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MITOCHONDRIAL GENE EXPRESSION IN HUMAN MONONUCLEAR CELLS By Monika D. Ruchała, M.S. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University. Virginia Commonwealth University, 2014. Director: Dr. James P. Bennett Jr, M.D., Ph.D., Bemiss Professor Departments of Neurology, Psychiatry and Physiology and Biophysics Adult neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), have been intensively studied in recent years in pursuit of mechanisms responsible for origin and progression. One emerging theme is mitochondrial energetic deficiency as a mechanism of neuronal death. Recent descriptions of protocols to generate induced pluripotent stems cells (iPSCs) from living patients offer the potential to create unique disease models. This model can potentially lead to crucial advances in developing treatment options for a wide variety of neurodegenerative diseases. In this thesis, we attempt to induce iPSCs from mononuclear cells (MNC) in peripheral blood acquired from patients with ALS and healthy control (CTL) subjects, and analyze their mitochondrial genomes. The reprogramming of MNC to yield iPSC was done by nucleofection of an episomal plasmid pEB­ C5, expressing OriP sequences of the Epstein­Barr and five reprogramming transgenes Oct4, Sox2, Klf4, c­Myc and Lin28. We investigated the expression of mitochondrial DNA genes, ND2, ND4, COXIII and 12s rRNA in the ALS and CTL MNC before and after their culturing. The results implicate deregulated mitochondrial bioenergetics as a characteristic of ALS. Future work will establish whether these abnormalities in mitochondrial bioenergetics persist in iPSC’s and iPSC-derived neurons from ALS subject

The inner membrane of mitochondria harbors the complexes of the respiratory chain and the ATP synthase, which perform the key metabolic process oxidative phosphorylation. These complexes are composed of subunits from two different genetic origins: the majority of constituents is synthesized on cytosolic ribosomes and imported into mitochondria, but a handful of proteins, which represent core catalytic subunits, are encoded in the organellar DNA and translated on mitochondrial ribosomes. Using yeast as a model organism, I investigated the mitochondrial ribosomal tunnel exit, the region of the ribosome where the nascent chain emerges and that in cytosolic ribosomes serves as a platform to bind biogenesis factors that help the newly synthesized protein to mature. This study provided insights into the structural composition of this important site of mitochondrial ribosomes and revealed the positioning of Cbp3 at the tunnel exit region, a chaperone required specifically for the assembly of the bc1 complex. In my further work I found that Cbp3 structurally and functionally forms a tight complex with Cbp6 and that this complex exhibits fundamental roles in the biogenesis of cytochrome b, the mitochondrially encoded subunit of the bc1 complex. Bound to the ribosome, Cbp3-Cbp6 stimulates translation of the cytochrome b mRNA (COB mRNA). Cbp3-Cbp6 then binds the fully synthesized cytochrome b, thereby stabilizing and guiding it further through bc1 complex assembly. The next steps involve the recruitment of the assembly factor Cbp4 to the Cbp3-Cbp6/cytochrome b complex and presumably acquisition of two redox active heme b cofactors. During further assembly Cbp3-Cbp6 is released from cytochrome b, can again bind to the ribosome and activate further rounds of COB mRNA translation. The dual role of Cbp3-Cbp6 in both translation and assembly allows the complex to act as a regulatory switch to modulate the level of cytochrome b synthesis in response to the bc1 complex assembly process. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>