Browsing by Subject "trehalose"

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    DMSO-Free Cryopreservation of Therapeutic Cells by Agarose Hydrogel Encapsulation
    (2023-07) Wang, Mian
    In recent decades, the rapid advancement of cell therapy and regenerative medicine has generated an urgent need for efficient and safe cell/tissue cryopreservation techniques on both laboratory and industrial scales. The current use of dimethyl sulfoxide (DMSO) as a cryoprotective agent (CPA) presents toxicity concerns, prompting the development of alternative methods. The primary objective of this research is to develop a novel cryopreservation method for therapeutic cells using hydrogel encapsulation, which aims to minimize cryoinjuries while eliminating the need for toxic penetrating CPAs such as DMSO. The dissertation consists of three main parts. In the first part, molecular changes associated with warming injury in cryopreserved human white blood cells (WBCs) were analyzed. It was observed that slow warming led to irreversible dehydration of cell membrane lipids and denaturation of cellular proteins. Also, WBCs were found to be very susceptible to kinetic processes during warming, including eutectic crystallization/melting, devitrification, and ice recrystallization. The second part focuses on the development of an encapsulation cryopreservation method using the combination of agarose hydrogel and trehalose as an alternative to membrane-permeable CPAs. A comprehensive analysis of the kinetic and thermodynamic changes within the agarose-trehalose hydrogel during freeze/thaw was conducted. The combination of agarose and hydrogel was found to reduce ice phase volume with a less ordered structure, eliminate eutectic crystallization and melting, and inhibit ice recrystallization during warming. The third part of the dissertation involves the validation of the DMSO-free hydrogel encapsulation method for the cryopreservation of natural killer (NK) cells. High post-thaw cell viability was achieved, while decreased viability with compromised cytotoxicity was observed after cells were extracted from the hydrogel. Ongoing efforts are focused on optimizing the cell extraction process from the hydrogel.
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    Regulation of insulin signaling in Drosophila melanogaster
    (2015-08) Kim, Jung
    Secreted ligands of the insulin family promote cell growth and maintain sugar homeostasis. Insulin release is tightly regulated in response to dietary conditions, but how insulin producing cells (IPCs) coordinate their responses to distinct nutrient signals is unclear. Here I show that regulation of insulin secretion in Drosophila larvae has been segregated into distinct branches: circulating sugars selectively promote the release of Drosophila insulin-like peptide 3 (Dilp3), whereas amino acids selectively promote secretion of Dilp2. Dilp3 is uniquely required for sugar-mediated activation of TOR signaling and suppression of autophagy in the larval fat body. Sugar levels are not sensed directly by the IPCs, but rather by the adipokinetic hormone (AKH)-producing cells of the corpora cardiaca, and I demonstrate that AKH signaling is required in the IPCs for sugar-dependent Dilp3 release. Thus, IPCs integrate multiple cues to regulate secretion of distinct insulin subtypes under varying nutrient conditions. The sensitivity of insulin signaling determines the activity of insulin in the presence of insulin, and misregulation of insulin sensitivity leads to metabolic diseases such as type-2 diabetes. Mechanical stress is a known regulator of insulin sensitivity, but the mechanisms by which mechanical stress regulates insulin sensitivity are unclear. Here, I showed that mechanical stress is required for activation of insulin signaling in the Drosophila larval fat body both ex vivo and in vivo. Interestingly, mechanical stress affects most of components in the insulin pathway: localization of insulin receptor (InR), chico, and lnk, and the activities of PI3K, AKT, and TOR. I demonstrated that integrin signaling, previously shown to sense mechanical stress, is necessary for the insulin- and mechanical stress-dependent activation of TOR. Together, my data suggest that mechanical stress sensed by integrin signaling regulates insulin sensitivity by altering upstream components of insulin signaling, such as InR, chico and lnk.