Browsing by Subject "Mammalian cells"

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    Advancements In Microfluidics For Biotechnology Applications
    (2018-10) Agrawal, Pranav
    Microfluidic technology has made a huge impact in the field of biotechnology and life sciences. The advancements can be categorized into three aspects: understanding of physical phenomena at the microscale; development of tools for easy integration of different phenomena; and devising systems for various applications. This thesis highlights the ability of microfluidic technology in manipulating different biological entities by fabricating small feature sizes. In particular, we have focused on the development of new processes for three biotechnology applications – (i) long DNA sample preparation for genomic; (ii) delivery of genetic delivery vehicles for gene and cell therapy; and (iii) an in vitro model to study human gut. Each of these systems is developed in close collaboration with potential users and is aimed towards easy integration with the existing workflow. Long-read genomic applications such as genome mapping in nanochannels require long DNA that is free of small-DNA impurities. Chapter 2 reports a chip-based system based on entropic trapping that can simultaneously concentrate and purify a long DNA sample under the alternating application of an externally applied pressure (for sample injection) and an electric field (for filtration and concentration). In contrast, short DNA tends to pass through the filter owing to its comparatively weak entropic penalty for entering the nanoslit. The single-stage prototype developed here, which operates in a continuous pulsatile manner, achieves selectivity of up to 3.5 for λ-phage DNA (48.5 kilobase pairs) compared to a 2 kilobase pair standard based on experimental data for the fraction filtered using pure samples of each species. The device is fabricated in fused silica using standard clean-room methods, making it compatible for integration with long-read genomics technologies. Non-viral delivery vehicles are becoming a popular choice to deliver genetic materials for various therapeutic purposes, but they need engineering solution to improve and control the delivery process. In Chapter 3, we demonstrate a highly efficient method for gene delivery into clinically relevant human cell types, such as induced pluripotent stem cells (iPSCs) and fibroblasts, reducing the protocol time by one full day. To preserve cell physiology during gene transfer, we designed a microfluidic strategy, which facilitates significant gene delivery in short transfection time (<1 minute) for several human cell types. This fast, optimized and generally applicable cell transfection method can be used for rapid screening of different delivery systems and has significant potential for high-throughput cell therapy applications. Microfluidic in vitro models are being developed to mimic individual or combination of various human organ functions for systematic studies, and for better predictive models for clinical studies. In Chapter 4, we outline a microfluidic-based culture system to study host-pathogen interaction in the human gut. We demonstrate that the infection of Enterohemorrhagic Escherichia coli (EHEC) in epithelial cells are oxygen dependent and can be used to prolong co-culture of bacterial and epithelial cells. This work presents a large scope to study the factors influencing the infection, especially the commensal microbiome in the human gut. Overall, this thesis shows how the microfluidic system can be useful in solving real-life problems and envision further advancements in the field of biotechnology.
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    Investigation of proteins that interact with NAADP-Gated two-pore channels.
    (2012-02) Moshier, Yaping Lin
    All living organisms respond to environmental stimuli by eliciting a sequence of signaling cascades, many of which converge in regulating [Ca2+]cyt via intracellular Ca2+ stores. Three agonist-mediated second messengers have been identified, including inositol 1,4,5-trisphosphate (IP3), cyclic ADP ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP). NAADP is the most potent calcium mobilizer identified to date, and unlike IP3 and cADPR that target ER Ca2+ stores, NAADP-mediated Ca2+ response is restricted to acidic Ca2+ stores. Several candidate Ca2+ channels expressed in the endolysosomal system have been proposed to be gated by NAADP, with recently two-pore channels (TPCs) emerging as NAADP targets. My research project utilized a radioactive photoactivable NAADP analogue, 32P-5 azido-NAADP (32P-5N3-NAADP), to perform an unbiased assay in a variety of model systems, attempting to verify NAADP targets via a direct crosslinking approach. My results revealed that 5N3-NAADP labeled protein candidate(s) were significantly smaller than predicted sizes of TPC proteins (85-100kDa) in all systems examined (22/23kDa doublet in mammals, 41kDa in sea urchin). Further, the labeling pattern and intensity of the NAADP-targeted doublet remained unchanged in TPC-overexpressing cells. Surprisingly, photoaffinity labeling (PAL) of the high affinity NAADP targets was preserved in TPC-knockout pancreatic samples, further suggesting that NAADP binding and Ca2+ release are mediated by distinct protein identities. My data contradicts current models that TPCs are direct NAADP receptors, implying an alternative cellular binding partner for NAADP on endolysosomes that then serves to indirectly regulate TPC activity. This is an important revision of current dogma, and crucial for rational design of drugs that may modulate NAADP activity. Such therapeutics may be important in disorders (diabetes, lysosomal storage disorders, and neuronal excitotoxicity) where NAADP signaling is pathologically perturbed.