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Item Investigating the metalloproteome of bacteria and archaea(Montana State University - Bozeman, College of Letters & Science, 2024) Larson, James Daniel; Chairperson, Graduate Committee: Brian Bothner; This is a manuscript style paper that includes co-authored chapters.Metalloproteins are proteins that rely on a bound metal for activity and comprise 30-50% of all proteins which are responsible for catalyzing imperative biological functions. Understanding the interplay between essential and toxic metals in the environment and the metalloproteins from an organism (metalloproteome) is important for a fundamental understanding of biology. A challenge in studying the metalloproteome is that standard proteomic methods disrupt protein-metal interactions, therefore losing information about protein- metal bonds required for metalloprotein function. One of the focuses of my work has been to develop a non-denaturing chromatographic technique that maintains these non-covalent interactions. My approach for investigating the native metalloproteome together with leading- edge mass spectrometry methods was used to characterize microbial responses to evolutionarily relevant environmental perturbations. Arsenic is a pervasive environmental carcinogen in which microorganisms have naturally evolved detoxification mechanisms. Using Escherichia coli strains containing or lacking the arsRBC arsenic detoxification locus, my research demonstrated that exposure to arsenic causes dramatic changes to the distribution of iron, copper, and magnesium. In addition, the native arsRBC operon regulates metal distribution beyond arsenic. Two specific stress responses are described. The first relies on ArsR and leads to differential regulation of TCA-cycle metalloenzymes. The second response is triggered independently of ArsR and increases expression of molybdenum cofactor and ISC [Fe-S] cluster biosynthetic enzymes. This work provides new insights into the metalloprotein response to arsenic and the regulatory role of ArsR and challenges the current understanding of [Fe-S] cluster biosynthesis during stress. Iron is an essential and plentiful metal, yet the most abundant iron mineral on Earth, pyrite (FeS2), was thought to be unavailable to anaerobic microorganisms. It has recently been shown that methanogenic archaea can meet their iron (and sulfur) demands solely from FeS2. This dissertation shows that Methanosarcina barkeri employs different metabolic strategies when grown under FeS2 or Fe(II) and HS- as the sole source of iron and sulfur which changes the native metalloproteome, metalloprotein complex stoichiometry, and [Fe-S] cluster and cysteine biosynthesis strategies. This work advances our understanding of primordial biology and the different mechanisms of iron and sulfur acquisition dictated by environmental sources of iron and sulfur.Item Investigation of octopamine-glutamate dual transmission neurons(Montana State University - Bozeman, College of Letters & Science, 2020) McKinney, Hannah Margaret; Chairperson, Graduate Committee: Steven R. Stowers; Lewis Sherer, Jessica L. Williams, Sarah Certel and Steven R. Stowers were co-authors of the article, 'Characterization of drosophila mimic-converted octopamine receptor GAL4 lines' in the journal 'Journal of Comparative Neurology' which is contained within this dissertation.; Dissertation contains a paper of which Hannah Margaret McKinney is not the main author.Dual transmission, or the ability of a neuron to signal with more than one neurotransmitter, is now a well-established phenomenon in the field of neuroscience. However, many questions about this type of signaling process still remain with regards to its mechanisms and its impacts on neural circuitry and organism behavior. In particular, the mode of neurotransmitter release from synaptic vesicles can have significant profoundly affects elements on neural circuitry and, subsequently, on behaviors of an organism. In Drosophila melanogaster, a particular subset of neurons important for the behaviors of courtship and aggression signal with the neuromodulator octopamine and the excitatory neurotransmitter glutamate. Whether these two neurotransmitters are released simultaneously (co-release) or are housed for separate synaptic release (cotransmission) is unknown. The mechanism of release for these neurotransmitters in this population of neurons is investigated here through the development of synaptic vesicle visualization tools, synaptic vesicle isolation, and an examination of the expression of octopamine and glutamate receptors; I explored the hypothesis that receptor expression downstream of dual transmitting neurons will provide information about the co-release or co-transmission of octopamine and glutamate. Results from these experiments demonstrated release of octopamine and glutamate from the same synaptic site, with some variation, and a significant amount of presynaptic receptor expression. The results indicate these dual transmission neurons may release octopamine and glutamate at the same synapse for both post-synaptic signaling as well as pre-synaptic signal modulation.