generation. This thesis consists of four major chapters: the recognition of vascular endothelial growth factor receptors and of anti-angiogenic oligopeptide receptors on endothelial cell surfaces, mechanical interaction between endothelial cells and pericytes that encompass capillary blood vessels; cell-matrix contact via focal complexes; and leukemia cells rolling on endothelial cell surfaces and P-selectin-conjugated glass substrata.(cont.) This thesis also includes appendices that detail the effect of force transducer stiffness on the measurement of unbinding force, nerve cell imaging to I observe the connection between axons and dendrites, and chemomechanical characterization of polyelectrolyte multilayers, biodegradable hydrogels, and biological glues. In Chapter 2, transmembrane receptors on endothelial cell surfaces are mapped and associated binding kinetics/thermodynamics of ligand-receptor pairs are quantified via AFM functionalized force imaging or single-molecule recognition imaging. Functionalized force imaging is then used to identify unknown receptors, receptors for an oligopeptide isolated from tissue inhibitor of metalloproteinase-2, called Loop 6. In Chapter 3, mechanical stress by pericytes that envelop capillary blood vessels is quantified, demonstrating that pericytes exert significant mechanical strain on the extracellular environment. In Chapter 4, picoNewton-scale force dynamics at fibroblasts' focal complexes, measured in real-time through cell force spectroscopy, demonstrates that cells exert mechanical force that can speed the rupture of ligand-receptor pairs in focal complexes during migration and adhesion to underlying substrata. The last part of this thesis, Chapter 5, discusses the role of actin-mediated force in leukemia cell rolling on endothelial cell surfaces. The measurement of picoNewton-scale force dynamics using cell force spectroscopy suggests that, in addition to drag force exerted by blood flow, cytoskeletal force dynamics contribute to the cell rolling process.(cont.) Together, these studies from the single-molecule to whole-cell level detail the strong coupling between mechanical force and ligand-receptor reaction kinetics.by Sunyoung Lee.Ph.D

Quantifying effects of substrata chemomechanical properties on eukaryotic and prokaryotic cell adhesion and morphology

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2008.Includes bibliographical references (p. 193-201).It is now widely accepted that cells are capable of processing both mechanical and chemical signals from the extracellular environment. Exactly how these two factors affect the cell biology in the context of physiological circumstances is an area of intense interest that has given rise to an entire field of study called cell mechanotransduction. The unambiguous decoupling of mechanical and chemical properties that stimulate cell development and phenotypic change is challenging from an experimental standpoint. This thesis describes some of the first studies of chemomechanical coupling arising from anchorage-dependent forces between cells and a versatile class of chemically and mechanically tunable polymer thin films, termed polyelectrolyte multilayers. Specifically, investigation of the effects of extracellular chemomechanical stimulation on cell morphology and adhesion in the eukaryotic cells such as vascular endothelial cells and fibroblasts; and the adhesion of prokaryotic cells S. epidermidis and E. coli are presented. Endothelial cells (EC) comprise a major portion of the cell population in the human body. Because of the extensive distribution of endothelial cells in various tissues, they function across a broad range of mechanical and chemical environments. Furthermore, a general understanding of how mechanical forces contribute to the development of cellular function is an important aspect in the development of therapeutic techniques and materials capable of addressing a wide spectrum of human diseases and injuries. Cell adhesion to extracellular matrices and tissues can be indicative of underlying molecular processes in both healthy and disease states.(cont.) Through the use of a mechanically tunable class of polymer thin films called polyelectrolyte multilayers (PEMs) developed by Rubner et al., we have demonstrated that the adhesion and morphology of human microvascular endothelial cells depend directly on the mechanical stiffness of these synthetic substrates, as quantified by the nominal elastic modulus E. Characterization of the mechanical properties and surface features of PEMs is attained via scanning probe microscopy (SPM) and SPM-enabled nanoindentation. Typical cellular response to increased substrata stiffness includes increased number of cells adhered per unit substratum area. We have further demonstrated that the chemical and mechanical signals imposed at the cell-substrata interface can be decoupled, thereby providing two independent parameters capable of controlling cell behavior. This capacity of the cell to sense and/or exert chemical and mechanical forces, in addition to initiating a sustained molecular response, is termed the chemomechanical response element. Finally, adhesion dependent mechanosensation in bacteria is explored, with respect to the chemomechanical response elements common to eukaryotic and prokaryotic cells. Potential applications towards the development of therapeutic materials and compounds for treatment of various disease states are discussed, with particular attention to limiting hospital acquired infections.by Michael Todd Thompson.Ph.D

In Situ Examination of Nanoscale Reaction Pathways in Battery Materials

In order to engineer less expensive and more energy-dense batteries, new materials that can reliably store and transport active ions must be first developed. However, these materials are known for their poor reversibility due to large morphological changes during cycling. To maximize reversibility during charge and discharge, we must be able to understand and control the electrochemical reaction mechanisms of these new electrode materials. This dissertation uses in situ experiments, primarily in situ transmission electron microscopy (TEM), to understand the nanoscale reaction pathways in various high-capacity electrode materials during reactions with Li+, Na+, and K+ ions. Upon reacting with alkali-metal ions, these electrode materials often exhibit much higher specific storage capacities than conventional Li-ion battery electrode materials. In addition, these types of materials can also be used in lower-cost sodium- and potassium-based systems. Hence, they could replace electrode materials in Li-ion batteries, which would make possible engineering batteries with higher specific energy. However, the more substantial volumetric changes that these electrode materials undergo during reaction cause a significant decrease in the capacity retention. This decrease in the capacity retention is caused by the mechanical fracture of the active material and continuous growth of the solid-electrolyte interphase (SEI) on the surface of the anode particles, which both lead to very low cyclability of these systems. If these battery systems are to be improved, it is critical to understand both how the larger Na+ and K+ ions affect the nanoscale phase transformations during these reactions and how to engineer high capacity battery materials with high coulombic efficiency and longer cycle life. As part of the research described in this dissertation, studies on the Cu2S and FeS2 active materials were conducted to examine the effect that larger alkali metal ions have on the reaction mechanisms of large-volume-change materials. Evidence obtained from extensive in situ and ex situ experiments suggests that the larger volume changes associated with the sodium/potassium reactions indicate that the different reaction pathways affect the materials behavior. This altered reaction behavior results in a more stable morphology for the overall cycling of the electrode material. In an effort to aid the engineering of a high capacity battery material with longer cycle life, a study was conducted on Sb nanocrystal electrode materials that exhibited stable electrochemical behavior. This study demonstrated that small spherical particles naturally formed uniform internal voids that were easily filled and vacated during cycling. This was found to be due to the resilient lithiated oxide layer that formed after the first lithiation and subsequently prevented shrinkage during delithiation. A chemomechanical model describing the void formation was developed; this model can serve as a tool to guide the creation of oxide or other shells that enable alloying materials to undergo voiding transformations in situ. When reacting with alkali ions of different sizes, all of these materials (Cu2S, FeS2, and Sb) exhibited counter-intuitive phase evolution and mechanical degradation behavior. The findings indicate that, thanks to their high energy density, large-volume-change materials could make possible the development of next-generation batteries, whether they be Li-ion batteries or batteries with other chemistries that undergo complex morphological changes.Ph.D

Electrocatalytic reaction driven flow

Bimetallic Pt-Au nanorods in form of microswimmers within an aqueous solution exhibit self-propulsion that is powered by self-electrophoresis. This bimetallic Pt-Au system can be immobilized to generate convective fluid flow thereby acting as a micropump. In this work, a combined experimental and numerical approach was used to investigate the key elements, including the self-induced electric field, the proton gradients, and the reaction kinetics that impact the chemomechanical actuation of the Pt-Au electrocatalytic system. The findings contribute towards the fundamental understanding of fluid flow powered by an electrocatalytic micropump that applies to mass transport enhancement in systems

A survey of physical methods for studying nuclear mechanics and mechanobiology

It is increasingly appreciated that the cell nucleus is not only a home for DNA but also a complex material that resists physical deformations and dynamically responds to external mechanical cues. The molecules that confer mechanical properties to nuclei certainly contribute to laminopathies and possibly contribute to cellular mechanotransduction and physical processes in cancer such as metastasis. Studying nuclear mechanics and the downstream biochemical consequences or their modulation requires a suite of complex assays for applying, measuring, and visualizing mechanical forces across diverse length, time, and force scales. Here, we review the current methods in nuclear mechanics and mechanobiology, placing specific emphasis on each of their unique advantages and limitations. Furthermore, we explore important considerations in selecting a new methodology as are demonstrated by recent examples from the literature. We conclude by providing an outlook on the development of new methods and the judicious use of the current techniques for continued exploration into the role of nuclear mechanobiology

Chemomechanics of attached and suspended cells

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2012.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 (p. 173-184).Chemomechanical coupling in single eukaryotic animal cells is investigated in the con- text of the attached (substratum-adhered) and the suspended (free-floating) states. These dichotomous configurations determine behavioral differences and commonalities relevant to therapeutic reimplantation of stem cells and to our general under- standing of the cell as an animate material. Analytical, simulation, and experimental techniques are applied to key questions including: (1) How deep can mechanosensitive attached cells "feel" into the adjacent environment? (2) In what manner do suspended cells deform, absent the prominent actomyosin stress fibers that arise upon attachment to a rigid substratum? (3) What explains the remarkable mechanical heterogeney among single cells within a population? (4) Can we leverage putative mechanical markers of useful stem cells to sort them before reimplantation in tissue generation therapies? Attached cells are found to barely detect an underlying rigid base more than 10 micrometers below the surface of a compliant coating. This conclusion, based on ex- tensions to the Boussinesq problem of elasticity theory, is validated by observations of cell morphology on compliant polyacrylamide coatings in a range of thicknesses. Analytical equations are developed for estimating the effective stiffness sensed by a cell atop a compliant layer. We also identify and consider conceptualizations of a "critical thickness," representing the minimum suitable thickness for a specific application. This parameter depends on the cell behavior of interest; the particular case of stem cell culture for paracrine extraction is presented as a case study. Suspended cells are found to exhibit no single characteristic time scale during de- formation; rather, they behave as power-law (or "soft glassy") materials. Here, optical stretching is used as a non-contact technique to show that stress fibers and probe-cell contact are not critical in enabling power-law rheological behavior of cells. Further- more, suspended cell fluidity, as characterized by both the hysteresivity of complex modulus and the power-law exponent of creep compliance, is found to be unaffected by adenosine triphosphate (ATP) depletion, showing that ATP hydrolysis is not the origin of fluidity in cells during deformation. However, ATP depletion does reduce the natural variation in hysteresivity values among cells. This finding, and the finding that changes in the power-law exponent and stiffness of single cells are correlated upon repeated loading, motivates study of how and why these parameters are coupled. To further explore this coupling, chemomechanical cues are applied to cell populations to elucidate the origin of the wide, right-skewed distribution of stiffness values that is consistently observed. The distribution and width are found to be not detectably dependent on cell-probe contact, cell lineage, cell cycle, mechanical perturbation, or fixation by chemical crosslinking. However, ATP depletion again reduces heterogeneity, now in the case of cell stiffness values. It is further found analytically that a postulated Gaussian distribution of power-law exponent values leads naturally to the log-normal distribution of cell stiffness values that is widely observed. Based on these connections, a framework is presented to improve our understanding of the appearance of mechanical heterogeneity in successively more complex assemblies of cell components. Two case studies are described to explore the implications of unavoidable intrinsic variation of cell stiffness in diagnostic and therapeutic applications. Finally, all the single-cell mechanical parameters studied so far (stiffness during creep and recovery, stiffness heterogeneity among cells, and power-law exponents in creep and recovery) are characterized in mesenchymal stem cells during twenty population doublings with the aim of developing a high-throughput sorting tool. How- ever, mechanical and structural changes that are observed in the attached state during this culture time are not observed after cell detachment from the substratum. The absence in the suspended state of these alterations indicates that they manifest themselves through stress fiber arrangement rather than cortical network arrangement. While optical stretching under the present approach does not detect mechanical markers of extended passaging that are correlated with decreased differentiation propensity, the technique is nevertheless found capable of investigating another structural transition: mechanical stiffening over tens of minutes after adherent cells are suspended. This previously unquantified transition is correlated with membrane resorption and reattachment to the cortex as the cell "remodels" after substratum detachment. Together, these quantitative studies and models of attached and suspended cells de- fine the extremes of the extracellular environment while probing mechanisms that con- tribute to cellular chemomechanical response. An integration of the results described above shows that no one existing model can describe cell chemomechanics. However, the cell can be usefully described as a material -- one in which animate mechanisms such as active contraction will generally, but not invariably, need to be considered as augmenting existing viscoelastic theories of inanimate matter.by John Mapes Maloney.Ph.D

University of Arkansas, Chemistry and Biochemistry Department Graduate Student Publications, 2018- November 2023. 39p.

This report provides a compilation of the research publications by the Chemistry and Biochemistry (CHBC) Graduate students for the period: 2018-November 2023. It includes publications by the CHBC graduates and those where a CHBC faculty was the main advisor. It includes a summary of the research. The listing is organized according to type of publications within specific years

Viscoelasticity Acts as a Marker for Tumor Extracellular Matrix Characteristics

Biological materials such as extracellular matrix scaffolds, cancer cells, and tissues are often assumed to respond elastically for simplicity; the viscoelastic response is quite commonly ignored. Extracellular matrix mechanics including the viscoelasticity has turned out to be a key feature of cellular behavior and the entire shape and function of healthy and diseased tissues, such as cancer. The interference of cells with their local microenvironment and the interaction among different cell types relies both on the mechanical phenotype of each involved element. However, there is still not yet clearly understood how viscoelasticity alters the functional phenotype of the tumor extracellular matrix environment. Especially the biophysical technologies are still under ongoing improvement and further development. In addition, the effect of matrix mechanics in the progression of cancer is the subject of discussion. Hence, the topic of this review is especially attractive to collect the existing endeavors to characterize the viscoelastic features of tumor extracellular matrices and to briefly highlight the present frontiers in cancer progression and escape of cancers from therapy. Finally, this review article illustrates the importance of the tumor extracellular matrix mechano-phenotype, including the phenomenon viscoelasticity in identifying, characterizing, and treating specific cancer types

Chemomechanical regulation of integrin activation and cellular processes in acidic extracellular pH

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012.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 (p. 162-176).It is well established that extracellular pH (pHe) becomes acidic in several important physiological and pathological contexts, including the tumor and wound microenvironments. Although it is known that acidic pHe can have profound effects on cell adhesion and migration processes integral to tumor progression and wound healing, the molecular mechanisms underlying the cellular responses to acidic pHe are largely unknown. Transmembrane integrin receptors form a physical linkage between cells and the extracellular matrix, and are thus capable of modulating cell adhesion and migration in response to extracellular conditions. In this thesis, computational and experimental approaches are used to investigate the role of acidic extracellular pH in regulating activation and binding of integrin [alpha]v[beta]3, and to characterize the consequences for downstream subcellular- and cellular-scale processes. Molecular dynamics simulations demonstrate that opening of the integrin [alpha]v[beta]3 headpiece occurs more frequently in acidic pHe than in normal pHe, and that this increased headpiece opening can be partially attributed to protonation of ASP[beta]127 in acidic pHe. These computational data indicate that acidic pHe can promote activation of integrin [alpha]v[beta]3. This is consistent with flow cytometry and atomic force microscope-enabled molecular force spectroscopy experiments, which demonstrate that there are more activated [alpha]v[beta]3 receptors on live [alpha]v[beta]3 CHO-B2 cell surfaces at acidic pHe than at normal pHe 7.4. Put together, these atomistic- and molecular-level data suggest a novel mechanism of outside-in integrin activation regulation by acidic extracellular pH. Next, the consequences of acid-induced integrin activation for subcellular- and cellular-scale processes are investigated. Kymography experiments show that [alpha]v[beta]3 CHO-B2 cell membrane protrusion lifetime is increased and protrusion velocity is decreased for cells in pHe 6.5, compared to cells in pHe 7.4. Furthermore, [alpha]v[beta]3 CHO-B2 cells in pHe 6.5 form more actin-integrin adhesion complexes than cells in pHe 7.4, and acidic extracellular pH results in increased cell area and decreased cell circularity. Cell migration measurements demonstrate that [alpha]v[beta]3 CHO-B2 cells in pHe 6.5 migrate slower than cells in pHe 7.4, and that the fibronectin ligand density required for peak migration speed is lower for cells in pHe 6.5. Together, these data show that acidic pHe affects subcellular- and cellular-scale processes in a manner that is consistent with increased integrin activation in this condition. Finally, the migration behavior of [alpha]v[beta]3 CHO-B2 cells, bovine retinal microvascular endothelial cells, and NIH-3T3 fibroblasts in an extracellular pH gradient is investigated. Results demonstrate that NIH-3T3 fibroblasts do not exhibit directional preferences in the pHe gradient, but that [alpha]v[beta]3 CHO-B2 cells and bovine retinal microvascular endothelial cells migrate preferentially toward the acidic end of the gradient. These data suggest that acidic extracellular pH may serve as a cue that directs migration of angiogenic endothelial cells to poorly vascularized regions of tumors and wounds. Overall, this thesis research results in multiscale, in-depth understanding of extracellular pH as a critical regulator of cell function, with associated implications for tumor growth, wound healing, and the role of proton pumps in cell migration.by Ranjani Krishnan Paradise.Ph.D