Lakin-Thomas, PatriciaMudiyanselage, Lalanthi Kumari Ratnayake Ratnayake2020-05-112020-05-112019-092020-05-11https://hdl.handle.net/10315/37417Circadian rhythms are found in all domains of life and control many physiological processes. Transcription/translation feedback loops (TTFLs) are important molecular mechanisms in circadian systems. In the model eukaryote Neurospora crassa, TTFL includes FRQ (frequency) and WCC (white-collar complex). Rhythms have been observed in the absence of a functional FRQ/WCC TTFL. These are called FRQ-less rhythms which are assumed to be driven by one or more FRQ-less oscillators (FLOs). Through UV mutagenesis former lab members have found a mutation (uv90) that compromises FRQ-less rhythms and also affects rhythmicity when FRQ is functional, demonstrating the close integration between the TTFL and FLO(s). My PhD dissertation research is focused on the uv90 mutation. The gene it affects has now been identified as gene number NCU05950. The uv90 mutation was characterized as a deletion of 35,128 nucleotides in chromosome VI. NCU05950 was identified as a homologue of the conserved nutrient-sensing TOR pathway proteins EGO1 in yeast and LAMTOR1 in mammals. We have named the N. crassa protein as VTA (Vacuolar-TOR-Associated protein). GFP-tagging of VTA showed it is anchored to the outer vacuolar membrane. Deletion of putative acylation sites destroys this localization as well as the proteins function in rhythmicity, demonstrating that vacuolar localization of VTA is required for clock function. The NCU05950 deletion mutant is defective in its growth response to different amino acids and different concentrations of glucose, confirming its function in nutritional sensing. A FLAG fusion of NCU05950 exhibited nearly constant levels of protein across two circadian cycles. Northern blot results using a probe for the wild type VTA showed arrhythmic transcript levels. IP-MS analysis showed interaction of VTA with other TOR pathway proteins. Deletion mutants of other N. crassa TOR pathway associated genes are defective in their response to different nutritional conditions and showed altered conidiation rhythms. My findings establish a connection between the TOR pathway and circadian rhythms. I conclude that VTA is a key protein in the complex that anchors TOR to the vacuole. It plays a role in maintaining circadian rhythmicity by integrating networks of metabolism and TTFLs to construct a complete circadian system.Author owns copyright, except where explicitly noted. Please contact the author directly with licensing requests.Cellular biologyElectronic Thesis or Dissertation2020-05-11Neurospora crassaCircadian rhythmsTOR signallingLAMTOREGOFRQ-less rhythmsTarget of RapamycinNutrient SensingFilamentous fungi