Molecular machinery and manufacturing with applications to computation

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Architecture, 1991.Vita.Includes bibliographical references (p. 469-487).by K. Eric Drexler.Ph.D

Neuronal dynamics and connectivity analysis of neuronal cultures on multi electrode arrays

Despite a number of attempts over the past two decades, research into reliable, controlled induction of long term evoked responses, mimicking low level learning and memory in dissociated cell cultures remains challenging. In addition, a full understanding of the stimulus-response relationships that underlie synaptic plasticity has not yet been achieved, and many of the underlying principles remain largely unknown. Plasticity studies have been predominantly limited to low density Multi/Micro Electrode Arrays (MEAs). With the advent of complementary metal-oxide-semiconductor (CMOS) based High-Density (HD) MEAs, unprecedented spatial and temporal resolution is now possible. In this thesis, an attempt to bridge the gap between studies of neural plasticity and the use of CMOS based HD-MEAs with thousands of electrodes, is reported. Additionally, since such HD-MEAs generate a large volume of data and require advanced analytics to efficiently process and analyse recordings, computational tools and novel algorithms to infer connectivity during plasticity have been developed. The study showed that the responsiveness, stability and initial firing rate of neuronal cultures are the deciding factors to reliably induce evoked responses. With multi-site stimulation, sustained long term potentiation was achieved, which was validated both by evoked response plots and overall firing rates measured at five different time points - before and after repeated stimulation, and at a three day time points. In contrast, while depression responses were observed, it was found that the effects were not sustained over many days. The findings of the study suggest that appropriate selection of neuronal cultures is crucial for inducing desired evoked responses and criteria for this have been developed. Furthermore, it is concluded that the initial responses to test stimuli can be used to determine whether potentiated or depressed responses are to be expected. To analyse the recordings, pipeline of computational tools was developed. Firstly, neuronal synchrony metrics were adapted for the first time for large HD-MEA recordings and shown to correspond effectively to the firing dynamics. To analyse functional connectivity, an information theoretic approach, Transfer Entropy(TE), was utilised. The method showed accurate estimation of functional connectivity with mid 80th percentile accuracy on simulated data. A superimposition method was proposed to enhance confidence in the connectivity estimation. To statistically evaluate connectivity estimation, a new surrogate method, based on ISI distribution approach, was proposed and validated with a simulated Izhikevich network. The method achieved improved accuracy, compared to the existing ISI shuffling method. This newly developed method was later utilised to infer connectivity and refine connections during the learning process of real neuronal cultures over many days of stimulation. The connectivity inference corresponded accurately to both the spontaneous and stimulated networks during evoked responses and the proposed method permitted observation of the evolution of connections for the potentiated network

MOLECULAR DESIGN STRATEGIES AND IMAGING OF ELECTRONIC PEPTIDE NANOMATERIAL SCAFFOLDS

Peptide-pi conjugated materials represent a powerful class of supramolecular materials with the unique ability to bridge the traditionally disparate realms of electronics and biological settings. The modular nature of peptide synthesis has facilitated systematic investigations into the impacts of amino acid composition for material properties. This dissertation presents design strategies to achieve new conformations and electronic outcomes within these peptide materials, efforts to characterize mixed peptide assemblies on the nanoscale and attempts to employ them in biological settings. In chapter 1 the state of organic electronics and its extensions to bioelectronics is introduced. Previous efforts from our lab to control peptide-pi-peptide scaffolds through monomer design and assembly trigger are discussed. Then, the fundamentals of electron microscopy are reviewed along with electron specimen interactions and their use in characterizing nanomaterials. In chapter 2, constitutional isomerism brought about by swapping regimes of unique hydrophobicity and the influence on physical properties, morphology, and electronic communication among pi-cores is examined. The addition of a purely hydrophobic alkyl tail is also discussed wherein access to a range of nanoarchitectures without diluting the impacts of constitutional isomerism is demonstrated. Chapter 3 details efforts to use bromine and sulfur as elemental indicators of co-assembly or self-sorting and examine the nanoscopic details of mixed assemblies by way of STEM-EDS mapping. This characterization method is applied to statistically mixed co-assemblies, self-sorted structures based on exploiting subtle pKa differences as previously reported by our lab, and a novel self-sorting paradigm brought about by tailoring specific monomer-monomer interactions. In chapter 4, the field of diphenylalanine research and extensive efforts to effectuate control over the assembly of this unique dipeptide are discussed. iii Efforts to control the assembly of diphenylalanine by single fluorine substitution at the beta-carbon are explored wherein preliminary studies suggest that this relatively small change in structure can have dramatic impacts on assembly. Chapter 5 is concerned with biologically relevant nanomaterials and the promise of peptide-pi conjugates in 3D cell culture applications. Both failed and successful attempts to prepare a hydrogel scaffold containing attributes known to guide neuronal stem cells toward neuronal lineages are presented. Finally, promising efforts to translate peptide-pi-peptide nanomaterials to in vivo stroke recovery models are detailed

Numerical modeling of F-.Actin bundles interacting with cell membranes

Actin is one of the most aboundant proteins in eukaryotic cells, where it forms a dendridic network (cytoskeleton) beneath the cell membrane providing mechanical stability and performing fundamental tasks in several functions, including cellular motility. The first step in cell locomotion is the protrusion of a leading edge, for which a significant deformation of the membrane is required: this step relies essentially on the forces generated by actin polymerization pushing the plasma membrane outward. Different types of structures can emerge from the plasma membrane, like lamellipodia (quasi-2d actin mesh) and filopodia (parallel actin bundles). The main topic of the research project is the dynamics of bundles of parallel actin filaments growing against barriers, either rigid (a wall) or flexible (a membrane). In the first part of the thesis, the dynamic behavior of bundles of actin filaments growing against a loaded wall is investigated through a generalized version of the standard multi filaments Brownian Ratchet model in which the (de)polymerizing filaments are treated not as rigid rods but as semi-flexible discrete wormlike chains with a realistic value of the persistence length. A Statistical Mechanics framework is built for bundles of actin filaments growing in optical trap apparatus (harmonic external load) and several equilibrium properties are derived from it, like the maximum force that the filaments can exert (stalling force) or the number of filaments in contact with the wall. Besides, Stochastic Dynamic simulations are employed to study the non-equilibrium relaxation of the bundle of filaments growing in the same optical trap apparatus, interpreting the system evolution by a suitable Markovian approach. Thanks to the observed time scale separation between the wall motion and the filament size relaxation, the optical trap set-up allows to extract the full velocity-load curve V(F) -- the velocity at which the obstacle moves when subject to the combined action of the polymerizing filaments and the external load F -- from a single experiment. The main finding is the observation of a systematic evolution of steady non-equilibrium states over three regimes of bundle lengths L. A first threshold length Λ marks the transition between the rigid dynamic regime (L Λ), where the velocity V(F,L) is an increasing function of the bundle length L at fixed load F, the enhancement being the result of an improved level of work sharing among the filaments induced by flexibility. A second critical length corresponds to the beginning of an unstable regime characterized by a high probability to develop escaping filaments which start growing laterally and thus do not participate anymore to the generation of the polymerization force. This phenomenon prevents the bundle from reaching at this critical length the limit behavior corresponding to Perfect Load Sharing. In the second part of the thesis, filaments growing against a flexible, deformable membrane are studied by means of Langevin dynamics simulations; the membrane is discretized into a dynamically triangulated network of tethered beads, while the filaments are described as chains of bonded monomers. Both the monomers in the filaments and the membrane beads, which interact with each other via a purely repulsive potential, are followed in space and time integrating its equations of motion with a second order accurate scheme. The elastic properties of the membrane are studied in detail via several methods, showing an unprecedentent level of agreement among them. The onset of filopodial protrusions is observed for N>1 filaments growing from beneath the membrane and pushing it upwards, with a velocity which is systematically larger for flexible filaments than for rigid ones. Since filaments are wrapped by the membrane in the protrusion, escaping filaments are not predicted nor observed in this case

The regulation and induction of clathrin-mediated endocytosis through a protein aqueous-aqueous phase separation mechanism

La morphologie des cellules et leurs interactions avec l’environnement découlent de divers procédés mécaniques qui contribuent à la richesse et à la diversité de la vie qui nous entoure. À titre d’exemple, les cellules mammifères se conforment à différentes géométries en fonction de l’architecture de leur cytosquelette tandis que les bactéries et les levures adoptent une forme circulaire par turgescence. Je présente, dans cette thèse, la découverte d’un mécanisme de morphogénèse supplémentaire, soit la déformation de surface cellulaire via l’assemblage de protéines par démixtion de phases aqueuses non miscibles et l’adhésion entre les matériaux biologiques. J’expose de façon spécifique comment ce mécanisme régule le recrutement et le mouvement dynamique des protéines qui induisent l’invagination de la membrane plasmique lors de l’endocytose clathrine-dépendante (CME). Le phénomène de démixtion des protéines dans le cytoplasme est analogue à la séparation de phase de l’huile en solution aqueuse. Il constitue un mécanisme cellulaire important et conservé, où les protéines s’agglomèrent grâce aux interactions intermoléculaires qui supplantent la tendance du système à former un mélange homogène. Plusieurs exemples de compartiments cellulaires dépourvus de membrane se forment par démixtion de phase, tels que le nucléole et les granules de traitement de l’ARN [1-6]. Ces organes ou compartiments dénommés NMO, du terme anglais « non-membranous organelles », occupent des fonctions de stockage, de traitement et de modification chimique des molécules dans la cellule. J’explore ici les questions suivantes : est-ce que les NMO occupent d’autres fonctions à caractère morphologique ? Quels signaux cellulaires régulent la démixtion de phase des protéines dans la formation des NMO ? Fondée sur la physique mécanique du contact entre les matériaux, j’émets l’hypothèse que des compartiments cellulaires nanoscopiques, formés par démixtion de phase, génèrent des forces mécaniques par adhésion interfaciale. Le travail mécanique ainsi obtenu déforme le milieu cellulaire et les surfaces membranaires adjacents au NMO nouvellement créé. Le but de mon doctorat est de comprendre comment les cellules orchestrent, dans le temps et l’espace, la formation des NMO associés au CME et comment ceux-ci génèrent des forces mécaniques. Mes travaux se concentrent sur les mécanismes de démixtion de phase et d’adhésion de contact dans le processus d’endocytose chez la levure Saccharomyces cerevisiae. Pour enquêter sur le rôle des modifications post-traductionnelles dans ces mécanismes, nous avons premièrement analysé la cinétique de phosphorylation des protéines en conditions de stress. Mes résultats démontrent que le recrutement et la fonction de certaines protéines impliquées dans le CME se régulent via des mécanismes de phosphorylation. Outre les processus de contrôle post-traductionnel, nous avons élucidé le rôle des domaines de faible complexité dans l’assemblage de plusieurs protéines associées avec le CME. De concert avec les modifications de phosphorylation, des domaines d’interaction protéine-protéine de type PrD (du terme « prion-like domains ») modulent directement le recrutement des protéines au sein des NMO associés au CME. La nature intrinsèquement désordonnée de ces PrD favorise un mécanisme d’assemblage des protéines par démixtion de phase tel que postulé. Finalement, mes travaux confirment que la formation de ces NMO spécifiques génère des forces mécaniques qui déforment la membrane plasmique et assurent le processus de CME. D’un point de vue fondamental, mes recherches permettent de mieux comprendre l’évolution d’une stratégie cellulaire pour assembler des compartiments cellulaires sans membrane et pour fixer les dimensions biologiques associées au CME. De manière plus appliquée, cette étude a le potentiel de générer des retombées importantes dans la compréhension et le traitement de maladies neurodégénératives souvent associées à une séparation de phase aberrante et à la formation d’agrégats protéiques liés à la pathologie.Evolution has resulted in distinct mechanical processes that determine the shapes of living cells and their interactions with each other and with the environment. These molecular mechanisms have contributed to the wide variety of life we observe today. For example, mammalian cells rely on a complex cytoskeleton to adapt specific shapes whereas bacteria, yeast and plants use a combination of turgor pressure and cell walls to have their characteristic bloated form. In this dissertation, I describe my discovery of an unforeseen additional mechanism of morphogenesis: protein aqueous-aqueous phase separation and adhesive contact between biomaterials as a simple and efficient ways for cells to organize internal matter and accomplish work to shape internal structures and surfaces. I specifically describe how a fundamental process of phospholipid membrane and membrane-embedded protein recycling, clathrin-mediated endocytosis (CME), is driven by this mechanism. Analogous to water and oil emulsions, proteins, and biopolymers in general, can phase separate from single to a binary aqueous phase. For proteins that de-mix from the bulk environment, the intermolecular interactions (or cohesive energy) that favors protein condensation only needs to overcome the low mixing entropy of the system and represents a conserved and energy efficient cellular strategy [2, 3, 7, 8]. So far, various examples of phase separated cellular compartments, termed non-membranous organelles (NMOs), have been discovered. These include the nucleoli, germ line P granules and P bodies, to name a few [1-6]. NMOs are involved in many conserved biological processes and can function as storage, bioreactor or signaling bodies. Cells use phase separation as a scheme to organize internal matter, but do NMOs occupy other complex functions, such as morphogenesis? What specific signals trigger protein phase separation? Based on mechanical contact theory, I proposed that hundreds of nanometer- to micron-scale phase separated bodies can deform the cellular environment, both cytoplasm and membranes, through interfacial adhesion. I studied how mechanical contact between a phase-separated protein fluid droplet and CME nucleation sites on membranes drive endocytosis in the model organism budding yeast, Saccharomyces cerevisiae. Specifically, this dissertation describes first, my investigations of post-translational modifications (phosphorylation) of several CME-mediating proteins and the implications of these modifications in regulating CME. I then describe how my efforts to understand what was distinct about the proteins that are phosphorylated led me to propose their phase separation into droplets capable of driving invagination and vesicle formation from plasma membrane. I used fluorescence microscopy, mass spectrometry and micro rheology techniques to respectively determine the spatiotemporal dynamics, phosphorylation modifications and material properties of coalesced CME-mediating proteins. I further investigated how phase separation of these proteins might generate mechanical force. I demonstrate that changes in the phosphorylation of some endocytic proteins regulates their recruitment to CME nucleation sites. We achieved reliable predictions of functional phosphosites by combining information on the conservation of the post-translational modifications with analysis of the proportion of a protein that is dynamically phosphorylated with time. The same dynamically phosphorylated proteins were enriched for low amino acid compositional complexity “prion-like domains”, which we demonstrated were essential to these proteins undergoing aqueous-aqueous phase separation on CME nucleation sites. I then demonstrate how phase separated droplet can produce mechanical work to invaginate membranes and drive CME to completion. In summary, I have discovered a fundamental molecular mechanism by which phase separated biopolymers and membranes could apply work to shape each other. This mechanism determines the natural selection of spatial scale and material properties of CME. Finally, I discuss broader implications of this dissertation to mechanistic understandings of the origins of neurodegenerative diseases, which likely involve pathological forms of protein phase separation and/or aggregation

Design and Application of Wireless Body Sensors

Hörmann T. Design and Application of Wireless Body Sensors. Bielefeld: Universität Bielefeld; 2019