Cell-free protein synthesis (CFPS) using crude lysates has developed into a robust platform technology over the last 60 years to express numerous types of recombinant proteins. The open-nature, elimination of reliance on cell viability, and focus of all energy towards production of the protein of interest represent substantial advantages of CFPS over in vivo protein expression methods. CFPS has provided new opportunities across a series of research fields that include metabolic engineering, therapeutic and vaccine development, education, biosensors, and many more. In recent years, optimizations of CFPS have even allowed the platform to reach the industrial level of protein production. Although there have been many advancements toward CFPS development, the democratization of the platform to a wide variety of educational, research, and industrial institutions has lacked due to an absence of resources for new users as well as a limited number of developments toward redesigning the tedious and time-consuming protocols to generate robust cell extract. To address these challenges to CFPS implementation, a comprehensive review spanning numerous cell lines with their respective applications, methodologies, and reaction formats were provided in addition to detailed protocols outlining the process of going from E. coli cells to a completed CFPS reaction. Together, these resources provide the scientific community with easily accessible resources for CFPS implementation. Moreover, the aforementioned protocols were redesigned from a four-day process into one that may be completed in under 24-hour’s time with very little researcher oversight. The resulting workflow maintained the robustness of prior methods but generated 400% more extract compared to traditional methods via a set-it-and-forget-it approach. To date, the works presented herein have garnered tremendous viewership from the CFPS research community with a substantial following among all three of the articles. Moving forward, I anticipate that these works will continue to bring new users into the CFPS field through the ease of access to these resources and through the advance of the simplistic and reproducible new workflow for preparation of robust E. coli cell extract.

Purpose: To determine the effect of four postexercise beverages, differing in macronutrient content, on metabolic response to an acute resistance exercise bout. Methods: Forty male subjects performed five sets of eight repetitions at 80% 1RM for leg press and leg extension, and then consumed one of four postexercise beverages (Placebo, PL: a carbohydrate-electrolyte beverage, CE; or one of two milk-based beverages, MILK 1: 1% chocolate milk; MILK 2: a high protein milk beverage). Indicators of muscle protein synthesis (MPS) were assessed before and 1-hr after consuming a postexercise beverage. Muscle protein degradation (MPD) was examined the day before and the day of exercise. Results: No significant differences were found among groups in MPS. The resistance exercise bout increased the amount of eIF4E-eIF4G by 4.5% 1-hr postexercise (p<0.05) without affecting the amount of eIF4E-4E-BP1. One hour after beverage consumption, serum total amino acid concentration increased for MILK 1 (p=0.003) and MILK 2 (p<0.001) but decreased for CE (p=0.028) and PL (p=0.276). Consumption of MILK 1, MILK 2, and CE significantly increased circulating levels of serum insulin (p<0.001). Serum growth hormone increased 3-fold as a result of the exercise bout but fell to baseline for all groups by 60 min (p<0.001). Conclusion: The resistance exercise bout was anabolic as shown by the increase in the active eIF4E-eIF4G complex and serum growth hormone. Consumption of MILK 2 led to the most optimal environment for muscle anabolism; however, none of the experimental beverages influenced the measured indicators of muscle protein translation 1-hr after ingestion. / Master of Science

Resistance exercise (RE) training is a well-known and effective method for promoting increases in muscle mass and strength. A single bout of RE induces physiological disturbances that require coordinated activation of the immune system and intramuscular signaling in order to return the tissue to homeostasis and adapt to the RE challenge. On the other hand, acute binge alcohol consumption can affect the immune response to an inflammatory challenge, intramuscular anabolic signaling, and muscle protein synthesis, and the effects of alcohol on these processes are opposite that of RE. Furthermore, individuals who report more frequent exercise also report a greater frequency of binge drinking. However, few investigations exist regarding the effects of binge alcohol consumed after a bout of RE on RE-induced physiological changes and performance recovery. Therefore, the overarching purpose of the investigations contained within this dissertation was to investigate the effect of alcohol consumed after RE on the RE-induced changes in mTOR pathway signaling, muscle protein synthesis, inflammatory capacity, strength recovery, and power recovery. Although RE increased mTOR pathway signaling and inflammatory capacity after exercise and reduced maximal strength and explosive power the day after exercise, we observed no effects of alcohol (1.09 g ethanol∙kg-1 lean body mass, designed to result in a peak blood alcohol concentration of approximately 0.12 g∙dl-1) consumed after RE on mTOR pathway signaling, 24-hour rates of muscle protein synthesis, inflammatory capacity, or strength and power recovery in resistance-trained individuals.

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