Browsing by Author "Amrein, Matthias"
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- ItemOpen AccessAnalyzing the Nanomechanical Oscillations of Brain Tumor Cells using Optical Tweezers(2019-01-02) Ghandorah, Salim; Amrein, Matthias; Ghasemloonia, Ahmad; Green, Francis H. Y.; Mahoney, Douglas J.Under the microscope, cells manifest nanomechanical oscillations (also known as vibrations and fluctuations). While the exact source of these oscillations is unknown, they may reflect the numerous inherent active processes of living cells. The sum of these active processes of cell characterizes its phenotype and function. Therefore, we hypothesize that the oscillation spectrum is unique for each cell type, and reflective of its functional state, allowing phenotypically-different cell types to be differentiated and functional changes in the cells to be followed. To test this hypothesis, we employed a novel, highly sensitive tool, optical tweezers, to record the oscillations of two brain tumor cells and applied advanced multivariate analysis to statistically evaluate the difference between the oscillation spectra. Two main sub aims were addressed; (1) To find optimal conditions for the optical tweezers setup for single cell oscillation measurements, and (2) To establish statistical methods to differentiate cells based on their oscillation patterns. Our results showed feature-rich spectra of different cell types over a frequency range from a few Hertz (Hz) to 50 kHz. The multivariate analysis generated two separate clusters for each cell type. The analysis also allowed evaluation of the spectra for features that are strongly associated with differences and the features that are common among cells. My thesis expands the current knowledge of cellular oscillations. While previous studies demonstrated a correlation between the metabolic activity of cells and overall magnitude of oscillations, spectral decomposition was either not reported or showed few, if any, cell-specific frequencies. In conclusion, the recording of oscillation spectra of single cells using optical tweezers, followed by multivariate statistical analysis is a promising method to differentiate cell types and follow cellular functions in real-time.
- ItemOpen AccessBiophysical Characterization of Nanoparticle Interactions with Lung Surfactant Models for Enhanced Pulmonary Drug Delivery(2017) Lai, Patrick; Prenner, Elmar J.; Edwards, Robert Allan; Amrein, Matthias; Heyne, Belinda; Lavasanifar, AfsanehInhalable nanoparticles for drug delivery has shown promising results in the treatment of lung disease. The impact of these small sized drug carriers on the lungs is remains unclear. One of the first barriers that nanoparticles will encounter upon inhalation is the lung surfactant that lines each alveolus. This is a single molecule layer of lipids and proteins that forms at the air-water interface on top of the alveolar lining fluid. Its main role is to lower surface tension preventing the collapse of the alveoli during the breathing cycle. Impairing surfactant function results in collapse of the alveolar sacs leading to respiratory distress. The aim of the thesis was to use in vitro studies to identify which components of lung surfactant that are potentially impacted or influenced by the presence of nanoparticles. This was conducted using a model system for lung surfactant made up for phosphatidylcholine, phosphatidylglycerol and the neutral lipid cholesterol. This was compared to a clinical surfactant BLES that is used in surfactant replacement therapy. Surface activity was measured using a Langmuir trough with two Teflon barriers to mimic the air-water interface and compress the surfactant monolayer. Imaging of the monolayer was conducted with Brewster angle microscopy to visualize changes in the organization of the surfactant models. Gelatin and polyisobutylcyanoacrylate nanoparticles are both biocompatible materials that have been tested for inhalable drug delivery and are used in these experiments. In vitro testing was done to evaluate three different methods of adding nanoparticles to the surfactant monolayer to develop a better in vitro model to study nanoparticle surfactant interactions. These nanoparticles were either mixed with surfactant before application on the trough, added to the subphase or sprayed from the air as dry powder. Cholesterol was found to play a major role in nanoparticle-surfactant interactions by enhancing the formation of spike-like structures from the surfactant monolayer. The Langmuir trough was shown to be a useful tool for studying in vitro interactions. Further optimization of the spraying dry powder nanoparticles onto the Langmuir trough can potentially be a useful tool in vitro tool to predict potentially harmful in vivo effects
- ItemOpen AccessBronchial Thermoplasty and Airway Remodeling in Severe Asthma(2016) Laing, Austin; Kelly, Margaret; Proud, David; Green, Francis; Amrein, MatthiasIncreased airway smooth muscle (ASM) is a characteristic of severe asthma and strongly associated with airway hyperresponsiveness. Bronchial Thermoplasty (BT) aims is a new technique in which heat is applied to the airway. We hypothesize that BT causes reduced ASM in patients with severe asthma. We investigated the effect of BT on the airway in patients with severe asthma, acute effects in a pig model and the effect of hyperthermia on ASM and bronchial epithelial cells. We found a significant reduction in ASM and goblet cells at 6 weeks and 1 year post-BT, respectively. Acute effects of BT in the pig airway suggested a sterile injury pattern. Exposure to 65oC produced morphological changes in ASM and epithelial cells. We conclude that there is evidence of ASM and goblet cell reduction in response to BT but further confirmation awaits full analysis of the study in asthmatics.
- ItemOpen AccessCell Vibrational Profiling (CVP) Using Optical Tweezers to Improve Tumour Diagnosis: A Novel Methodological Approach(2021-06-29) Topham, Jared James; Amrein, Matthias; Green, Francis; Farshidfar, FarshadCellular oscillations have long been recognized and previously suggested as a powerful real-time diagnostics tool for identifying and differentiating biological specimens. The oscillatory signals emitted by various biological samples are thought to be due to metabolic processes inherent to the measured sample. Various methods to measure oscillatory signals have used atomic force and dark-field microscopy accompanied by traditional interpretative methods such as Root Mean Square, Fast Fourier transform, time-domain analysis, and power spectral density. While these approaches were fundamental to the recognition of the field of cellular oscillations, the instrumentation lacked sensitivity and resolution, leading to difficulties in (1) analyzing complete frequency spectra of more than a few Hz, (2) providing empirically tested methodological approaches, and (3) statistically differentiating and/or determining distinct frequency regions for a cell-type of interest. This thesis sought to build and test a novel methodological approach to measuring and determining cellular vibrational frequency profiles. We hypothesized, each cell line (NMSCs, HEKa, and A549) exhibited unique and measurable vibrational frequency profiles reflective of their intrinsic cellular processes, measured via optical tweezers. We disclosed our efforts to improve the methodology by (1) optimizing experimental set-ups, looking specifically at sample preparation (implementation of microfluidic chambers, synchronization of cell cycles, and changes to media viscosity), and (2) analyzing data through the implementation of multivariate statistical analysis achieved through our newly developed software: VibrationScanner. Our results capitalized on our discoveries to establish a new method, cell vibrational profiling, in hopes that it may become a high-throughput, cost-effective, and sensitive technique to reliably identify biological functions and frequency components of cells.
- ItemOpen AccessCharacterization of Viscoelastic Materials Using Atomic Force Microscopy(2017) Hoorzad, Hamid; Kim, Seonghwan; Du, Ke; Egberts, Philip; Amrein, MatthiasAtomic Force Microscopy (AFM) is a versatile method for nanoscale measurement of the properties of materials. While many methods have been developed to utilize AFM for nanoscale characterization of stiffness, damping characterization has remained less explored. In this research, we present a method for measuring stiffness and damping using Contact Resonance Atomic Force Microscopy (CR-AFM) and Modal Finite Element Analysis (FEA). To do so we compare resonance peaks and quality factors obtained from FEA and CR-AFM and we adjust the parameters until there are in good agreement. We use this method to measure the stiffness and damping of several polymers.
- ItemOpen AccessDoes Calcium Interact With Titin’s Immunoglobulin Domain in Cardiac Muscle?(2009) DuVall, Michael; Amrein, Matthias; Gifford, Jessica; Herzog, Walter
- ItemEmbargoEfficient Clearance of Inhaled Nanoparticles Depends on Strong Adhesion to the Epithelium: The Role of Hydrophilicity, Coating, Size, and Shape(2023-09-14) Bogari, Nawaf Nasir; Amrein, Matthias; Green, Francis; Yates, Robin; Shi, Yan; Schriemer, David; Hubbs, AnnHealth alerts regarding high levels of fine particles in ambient air are increasingly common, reflecting a significant worldwide crisis. These particles contribute substantially to premature death, a problem only expected to grow. Among these, nanoparticles pose a particular threat, linked to respiratory diseases such as chronic obstructive pulmonary disease (COPD), asthma, and cancer as well as extrapulmonary effects. The health implications of nanoparticles differ, rendering simple particle concentration nearly meaningless. In this thesis, we focused on understanding the accumulation of nanoparticles in the lung, which is a critical aspect of their toxicity. We developed in-vitro assays to predict whether particles remain in the alveolar lumen to be cleared through alveolar and airway pathways, and which particles traverse the alveolar epithelium to induce interstitial lung disease or enter the blood and lymphatic vessels to cause systemic effects. We hypothesized that the fate of inhaled nanoparticles in the alveolar lung primarily depends on their adhesion strength to alveolar epithelium and the physical/chemical characteristics of the particle. To test this, we employed atomic force microscopy (AFM) for adhesion force measurement, transmission electron microscopy (TEM) to correlate adhesion strength to nanoparticle uptake, and confocal laser scanning microscopy (CLSM) to study nanoparticle translocation. Characteristics of nanoparticles, including protein corona coating, alveolar surfactant coating, hydrophobicity/hydrophilicity, size and geometry were studied in the presence or absence of pharmacological blockers to determine endocytic mechanism(s) involved in the interaction with the epithelium. Results showed that the adhesion of nanoparticles to the epithelial cells was an active process, and the strength of adhesion to the epithelium correlated directly to their uptake and transcytosis. Amorphous silica nanoparticles (ASN) with 15 nm in diameter were found to adhere strongly and translocate across the epithelium, whereas nanocarbon black particles 15 nm in diameter (nCB15) exhibited weak adhesion and remained in the alveolar lumen. Interestingly, commonly studied zeta potential had no influence on the interaction, whereas particle coating with surfactant increased their potential to accumulate in the alveolar lumen. Rendering ASN particles hydrophobic reduced their adhesion to the epithelium. The size of nanoparticles was linked to how cells perceived nanoparticles, with particles larger than 150 nm being endocytosed in a clathrin-enhanced mechanism, while particles less than 150 nm were taken up in a caveolin-enhanced mechanism. The epithelium did not show a preferred endocytic mechanism with respect to clathrin or caveolin for silica nanorods (SNR) yet showed a cytotoxic response to these elongated particles. This work contributes to the development of an effective framework for assessing the potential risks of inhaled nanoparticles, and our novel approach of categorization can support public health policies aiming to reduce exposure to nanoparticles in various environments.
- ItemOpen AccessIdentification and Characterization of Moesin-, PIP2-mediated Solid Particle Phagocytosis(2018-08-09) Tu, Zhongyuan; Shi, Yan; Yates, Robin Michael; Amrein, Matthias; Prenner, Elmar J.; Botelho, RobertoPhagocytosis is the defining feature of professional phagocytes of the innate immune system. This function is typically carried out by phagocytic receptors on the cell surface. These receptors can mediate binding and engulfment of solid particles. However, these phagocytic receptors have evolved very recently in history comparing to phagocytosis as a conserved cellular function. This suggests a primordial form of phagocytosis might exist. Years ago, our laboratory uncovered an expected phagocytic mechanism that solid particle can bind to membrane lipids on phagocytes to trigger lipid sorting. Consequently, this can lead to phagocytosis akin to FcγR-based phagocytosis regarding its dependence on Immunoreceptor Tyrosine-based Activation Motif (ITAM), Src-family kinases, Syk, and phosphoinositide 3-kinase (PI3K). Based on these findings, we proposed a hypothetical mechanism for solid particle phagocytosis termed “Signaling Equivalent Platform” (SEP). In short, membrane engagement with solid structures, either via ligand/receptor binding or merely being stabilized by an approaching solid surface will lead to a shared downstream pathway with the same dependence on ITAM and Syk. Both modes of phagocytosis are equivalent for its activation by solid structures. However, the identity of the ITAM-containing molecule and the exact involvement of lipid during solid particle phagocytosis under SEP is still unclear. This thesis serves to strengthen the idea of SEP by identifying the ITAM-containing molecule and further characterizing the involvement of the ITAM-containing molecule and lipids during solid particle phagocytosis. We used a generic ITAM sequence as a probe and identified moesin as the ITAM-containing molecule from the mouse genome. We further demonstrated that a solid structure binding to the cell surface leads to autonomous accumulation of phosphatidylinositol 4, 5-bisphosphate (PIP2) to the site of contact, which attracts moesin, a conserved structural linker, to the plasma membrane. Moreover, Moesin, via its ITAM, is sufficient to activate phagocytic programming including Syk and downstream signaling that is virtually identical to that initiated by Fcγ receptors. Bioinformatic analysis suggested that this moesin-mediated signaling predates modern Fcγ and immune receptors. This thesis, therefore, reveals an evolutionarily conserved moesin-, PIP2-mediated signaling platform for the evolutionarily conserved phagocytosis that provides essential components for modern ITAM-based signaling cascades.
- ItemOpen AccessInteraction of Silica Nanoparticles with Primary Alveolar Epithelial Cells(2015-09-01) Bogari, Nawaf N.; Amrein, MatthiasThe fate of nanoparticles reaching the alveolar lung is not completely understood. Clearance of these particles has been ascribed to the alveolar macrophages scavenging this region. In contrast, in my thesis, I show in vitro, that particles interact more strongly with the alveolar epithelial cells (AECs) than alveolar macrophages (AMs), are then taken up efficiently and transported across the cells and released on the basolateral side. First, I developed a protocol for obtaining a pure culture of rat AEC. Single cell force spectroscopy showed the AEC to respond strongly and in a clathrin-independent manner to the particles, unlike a host of control cells, including alveolar macrophages. Fluorescence light microscopy and total internal reflection fluorescence light microscopy (TIRF) demonstrated the transport to be novel, actin-dependent and microtubule independent. In summary, my thesis provides evidence of a second clearance mechanism in addition to AMs.
- ItemOpen AccessObservations in Lipid Membrane Systems(2015-09-18) Munro, Fay; Amrein, Matthias; Shi, YanThe plasma membrane is phase separated into a fluid (Ld) phase and a more ordered (Lo) phase. The latter exists as small “rafts” of specific lipid composition containing a host of signaling proteins. Lipid raft theory links the aggregation of rafts to a signaling event. In its current iteration, the theory is incomplete, as it does not explain how rafts form and how they aggregate and disperse again. These problems are addressed when the membrane is viewed as a critical system. Previous work using giant plasma membrane vesicles (GPMVs) used this framework to explain the dynamics of the rafts. We intended to show critical behavior as a factor for cell signaling. Critical behavior was not easily ascertained and further observations were made. We found a heterogeneous population with respect to the behavior of the vesicles. We categorized these observations with respect to the appearance of the lipid membrane to characterize the phenomenon.
- ItemOpen AccessThe Effects of Free Radical-induced Epoxidation and Peroxidation of Unsaturated Phospholipids on the Self-assembly of Functional Surfactant Film(2016) Al-Saiedy, Mustafa; Amrein, Matthias; Green, Francis; Ling, Chang-Chun; Prenner, ElmarThe pulmonary surfactant is a protein-lipid mixture that covers the air-water interface. It lowers the alveolar surface tension to near zero values. Surfactant dysfunction has been reported in acute lung injury, acute respiratory distress syndrome, and cystic fibrosis, among other inflammatory lung diseases. Surfactant achieves low surface tension by forming a tightly packed uniform film at the air-water interface and thus, eliminates the electrostatic forces between liquid molecules. As the film pressure (π) increase, surfactant explores a third dimension or multilayers. Monolayer-Multilayer interactions provide additional stability to resist the varying high film pressure at the air-water interface, therefore, preventing premature buckling of the interfacial film. The surfactant may become impaired during pulmonary inflammation, where free radicals degrade its components, whether by introducing epoxides and hydroperoxides to the tightly packed hydrocarbon tails of surfactant phospholipids or by fatty acyl tail fragmentation. In this thesis, we show that oxidation mediated surfactant inhibition is cholesterol dependent. We study surfactant film thermodynamic changes as a result of oxidation and cholesterol. Additionally, we show the structure-function relationship and the underlying importance of monolayer-multilayer anchoring. Moreover, we show that multilayer distribution and arrangements due to oxidized cholesterol species do not alter surfactant film function. Further, we modeled the surfactant film via mechanical finite element analysis software (ANSYS) to examine the role of multilayers. We predict that the mechanism of surfactant dysfunction demonstrated in vitro is relevant to disease state.
- ItemOpen AccessVibrational Profiling of Brain Cells and Tumours using Atomic Force Microscopy(2018-03-08) Nelson, Sultan; Amrein, Matthias; Green, Francis H. Y.; Sutherland, Garnette RoyNanoscopic mechanical vibration is observed as a periodic plasma-membrane fluctuation in living cells. The study of this physiological phenomenon is an emerging field of research. All prior experimental work has been limited to single cell study, including erythrocytes (1,2), leukocytes (3), and cardiomyocytes (4). Moreover, the intensity of fluctuation has been shown to be indicative of cells’ overall metabolic activity (5,6). The fluctuation can be modulated using pharmacological blockers (7,8), thus excluding stochastic Brownian motion as the sole explanation. Given our main interest in the potential clinical application of this phenomenon for qualitative and efficient brain tumor identification, we examined vibration waves emanating from cultured cells, and tissues harvested from the brains of newborn rats, as well as from brain tumors and neocortex specimens. In this research project, we first developed a novel atomic force microscope-based (AFM-based) mechanical vibration detection method and a custom-written MATLAB vibration signal analysis algorithm. The AFM system used in this report utilized sensitive cantilevers (probes) to enhance the signal-to-noise ratio, and to improve overall performance. The method was also designed to detect cellular vibration without direct physical contact between the sample and the cantilever probe. Using this unique method, we recorded vibrations emitted from newborn rat hippocampus and cerebellum samples; these brain regions showed distinct vibration profiles. The effect of pharmacological agents on the tissue samples examined suggested synaptic activity is the major contributor to the subtle vibrations observed. For assessment of potential clinical applications of the method, we examined human surgical brain biopsy samples. Malignant astrocytoma tissue samples vibrated with markedly different frequency peaks and amplitude, compared to tissue from meningioma or normal lateral temporal cortex, thus providing a quantifiable measurement to accurately distinguish the three types of tissues. Lastly, we developed a method to convert cellular oscillation signals into sound within the frequency range of normal human hearing to provide medical specialists with a way to differentiate tumors from healthy brain tissue without the need for extensive, complex vibration, spectral analysis training. Evidence attested through this project has shown that vibrational profiling of cells and tissues can be adopted for simultaneous mapping of neuronal and metabolic activity in the brain. Further, the results of this research may have translational clinical implications as a prompt diagnostic technique, which may aid clinicians in discerning between healthy and cancerous tissues in real-time.