Browsing by Author "Kirienko, Natasha"

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    Uncovering Genes Responsible for Mitochondrial Maintenance and Surveillance
    (2023-07-27) Moreno, Armando; Kirienko, Natasha
    Mitochondria are key organelles for cellular health and metabolism and the activation of programmed cell death processes. Although pathways for regulating and re-establishing mitochondrial homeostasis have been identified over the past twenty years, the consequences of disrupting genes that regulate other cellular processes, such as division and proliferation on affecting mitochondrial function remain unclear. Using a cancer cell line mutation database, I developed a set of 139 genes in Caenorhabditis elegans predicted to play roles in mitochondrial maintenance or function. Disruption of a sample of genes from this set caused phenotypes consistent with mitochondrial dysfunction, including increased fragmentation of the mitochondrial network, abnormal steady-state levels of NADH, or ROS, or altered oxygen consumption. RNAi-mediated knockdown of these genes often also exacerbated α-synuclein aggregation in a C. elegans model of Parkinson’s disease. This gene set provides a foundation for identifying new mechanisms that support mitochondrial and cellular homeostasis and can be potentially used in targeted therapeutics of diseases such as cancer and neurodegenerative disease. In addition to the genes mentioned above, mitochondrial surveillance mechanisms are also essential for healthy mitochondrial function. These mechanisms are in place to ameliorate the deleterious effects of faulty protein import, increase in oxidative stress, and bioenergetic disruption. While some regulators for the ESRE (Ethanol and Stress Response Element) mitochondrial surveillance network have been identified in the past, a transcription factor specific to ESRE regulation has not been identified. Through a high-throughput screen, I discovered F23B12.7, a bZIP transcription factor, as an ESRE regulator. F23B12.7 is necessary for proper ESRE activation in C. elegans and is necessary for survival during Pseudomonas aeruginosa infection. This transcription factor is also necessary for protection against mitochondrial damaging agents such 1,10-Phenanthroline. F23B12.7 also shows regulation of both box C/D and box H/ACA snoRNP complexes (known for their role in ribosome biogenesis). This discovery opens a new avenue of research of the interplay between ribosomal biogenesis and mitochondrial health. Further understanding of these mitochondrial surveillance networks will also provide insight into how these mechanisms react to stress or infection and can be potential targets of treatment for mitochondrial related diseases.
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    Using C. elegans to Identify Novel Targets Against Multidrug-Resistant Bacteria
    (2020-07-06) Hummell, Nicholas Andrew; Kirienko, Natasha; Tao, Jane
    Antibiotic-resistant infections cause an average of 23,000 deaths per year. Due to dwindling commercial interest for antimicrobial discovery, novel methods for combating infection and developing new antimicrobials are desperately needed. Previously in our lab, we performed a high-throughput chemical screen to identify small molecules that rescued the nematode Caenorhabditis elegans from infection by P. aeruginosa. Of the hits identified, 5 (LK32, LK34, LK35, LK38 and LK56) were determined to be stimulators of host defense pathways while 5 additional hits (DMAQ-B1, CD437, carboplatin, oxaliplatin, and PSB-069) possessed a known target or bioactivity but had no previously reported antimicrobial activity against P. aeruginosa.. Using microarray analysis, RNAi knockdown of candidate pathways, transgene reporters, and infection assays with other pathogens, we made important observations concerning the mechanism of action and therapeutic repurposing potential for the compounds. Firstly, I identified a subunit of the Mediator complex, mdt-15, and the PMK-1/p38 MAPK pathway as necessary for rescue for LK56 and LK38 respectively, demonstrating that both pathways are amenable to immune stimulation. I have also found that some molecules can defend against E. faecalis and S. aureus as well as Pseudomonas while being largely non-toxic. Additionally, most molecules stimulated the activation of multiple innate immune pathways. These experiments showed the potential for development of broad-spectrum immune stimulants and identified promising pathways amenable to immune stimulation. They also highlighted potential for our LK molecules as tools for future studies of innate immune stimulation in C. elegans. For our bioactive compounds, I used similar C. elegans-based methods to generate a number of important conclusions: I confirmed the antimicrobial activity of CD437 against Gram-positive pathogens, observed a weakness of P. aeruginosa to platinum complexes, and established the naturally occurring insulin mimetic, DMAQ-B1, as a powerful antimicrobial agent. Although toxic, an existing non-toxic analog presents potential for further therapeutic optimization. Through these studies, I have utilized C. elegans as a powerful drug discovery tool to gain insight into mechanism and therapeutic utility of two groups of anti-infective molecules. I have shown the strength of our model in drug repurposing efforts as well as demonstrated therapeutic potential for immune stimulation as a promising approach to combatting the growing antimicrobial resistance crisis.