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Galactomyces Ferment Filtrate Suppresses Melanization And

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Galactomyces Ferment Filtrate Suppresses Melanization And Galactomyces Ferment Filtrate Suppresses Melanization and Oxidative Stress in Epidermal Melanocytes A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTORATE OF PHILOSOPHY (Ph.D.) in the Division of Pharmaceutical Sciences of the James L. Winkle College of Pharmacy by JàNay Karen Woolridge Cooper 2017 B.S. Biological Sciences University of Missouri, Columbia, MO, 2009 Committee Chair: Raymond E. Boissy, Ph.D. ABSTRACT Epidermal melanocytes are particularly susceptible to oxidative stress and cellular cytotoxicity in comparison to the other primary skin cell types due to their normal cell function of melanin synthesis which produces reactive intermediates and oxygen species as byproducts. This vulnerability encourages the development of oxidative driven diseases such as vitiligo, a depigmenting skin disorder resulting from the dysfunction or death of melanocytes consumed by biological stress and immune targeting. Galactomyces ferment filtrate (GFF, Pitera™) is a yeast derived extract comprised of a unique blend of vitamins, minerals, small peptides, and oligosaccharides. GFF is currently used as a moisturizing agent in cosmetics and has demonstrated anti-aging, barrier enhancing, and hypopigmenting capacities in skin. GFF may have a therapeutic utility that is uniquely applicable for vitiligo patients through diminishing melanization and coupled reactive oxygen species generation, while concurrently upregulating intracellular antioxidant activity. To commence this initiative, the fundamental biological mechanisms of GFF were investigated in human pigment cell cultures. The leadoff study assessed the effect of GFF on melanization in cultured human melanoma cells and normal human melanocytes. GFF successfully inhibited melanization in both long- and short- term treatment protocols. The activity of tyrosinase, the primary melanogenic enzyme that commands biochemical melanin synthesis, was also revealed to be diminished by GFF. mRNA expression of novel ion channels and transporters associated with established modulators of cellular and organelle pH were shown to be substantially downregulated in melanocytes treated with GFF; thus, prompting the investigation of pH within the melanosome, the specialized acidic ii organelle that hosts the melanogenesis process. ABCC9, TRPM6, TRPV1, SLC9A4, and SLC13A1 were the gene targets selected for further experimentation. Expression of these uncharacterized ion channels and transporters were found to be reduced in GFF treated melanocytes. Three of these pore proteins also exhibited significant colocalization with melanogenic enzymes and proteins, which implies subcellular localization to melanosomes. Overall, this data demonstrated that GFF efficaciously suppressed melanization in the human pigment cell model in part by deterring tyrosine hydroxylase activity. The new working hypothesis is that GFF significantly alters the pH of the melanosome counter to optimal tyrosine hydroxylase activity via downregulation of ion transport proteins. The second study evaluated the effect of GFF in dampening oxidative stress in normal epidermal melanocytes. GFF proved cytoprotective capabilities after sustaining the cell viability in melanocytes assaulted with 4-tertiary butylphenol, an apoptotic phenolic compound. GFF also suppressed reactive oxygen species generation in cells individually challenged with 4-tertiary butylphenol and ultraviolet radiation. mRNA expression from gene regulatory elements involved in oxidative stress response were shown to be modulated after treatment; thus, instigating an examination of the Nrf2-ARE pathway. GFF positively regulated the expression of the transcription factor Nrf2 and the established downstream phase II enzyme targets HO1, NQO1, and TXNRD1 in both long and short-term treatment protocols. These results begin to explain the mechanisms of action of GFF in the suppression of melanization and oxidative stress in epidermal melanocytes. iii iv ACKNOWLEDGMENTS For the revelation awaits an appointed time; it speaks of the end and will not prove false. Though it linger, wait for it; it will certainly come and will not delay. Habakkuk 2:3, NIV I give my utmost gratitude to my Heavenly Father for setting this path before me and equipping me with everything I needed to complete this journey. Thank you for picking me up out of every dark moment, fortifying the foundation of our faith relationship, and empowering my witness through this test. It wasn’t pretty, but I stand in awe and await the full completion of your work. To my parents, Eric and Renée Cooper, I love you and thank you for everything you’ve done throughout my life to get me to this moment. Mommi, you’ve always advised me to be the best in my field and command my destiny through education at the highest level. I am grateful for your vision and the tenacious spirit you instill in your children. Dad, I cannot begin to describe how invaluable your spiritual wisdom and guidance has been to my life and especially through these last six years. I am beyond appreciative for your presence and wholeheartedly share this victory with you by adopting your last name in hopes that I may bring it honor for the rest of my days. Thank you both for your refreshing encouragement, love, and care. Eric Jr., you are the comedic relief and true natural talent that brings completion to our household. I’m proud being your big sister. v To my beautifully, divine Purnell family: Pop, Mom, G, and YaYa. You are the light force behind everything. Since my birth, you created an environment that has enriched my deepest nature and personality. I am so proud to be a product of your love and provision. I miss you desperately and will carry your colorful memory with me in all that I do in this life. Thank you for helping me become. My best friends and family: Christopher Davis, Jessica Winslow, and Kendra Julion, sustaining this great effort would have been impossible without you. Christopher, thank you for supporting my dream from the beginning. Jessica, our spiritual sisterhood and like-mindedness continues to hearten my disposition and I am excited to travel this life path together. Kendra, thank you for being my respite from harsh realities. You each have upheld me through this journey in an essential manner. I am blessed to have you in my circle and will forever cherish our bonds. I sincerely appreciate the University of Cincinnati, the James L. Winkle College of Pharmacy, and the College of Medicine for this prime educational opportunity and the financial support. I especially thank my supervisory professor and committee chair Dr. Raymond Boissy for welcoming me in to the famed Boissy laboratory and allowing me to develop organically as a scientist. Your critical evaluations and enthusiasm have been motivational throughout the project, especially for my transition into the Doctorate program. I also thank my committee members: Dr. R. Randall Wickett, Dr. Ana Luisa Kadekaro, Dr. Gary Kelm, and Dr. Yuhang Zhang; collectively, you are an astounding group of scientists with extremely pertinent insight and feedback for this project that has been vital to its successes. I particularly thank Dr. Wickett for accepting into the Cosmetic Science program in addition to your multifaceted guidance in a variety of my student vi affairs. I also am inspired by Dr. Kadekaro; your passion for student education and mentorship as well as the thoroughness of your work embodies the scientist I aspire to become. I express gratitude to the entire UC Department of Dermatology for your amassed kindnesses and support throughout my time here. Amy Koshoffer, I am also grateful for your efforts in initiating my advancement as a scientist and teaching me 90% of the techniques and bioassays I know! I appreciate Dr. Zalfa Abdel-Malek and her staff Renny Starner and Viki Swope, whom I had the pleasure of working alongside in the laboratory, for all their assistance and instruction. Additionally, I wish to acknowledge Procter & Gamble, Co. and Dr. Tomohiro Hakozaki for the opportunity to work on such a dynamic project that has been particularly well suited to my scientific and professional interests in pigmentation biology and cosmetics. I am eternally thankful for the host of teachers, coaches, and mentors I have encountered throughout my lengthy scholastic journey for your joint contributions, with special thanks to the initiator Mrs. Sharon Dryden. I also have deep appreciation for Dr. Terri Hill, my mentor of twelve years and the staff at Terri L. Hill, M.D., PA. Thank you all for your support of my professional and educational endeavors beginning in my teenage years. To Mrs. Karen Henry, I cannot thank you enough for all your mentorship, care, and advocacy during my time at UC. I admire your intentionality and devotion to higher education and student affairs for both myself and the entire student roster of the Cosmetic Science program. You have been nothing but helpful and encouraging in the utmost manner. Finally, I thank a host of special friends who have enriched my life during my time in graduate school. Nikkita Womack, my dear friend and former teammate, thank you for your unwavering vii support, wise words, and precious friendship. Dr. Anne von Koschembahr, thank you for being such a considerate friend and motivator both personally and in the laboratory experience; I am inspired by your own educational journey and the determination you displayed
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