Master of Science (MS), Wright State University, 2021, Biological Sciences

The presynaptic protein alpha-synuclein encoded by SNCA is the primary component of Lewy bodies and Lewy neurites which are the histopathological hallmarks of Parkinson's disease. While the etiology of Parkinson's remains unclear, SNCA mutations and copy number variations are one of several genes linked to Parkinson's. The family of highly conserved chaperone proteins 14-3-3 have been shown to co-aggregate and share regions of 40% homology with alpha-synuclein. Mitochondrial dysfunction also plays a role in Parkinson's disease with abnormalities in mitochondrial respiration due to interference of complex I in the electron transport chain being found in Parkinson's patients. Alpha-synuclein increases fragmentation of mitochondria and accumulation of reactive oxygen species due to mitochondrial dysfunction which causes toxic oligomers of alpha-synuclein to form. In yeast, the molecular mechanisms of neurodegenerative disorders can be recapitulated because of the high conservation of cellular processes between eukaryotes and yeast. These mechanisms include the cytotoxic effects of human alpha-synuclein and its aggregate formation, as well as recapitulating the toxic phenotypes observed in human and transgenic animal models, allowing for genetic screenings for modifiers of alpha-synuclein. The drawback of prior yeast screenings is the restriction of the galactose induced GAL1 promoter. Our laboratory previously found a mutant gal3 allele that induces the GAL1 promoter for utilization under non-galactose conditions. I assessed the growth phenotype of an IntTox yeast strain with genome integrated 4-5 copies of the SNCA gene under the control of the GAL1 promoter by inducing SNCA expression under respiratory growth conditions using glycerol and ethanol as the carbon source. I demonstrated that the integrated IntTox SNCA strain recapitulated alpha-synuclein cytotoxicity under galactose and glycerol ethanol conditions allowing for comparative analysis of mitochondria depende (open full item for complete abstract)

Committee: Quan Zhong Ph.D. (Advisor); Lynn Hartzler Ph.D. (Committee Member); Paula Ann Bubulya Ph.D. (Committee Member) Subjects: Biology; Microbiology

Master of Science (MS), Wright State University, 2018, Biological Sciences

Optineurin, an autophagy adaptor protein genetically linked to Normal Tension Glaucoma and Amyotrophic Lateral Sclerosis, has been found in the pathological inclusions of various other neurodegenerative disorders, supporting an important role of optineurin in neurodegeneration. Using yeast as a model, we found that overexpression of optineurin drastically enhances the toxicity of the Parkinson's disease-causing protein, alpha-synuclein. Considering the conserved protein-protein interaction between optineurin and Ypt1, a yeast suppressor of the toxicity of both optineurin and alpha-synuclein, we hypothesize that cellular targets of optineurin underlie in the cytotoxicity and the enhancer effect. Using genome-wide yeast two-hybrid screens, we identified 97 yeast interacting proteins of optineurin and systematically tested their modifier effect on the toxicity of optineurin and alpha-synuclein. Given the neuroprotective effect of the mammalian homologs of Ypt1, the convergent pathways of the identified modifiers may represent conserved cellular perturbations induced by optineurin overexpression in higher eukaryotes.

Committee: Quan Zhong Ph.D. (Advisor); Mill Miller Ph.D. (Committee Member); Paula Bubulya Ph.D. (Committee Member) Subjects: Cellular Biology; Genetics; Neurosciences

Master of Science (MS), Wright State University, 2016, Biological Sciences

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative motor neuron disease that causes progressive paralysis and death by asphyxiation. There is no cure or effective treatment; however, previous research has identified several genes that appear related to the pathology of ALS. When mutated, these genes result in proteins that gain toxic functions and disrupt normal cellular processes. Fused in Sarcoma (hFUS) is a human transcription factor in the nucleus that binds to DNA and RNA. Mutations in hFUS are associated with both familial and sporadic cases of ALS, frontotemporal lobar degeneration (FTLD), and cancer. In ALS and FTLD, hFUS is mislocalized to the cytosol where it interacts with stress granules and forms aggregates. This aggregation and cytotoxicity has been previously studied in budding yeast; however, study in fission yeast may provide unique information. Fission yeast has several genetic advantages over budding yeast for modeling mammalian cell biology, such as 43% of genes contain introns and they posess a similar alternative splicing mechanism. Mammalian and fission yeast cells also both contain microRNA as well as similar cell growth cycles. In this project, I established a fission yeast model of hFUS and showed that hFUS is toxic when overexpressed in fission yeast. Both localization to the nucleus and mislocalization to the cytosol occurred during overexpression of hFUS. In addition, fission yeast homologues to previously identified budding yeast toxicity suppression proteins were able to suppress hFUS toxicity, suggesting the suppression mechanism is conserved.

Committee: Shulin Ju Ph.D. (Advisor); Labib Rouhana Ph.D. (Committee Member); Barbara Hull Ph.D. (Committee Member) Subjects: Cellular Biology