Date on Senior Honors Thesis

5-2025

Document Type

Senior Honors Thesis

Degree Name

B.S.

Department

Biology

Committee Chair

Michael Menze

Author's Keywords

Molecular Chaperones; Red Blood Cell (RBC) Preservation; Small Heat Shock Proteins; Desiccation Tolerance; Artemia francsicana; Liquid-Liquid Phase Separation (LLPS)

Abstract

The preservation of complex biological materials in the frozen state has been well established and plays a critical role in the advancement of clinical applications and the pharmaceutical industry. Cryopreservation of complex biological materials has several clinical applications, including vaccine preservation, bone marrow transplants, cell therapeutics, and increasing the shelf life of red blood cells (RBCs) for transfusion. However, many challenges are present, including the use of toxic protective agents and the financial burden of maintaining extremely low temperatures between 193.15 and – 77.6 K (Parihar, Kumar et al. 2023). Lyophilization, or freeze-drying, presents an opportunity to mitigate such challenges. However, dry-state storage of more complex water-loss-sensitive systems has yet to be achieved (Weng 2021). We turned to nature for strategies to help improve lyophilization outcomes and allow for storage at ambient temperatures. The animal extremophile Artemia franciscana, brine shrimp, can survive severe dehydration (desiccation) by upregulation of repair proteins that lessen the damage of water loss and during rehydration. One exceptional repair protein, the small heat shock protein (sHSP) p26, was the focus of this study. Protein expression and purification levels were optimized to study the characteristics of p26 that may allow for it to aid in protection during desiccation. We discovered that p26 physiochemical behavior is exceptionally salt-sensitive, likely promoting liquid-liquid phase separation (LLPS) and production of liquid condensates, which is believed to play a role in protection during drying. Characteristics of a secondary sHSP, ArHSP21, were also examined to compare the sequence and characteristics of p26 with ArHSP21 to understand how different molecular chaperones influence protection during stress. Additionally, molecular analysis was utilized to determine whether these proteins function independently or work together to enhance biomolecular protection. These sHSPs were identified as potential candidates to improve lyopreservation outcomes in other systems, including complex water-stress-sensitive biomolecules and systems.

Lay Summary

Currently, blood must be stored at 4oC and only lasts for up to 42 days before it must be discarded. Thus, proper storage equipment and continuous blood donations are necessary to maintain an adequate blood supply. This supply is critical because blood transfusions are the most common life-saving procedures in hospitals worldwide. Additionally, specific populations, including military personnel and underdeveloped countries, lack the proper equipment to maintain viable blood supplies. For instance, the military requires consistent access to blood for transfusions during combat. However, they cannot feasibly transport refrigerators to far-forward battlefields, limiting blood availability in life-threatening situations during combat. Freeze-drying blood to form a powder – similar to powdered milk - offers a potential solution to these issues, eliminating the need for refrigeration and extending blood's shelf life from days to years. Beyond blood transfusions, drying can potentially benefit vaccine preservation, cell therapies for personalized treatment, and bone marrow transplants.

This research focuses on understanding the mechanisms employed by the fairy shrimp Artemia franciscana, better known as sea monkeys, to allow them to survive drying in hopes that similar mechanisms could be used to successfully dry blood. The research focuses on p26 – a protein thought to aid in the stress protection of sea monkeys. This study aimed to optimize protein expression and purification to gain access to the protein for experimentation to determine the mechanism(s) by which p26 provides stress protection and better understand how this could be conferred to the dry preservation of biological materials such as red blood cells.

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