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- Zhang, Yuecheng
(författare)
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Glycosylation in cancer and infection : the role of sialic acid
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Sialic acids (SA), a group of nine-carbon backbone monosaccharides are abundantly expressed in vertebrates. They are usually linked to the terminal of glycan chains and play crucial roles in many biological processes, including cell adhesion, cell-cell interactions, immune modulation, cancer cell migration and invasion, as well as viral infections. To analyze and monitor SA expression, antibodies and glycan-binding lectins are typically used. However, high costs and poor stability limit the application in SA analysis. To overcome these drawbacks, an imprinting technique was used to synthesize an alternative SA receptor – SA molecularly imprinted polymers (SA-MIPs). Fluorescent molecules are embedded into the MIPs, facilitating the detection of MIPs binding to cells by flow cytometry and fluorescence microscopy. Firstly, core-shell SA imprinted MIPs were used to analyze SA expression in a panel of breast cancer cell lines. The SA expression of these cell lines was also tested by using the two glycan-binding lectins, MAL andSNA, which recognize α2,3 and α2,6 SA, respectively. Our results show that breast cancer cell lines express α2,3 and α2,6 SA dissimilarly, and hence present different SA-MIP binding patterns. The specificity of SA-MIPs was further verified by an inhibition assay using two pentavalent SA conjugates that interfere with the SAMIPs.Furthermore, the SA-MIP synthesis protocol has been improved by using silica-coated polystyrene particles. The polystyrene core particles are lighter and smaller, increasing MIP suspensibility and augmenting MIP-cancer cell interactions. The cancer cell binding properties and the specificity have been verified by using thirteen different cancer cell lines, showing that the SA-MIPs can be used as effective tools for SA expression analysis. The SA-MIPs were used to analyze the SA expression of in vitro cultured cells treated with soluble cytokines to mimic the tumor microenvironment. The SA expression of two cancer cell lines stimulated with soluble cytokines was analyzed by using lectins and SA-MIPs. The MIPbinding data correlated well with lectin staining results, demonstrating the potential of SA-MIPs to be used in the analysis of overexpressed SA in the tumor microenvironment. Furthermore, the involvement of SA in the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was assessed. The viral surface receptor-binding domain (RBD) recognizes and conjugates with receptors on host cells, triggering the infection. Although the interaction between the RBD and host cells has been extensively studied, the mechanism behind this reaction is not fully determined. In this study, the interaction between the viral RBD and a panel of human cell lines from tissues susceptible to viral infection was tested. Moreover, the role of SA in this interaction has also been tested and evaluated.
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| 2. |
- Periyannan Rajeswari, Prem Kumar
(författare)
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Droplet microfluidics for single cell and nucleic acid analysis
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Droplet microfluidics is an emerging technology for analysis of single cells and biomolecules at high throughput. The controlled encapsulation of particles along with the surrounding microenvironment in discrete droplets, which acts as miniaturized reaction vessels, allows millions of particles to be screened in parallel. By utilizing the unit operations developed to generate, manipulate and analyze droplets, this technology platform has been used to miniaturize a wide range of complex biological assays including, but not limited to, directed evolution, rare cell detection, single cell transcriptomics, rare mutation detection and drug screening.The aim of this thesis is to develop droplet microfluidics based methods for analysis of single cells and nucleic acids. In Paper I, a method for time-series analysis of mammalian cells, using automated fluorescence microscopy and image analysis technique is presented. The cell-containing droplets were trapped on-chip and imaged continuously to assess the viability of hundreds of isolated individual cells over time. This method can be used for studying the dynamic behavior of cells. In Paper II, the influence of droplet size on cell division and viability of mammalian cell factories during cultivation in droplets is presented. The ability to achieve continuous cell division in droplets will enable development of mammalian cell factory screening assays in droplets. In Paper III, a workflow for detecting the outcome of droplet PCR assay using fluorescently color-coded beads is presented. This workflow was used to detect the presence of DNA biomarkers associated with poultry pathogens in a sample. The use of color-coded detection beads will help to improve the scalability of the detection panel, to detect multiple targets in a sample. In Paper IV, a novel unit operation for label-free enrichment of particles in droplets using acoustophoresis is presented. This technique will be useful for developing droplet-based assays that require label-free enrichment of cells/particles and removal of droplet content. In general, droplet microfluidics has proven to be a versatile tool for biological analysis. In the years to come, droplet microfluidics could potentially be used to improve clinical diagnostics and bio-based production processes.
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