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Neuronal membrane disruption and mechanoporation are nanoscale damage mechanisms that critically affect brain cell viability during traumatic brain injury (TBI). These nanoscale cellular impairments are elusive in experiments and necessitate in silico approaches such as molecular dynamics (MD) simulations. Implementing MD, this research aims to investigate the effects of different key factors related to membrane deformation and damage, including force field resolutions, lipid compositions, and loading conditions. To examine the impact of force field resolution, MD deformation simulations were conducted on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) lipid bilayer membranes, using all-atom (AA), united-atom (UA), and coarse-grained Martini (CG-M) force fields. The mechanical responses of the three models progressively changed based on the coarse-graining level. The coarser systems exhibited lower yield stresses and failure strains, and higher mechanoporation damage. To study the influence of lipid components, tensile deformation was applied on seven lipid bilayers, each of which contained a different lipid type commonly found in human brain membrane. Larger headgroup structure, greater degree of unsaturation, and tail-length asymmetry decreased lipid packing, increased the area per lipid (APL), and decreased the failure strain of membrane. Lastly, the deformation behavior of a complex multicomponent MD bilayer (realistically representing human neuronal plasma membrane) under different strain rates and strain states was inspected. The yield stress increased with increasing strain rates and more equibiaxial strain states. Meanwhile, lower strain rates resulted in fewer but larger pores, as well as lower strain and APL at failure. Besides, more equibiaxial strain states exhibited more and larger pores, and lower failure strain. Similar failure APL was obtained regardless of strain states, suggesting that the membrane failed when reaching a critical APL value. In addition, the inclusion of cholesterol was shown to decrease the critical APL. The strain-state dependence results were then used to update the Membrane Failure Limit Diagram (MFLD) that indicates the planar strains for potential membrane failure. Overall, the study provides a non-invasive approach that aids in the current understanding of nanoscale neuronal damage dynamics and essential aspects affecting membrane mechanical responses, and furthermore lays the groundwork for future studies on brain injury biomechanics under various TBI scenarios.

<p>Peptides, specifically cell-penetrating peptides (CPP), have become wonderful research tools due to their enhanced stability, solubility, and ease of synthesis. They have been used for a wide range of biomedical applications, from insecticides to biosensors and drug-delivery scaffolds. The work presented in this dissertation characterizes the biophysical properties of two different CPPs. The first is the cationic amphiphilic polyproline helix (CAPH) peptide, P14LRR. In addition to cell penetration, this CPP has demonstrated broad spectrum antibacterial properties. Fluorescence polarization (FP) and SEC-MALS were conducted to understand the dissociation constant (KD) and oligomerization effects of P14LRR with respect to its putative molecular target in Staphylococcus aureus (S. aureus). A biotinylated derivative of this peptide was also used as a drug-delivery scaffold to transport fluorescently conjugated streptavidin into mammalian cells. A second CPP, DAN13, was also developed as a biosensor for phosphoinositide lipids, specifically PI(4,5)P2. This was effected through careful calibration using stacked supported lipid bilayers (SSLB) in combination with total internal reflection fluorescence (TIRF) microscopy. This was then used to determine the absolute densities and spatial distribution of PIP2 in live KRas mutant cells.</p>

No description available.

No description available.

Ziel der vorgestellten Arbeit war die Synthese von Nucleolipiden zur Lipophilisierung von Oligonucleotiden sowie deren Untersuchung im Hinblick auf ihre Wechselwirkung und Duplex-Bildung an horizontalen Lipidmembranen und verschiedenen Phasengrenzen zur Entwicklung eines neuartigen Bio-Chips für die RNA/DNA-Analyse. Mit der Synthese N(3)-prenylierter und 2’,3’-O-ketalisierter Pyrimidinbasen Uridin und Methyluridin wurden Nucleolipid-Bausteine dargestellt, die auch als terminale Kopfgruppen eines Oligonucleotid-Dodecamers den lipophilen Charakter dieser Oligonucleotid-Sequenz erhöhten. Für den Einsatz solcher LONs (Lipo-Oligonucleotide) in einer vereinfachten RNA/DNA-Analytik wurde eine Vielzahl von Lipo-Oligonucleotiden mit diversen Nucleolipid-Kopfgruppen synthetisiert und auf ihr Einlagerungsverhalten in künstliche Lipid-Bilayer untersucht. Fluoreszenz-spektroskopische Untersuchungen zeigten, dass alle Lipo-Oligonucleotide in der Lage sind, sich in künstliche Lipid-Bilayer einzulagern. Abhängig von der Struktur, der Länge und der Anzahl der C-Atom-Ketten dieser lipophilen Anker-Bausteine wurden die Geschwindigkeit und die Festigkeit der Verankerung im Lipid-Bilayer beeinflusst. Des Weiteren wurde die Hybridisierung von LONs mit komplementären Oligomeren an Lipidmembranen untersucht. Es konnte gezeigt werden, dass die im Bilayer verankerten Lipo-Oligonucleotide mit komplementären Oligomeren DNA-Duplexe bilden. Die hybridisierte DNA wurde nicht nur über einen kovalent gebundenen Cy5-Fluorophor am Gegenstrang nachgewiesen, sondern auch über den DNA-Interkalator SYBR Green I (SG). Am Beispiel von zwei Lipo-Oligonucleotiden (LON 20 und 23), die sich schnell und fest in der Bilayermembran verankern, konnte eine spontane Akkumulation dieser LONs an CHCl3/H2O sowie H2O/n-Decan Grenzflächen direkt nach der Probenzugabe beobachtet werden. Diese und andere Ergebnisse stützen den Einsatz von Lipo-Oligonucleotiden als Ziel-Oligomere in einem neuartigen RNA/DNA-Nachweisverfahren an Phasengrenzen.

Es ist eine allgemein akzeptierte Tatsache, dass der Kontakt von Proteinen mit synthetischen Materialien üblicherweise zur Proteinadsorption an der Materialoberfläche führt. Über den stattfindenden Prozess, insbesondere das Zusammenspiel zwischen Protein-Oberflächen-Wechselwirkungen und konformellen Änderungen der adsorbierten Proteine ist jedoch bisher nur wenig bekannt. In dieser Arbeit wird die ortsgerichtete Spinmarkierungstechnik (SDSL) auf die Strukturuntersuchung adsorbierter Proteine ausgeweitet. Diese nutzt das spezifische Einbringen einer spinmarkierte Seitenkette an gewünschte Positionen der Primärstruktur zur Analyse der Struktur und Dynamik diamagnetischer Proteine mittels der Elektronenspinresonanz(ESR)-Spektroskopie. Das globuläre Protein T4 Lysozym (T4L) wurde auf planare Modelloberflächen adsorbiert und strukturelle Änderungen in Abhängigkeit der physikalischen und chemischen Eigenschaften der Oberfläche verfolgt. Die spezifische Anbindung von T4L auf quarzgestützten zwitterionische Lipiddoppelschichten führt nur zu geringfügigen strukturellen Veränderungen des Proteins. Allerdings bildet sich eine makroskopisch geordnete Proteinschicht aus. Die Vorzugsrichtung der Proteine auf der Oberfläche kann durch Analyse der winkelabhängigen ESR-Spektren bestimmt werden. Die Wechselwirkung negativ geladener Oberflächen mit dem positiv geladenen T4L führt zu drastischeren Störungen der Proteinstruktur. Hierbei wird die Reaktion des Proteins auf den Kontakt mit einer fluiden quarzgestützten Lipiddoppelschicht, die das negativ geladenen Lipid Phosphatidylserin enthält, mit derer bei Adsorption auf einer ebenfalls negativ geladenen, jedoch rigiden Quarzoberfläche verglichen. Dass der Adsorptionsprozess auch das Substrat selbst beeinflussen kann, wird durch die Beobachtung einer Phasentrennung bei Proteinadsorption des Lipidgemischs aufgezeigt, das negativ geladene Lipide enthält. / Although it is commonly accepted that the exposition of proteins to man-made materials typically results in protein adsorption on the material surface, little is known about the interplay between the protein-surface interactions involved and the resulting conformational changes of the adsorbing protein. In this study the site-directed spin labeling (SDSL) approach has been extended to the investigation of proteins adsorbed to planar surfaces. The method involves the selective introduction of an artificial spin-labeled side-chain to a predefined residue of the amino acid sequence and allows the determination of the structure and dynamics of proteins by analysis of the electron paramagnetic resonance (EPR) spectra. The globular protein T4 Lysozyme (T4L) has been adsorbed to planar model surfaces to study the correlation between conformational changes of the protein and the physical and chemical properties of the surfaces. Tethering T4L to a planar quartz-supported zwitterionic lipid bilayer shows only minor changes in the structure of the protein. Furthermore, a macroscopic order of the adsorbed protein layer is proven by angular-dependent EPR spectra which allow the determination of the protein orientation. Offering surfaces that are net negatively charged to the highly positively charged T4L leads to the observation of more drastic conformational changes. Here, the conformation of T4L adsorbing to a fluid quartz-supported lipid bilayer containing negatively charged lipids is compared to the structure of T4L adsorbed to the negatively charged but rigid quartz surface. The adsorption process may also influence the substrate itself. This can be shown by the phase separation of the negatively charged lipid bilayer upon protein adsorption.

Indiana University-Purdue University Indianapolis (IUPUI) / It is increasingly recognized that mechanical properties of substrates play a pivotal role in the regulation of cellular fate and function. However, the underlying mechanisms of cellular mechanosensing still remain a topic of open debate. Traditionally, advancements in this field have been made using polymeric substrates of adjustable stiffness with immobilized linkers. While such substrates are well suited to examine cell adhesion and migration in an extracellular matrix environment, they are limited in their ability to replicate the rich dynamics found at cell-cell interfaces. To address this challenge, we recently introduced a linker-functionalized polymer-tethered multi-bilayer stack, in which substrate stiffness can be altered by the degree of bilayer stacking, thus allowing the analysis of cellular mechanosensitivity. Here, we apply this novel biomembrane-mimicking cell substrate design to explore the mechanosensitivity of C2C12 myoblasts in the presence of cell-cell-mimicking N-cadherin linkers. Experiments are presented, which demonstrate a relationship between the degree of bilayer stacking and mechanoresponse of plated cells, such as morphology, cytoskeletal organization, cellular traction forces, and migration speed. Furthermore, we illustrate the dynamic assembly of bilayer-bound N-cadherin linkers underneath cellular adherens junctions. In addition, properties of individual and clustered N-cadherins are examined in the polymer-tethered bilayer system in the absence of plated cells. Alternatively, substrate stiffness can be adjusted by the concentration of lipopolymers in a single polymer-tethered lipid bilayer. On the basis of this alternative cell substrate concept, we also discuss recent results on a linker-functionalized single polymer-tethered bilayer substrate with a lateral gradient in lipopolymer concentration (substrate viscoelasticity). Specifically, we show that the lipopolymer gradient has a notable impact on spreading, cytoskeletal organization, and motility of 3T3 fibroblasts. Two cases are discussed: 1. polymer-tethered bilayers with a sharp boundary between low and high lipopolymer concentration regions and 2. polymer-tethered bilayers with a gradual gradient in lipopolymer concentration.

Indiana University-Purdue University Indianapolis (IUPUI) / Carbon nanomaterials are widely produced and used in industry, medicine and scientific research. To examine the impact of exposure to nanoparticles on human health, the human airway epithelial cell line, Calu-3, was used to evaluate changes in the cellular proteome that could account for alterations in cellular function of airway epithelia after 24 h exposure to 10 μg/mL and 100 ng/mL of two common carbon nanoparticles, singleand multi-wall carbon nanotubes (SWCNT, MWCNT). After exposure to the nanoparticles, label-free quantitative mass spectrometry (LFQMS) was used to study differential protein expression. Ingenuity Pathway Analysis (IPA) was used to conduct a bioinformatics analysis of proteins identified by LFQMS. Interestingly, after exposure to a high concentration (10 μg/mL; 0.4 μg/cm2) of MWCNT or SWCNT, only 8 and 13 proteins, respectively, exhibited changes in abundance. In contrast, the abundance of hundreds of proteins was altered in response to a low concentration (100 ng/mL; 4 ng/cm2) of either CNT. Of the 281 and 282 proteins that were significantly altered in response to MWCNT or SWCNT, respectively, 231 proteins were the same. Bioinformatic analyses found that the proteins common to both kinds of nanotubes are associated with the cellular functions of cell death and survival, cell-to-cell signaling and interaction, cellular assembly and organization, cellular growth and proliferation, infectious disease, molecular transport and protein synthesis. The decrease in expression of the majority proteins suggests a general stress response to protect cells. The STRING database was used to analyze the various functional protein networks. Interestingly, some proteins like cadherin 1 (CDH1), signal transducer and activator of transcription 1 (STAT1), junction plakoglobin (JUP), and apoptosis-associated speck-like protein containing a CARD (PYCARD), appear in several functional categories and tend to be in the center of the networks. This central positioning suggests they may play important roles in multiple cellular functions and activities that are altered in response to carbon nanotube exposure. To examine the effect of nanotubes on the plasma membrane, we investigated the interaction of short purified MWCNT with model lipid membranes using a planar bilayer workstation. Bilayer lipid membranes were synthesized using neutral 1, 2-diphytanoylsn-glycero-3-phosphocholine (DPhPC) in 1 M KCl. The ion channel model protein, Gramicidin A (gA), was incorporated into the bilayers and used to measure the effect of MWCNT on ion transport. The opening and closing of ion channels, amplitude of current, and open probability and lifetime of ion channels were measured and analyzed by Clampfit. The presence of an intermediate concentration of MWCNT (2 μg/ml) could be related to a statistically significant decrease of the open probability and lifetime of gA channels. The proteomic studies revealed changes in response to CNT exposure. An analysis of the changes using multiple databases revealed alterations in pathways, which were consistent with the physiological changes that were observed in cultured cells exposed to very low concentrations of CNT. The physiological changes included the break down of the barrier function and the inhibition of the mucocillary clearance, both of which could increase the risk of CNT’s toxicity to human health. The biophysical studies indicate MWCNTs have an effect on single channel kinetics of Gramicidin A model cation channel. These changes are consistent with the inhibitory effect of nanoparticles on hormone stimulated transepithelial ion flux, but additional experiments will be necessary to substantiate this correlation.