Browsing by Author "Amrein, Matthias W."

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    Adhesion and uptake mechanisms of amorphous silica micro and nanoparticles in an in vitro model of human alveolar epithelial cells
    (2012) Tehranian, Sara; Jeje, Ayodeji A.; Amrein, Matthias W.
    With increasing application of nanoparticles in various fields including automotives, catalysis, cosmetics, electronics, food and textiles and higher likelihood of human exposure, it is important to understand how these particles interact with cells. Also in medicine, nanotechnology based strategies to enhance the transport of different agents into specific tissues, cells or cellular compartments, require an understanding of nanoparticle-cell interactions. Even with numerous publications in this field, there are still significant gaps in knowledge from particle characterization to initial particle-cell interactions and downstream events. The goal of this project is to address these issues systematically. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) are used to explore the links between initial adhesion of a particle to a cell and particle uptake. Amorphous silica is the model particle in this study because of its extensive applications. As the respiratory tract is a unique target for particles, an in vitro model of human type II alveolar epithelial cells (A549) is used. Adhesion and uptake mechanisms of amorphous silica micro and nanoparticles are compared and the effect of agglomeration on the interaction mechanisms is elucidated. Primary human lung fibroblasts are used as control. Transcytosis of the nanoparticles through A549 cells is also studied. The results show differences in the mechanisms involved in the adhesion and uptake of nanoparticle agglomerates and microparticles even when the size of the microparticle is in the range of the nanoparticle agglomerates size. Also, nanoparticle agglomerates appear to induce the same cellular responses as individual nanoparticles. A549 and primary human lung fibroblasts respond differently to nanoparticles. However, for microparticles, adhesion and uptake mechanisms are similar for both cell types. Transcytosis studies show that nanoparticles are able to cross A549 cells. TEM tomography reveals details of particle uptake and provides some leads on the endosomal escape of particles. The methods developed in this project open up a spectrum of investigations that otherwise would not be possible such as screening a large library of particles on different target cells. In addition to a fast turnover, this method allows "dissecting" the early steps of particle-cell interaction, including the biophysical reasons for adhesion and the events that trigger particle uptake.
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    Altered mechanical properties of titin immunoglobulin domain 27 in the presence of calcium
    (European Biophysics Journal, 2013-04) DuVall, Michael M.; Gifford, Jessica L.; Amrein, Matthias W.; Herzog, Walter
    Titin (connectin) based passive force regulation has been an important physiological mechanism to adjust to varying muscle stretch conditions. Upon stretch, titin behaves as a spring capable of modulating its elastic response in accordance with changes in muscle biochemistry. One such mechanism has been the calcium-dependent stiffening of titin domains that renders the spring inherently more resistant to stretch. This transient titin-calcium interaction may serve a protective function in muscle, which could preclude costly unfolding of select domains when muscles elongate to great lengths. To test this idea, fluorescence spectroscopy was performed revealing a change in the microenvironment of the investigated immunoglobulin domain 27 (I27) of titin with calcium. Additionally, an atomic force microscope was used to evaluate the calcium-dependent regulation of passive force by stretching eight linked titin I27 domains until they unfolded. When stretching in the presence of calcium, the I27 homopolymer chain became stabilized, displaying three novel properties: (1) higher stretching forces were needed to unfold the domains, (2) the stiffness, measured as a persistence length (PL), increased and (3) the peak-to-peak distance between adjacent I27 domains increased. Furthermore, a peak order dependence became apparent for both force and PL, reflecting the importance of characterizing the dynamic unfolding history of a polymer with this approach. Together, this novel titin Ig-calcium interaction may serve to stabilize the I27 domain permitting titin to tune passive force within stretched muscle in a calcium-dependent manner.
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    Electrochemical Biosensors Based on Thiol and N-Heterocyclic Carbene Self-Assembled Monolayers
    (2019-04-29) Mayall, Robert Matthew; Birss, Viola I.; Amrein, Matthias W.; Sutherland, Todd C.; Sanati-Nezhad, Amir; Mauzeroll, Janine
    Monitoring for the presence of biological threats is of great importance from both a healthcare and military perspective. Traditional monitoring technologies fall into two categories, detection, which relies on quick (<20 minute) sensing of a potential threat agent, and identification, which provides specific information about the nature of the threat agent but takes longer to perform (typically ca. 1 hour). The focus of this thesis is to develop novel sensor platforms capable of performing both detection and identification methodologies utilizing an electrochemical biosensor approach. The fabrication of a detection biosensor was based on the specificity of Toll-Like Receptors (TLRs), which are a class of proteins found in the innate immune system of animals. These proteins detect specific markers for classes of biological agents, with TLR-4 detecting the presence of Gram-negative bacteria. TLR-4 was tethered to a Au electrode via an alkylthiol self-assembled monolayer (SAM) using an oriented approach to mimic the natural conformation that the protein adopts in the immune system. This sensor system was shown to detect the presence of Gram-negative bacteria (from 1 to 105 cells/mL) in phosphate buffer solutions, while remaining insensitive towards both Gram-positive and viral challenges. This sensor was then modified in order to increase the measured currents and enable the transition to a deployable instrumentation system. The incorporation of trace levels of ferrocene moieties into the SAM was studied, producing a simple method to increase the currents by >3 orders of magnitude. When applied to the TLR-4 biosensor platform, no loss of sensitivity or specificity was observed. The higher currents allowed for the translation of the TLR-4 biosensor onto an inexpensive open-source potentiostat system that is field-deployable. A novel SAM chemistry, utilizing ultra-stable N-heterocyclic carbenes (NHCs), was also studied for use as a platform in the development of biosensors. A sensor capable of detecting intact measles virions, without any pretreatment steps, was developed, for the first time, using either NHC or comparable alkanethiol SAMs. While both systems showed excellent specificity, the NHC system produced significantly larger signals over the same range of measles virus concentrations as the alkanethiol system. Importantly, the sensors based on the NHC SAMs were able to detect the presence of measles virions after two weeks of storage, whereas the alkanethiol SAMs could not. Overall, it is clear that these sensors are strong candidates for the monitoring of biological threat agents. The oriented approach to protein attachment and the incorporation of ferrocene into the SAMs demonstrated excellent detection on par or better than other published work. This thesis also represents the first report of an electrochemical biosensor based on NHC SAMs, showing a significantly better response than comparable alkanethiol SAMs and far superior stability towards storage of a pre-fabricated sensor. Combined, the work of this thesis demonstrates that electrochemical biosensors hold great promise as sensors for both detection and identification purposes in healthcare and military settings.
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    Subversion of dendritic cell immunity to Cryptococcus gattii by a novel phagosomal F-actin cage structure
    (2020-04-28) Jamil, Khusraw; Mody, Christopher Hugh; Ganguly, Anutosh; Amrein, Matthias W.; Yates, Robin Michael; Yong, Voon Wee
    The highly virulent fungus, Cryptococcus gattii, emerged as a novel respiratory pathogen on Vancouver Island (British Columbia, Canada) nearly two decades ago and has spread to the surrounding regions encompassing the Pacific Northwest of United States, where there is an ongoing outbreak. C. gattii is a major cause of life-threatening cryptococcosis in immunocompetent individuals and has a mortality rate of up to 33%. Host immune response is a key determining factor for the development of cryptococcal disease. It is now recognized that evasion of host immune recognition is a hallmark of C. gattii pathogenesis, but the mechanism of immune evasion remains unclear. There is increasing evidence that C. gattii subverts dendritic cell (DC) activation to evade the protective T helper cell-mediated immunity. This thesis demonstrates that primary human DC can phagocytose C. gattii yeasts but trafficking to the late phagolysosome is blocked by retention of a filamentous actin (F-actin) cage on the phagosomes. Structural studies by super resolution microscopy revealed a novel, highly branched F-actin cage that physically interfered with lysosomal fusion. C. gattii F-actin cage promoted immune evasion by silencing the canonical RelA signaling of the NF-κB pathway required for DC costimulation and T cell activation. Disruption of the F- actin cage through targeted inhibition or by TNF-α signaling reprogrammed quiescent DC to immunocompetent antigen-presenting cells (APCs). Furthermore, the presence of phagosomal F-actin cage corresponded with the presence of C. gattii polysaccharide capsule. Acapsular mutant strains did not retain phagosomal F-actin and were remarkable at inducing DC activation and T cell proliferation. Collectively, our results have uncovered a unique mechanism of DC immune subversion by intracellular pathogens such as hypervirulent C. gattii. Manipulations of this mechanism can potentially inform novel therapeutic interventions against C. gattii.
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