Theses and Dissertations at Montana State University (MSU)
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Item Investigations of the gut-brain-metabolism axis in familial dysautonomia(Montana State University - Bozeman, College of Letters & Science, 2023) Cheney, Alexandra Marie; Co-chairs, Graduate Committee: Frances Lefcort; Valerie Copie; This is a manuscript style paper that includes co-authored chapters.Familial dysautonomia (FD), a neurodevelopmental and neurodegenerative disease primarily present in Eastern European Jewish populations, is a useful model system to explore the effects of neuronal dysregulation, particularly in the developing field of the gut-brain-metabolism axis. FD originates from a single genetic mutation in the ELP1 gene and differs from other neurological diseases, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and autism that are dependent on multiple factors. Metabolic and gut impairments have been observed in FD patients, but only symptom management has been pursued without further exploration into the underlying disease pathophysiology. To better understand how the gut environment changes as a result of neuronal dysregulation and how this impacts the gut-brain-metabolism axis in FD patients, several studies of both human and FD mouse model samples were undertaken. Serum and stool samples from FD patients and their relatives were analyzed for metabolic alterations using proton nuclear magnetic resonance (1H NMR)-based metabolomics. Stool samples from both a human cohort and FD mice were also analyzed for gut bacterial diversity via 16S rRNA gene sequencing. Additionally, stool metabolomes of FD mice were analyzed for metabolic alterations. The FD mouse model enabled us to explore how gut physiology changed during disease progression using gut histological methods and gut function assays. Our studies demonstrated significant changes in the metabolomes and gut microbiomes of FD patients compared to their healthy relative controls. Additionally, the FD mouse model, a pan-neuronal Elp1 conditional knock out, was sufficient to drive metabolic and gut microbiome changes, and impair gut barrier function compared to control mice. When FD mice cohabitated with healthy control mice and were able to exchange gut microbes via stool consumption, the cohoused FD mice improved in overall health and gut function. Our studies found that the gut microbiome and metabolome of cohoused FD mice were comparable to their cohoused control counterparts. Overall, this work has enhanced our understanding of the gut-brain-metabolism axis in Familial dysautonomia and has provided insights into underlying molecular mechanisms, which may be potential targets for therapeutic interventions, including the use of metabolic supplements and/or altering the gut microbiome.Item NMR hydrophilic metabolomic analysis of bacterial resistance pathways using multivalent quaternary ammonium antimicrobials in Escherichia coli and Bacillus cereus exposed to DABCO and mannose functionalized dendrimers(Montana State University - Bozeman, College of Letters & Science, 2021) Aries, Michelle Lynne; Chairperson, Graduate Committee: Mary J. Cloninger; This is a manuscript style paper that includes co-authored chapters.Novel antibiotics developed using a new scaffold are needed to combat the rising tide of antibiotic resistant bacteria. Multivalent antibiotics are a relatively new approach that have the potential to greatly increase the efficacy of antibiotics while making it difficult for bacteria to develop resistance. Dendrimers are an attractive framework for the multivalent presentation of antibacterial moieties. Quaternary ammonium compounds (QAC) are a positively charged class of membrane disruptors that are attracted to the large negative charge on phospholipid membranes. Nuclear magnetic resonance (NMR) metabolomics is a quantitative method used for comparison of metabolic profiles of wild type and mutated bacterial samples, enabling the study of bacterial response to antimicrobials. Proton (1 H) NMR hydrophilic metabolomics was used to study gram-negative and gram-positive bacteria upon exposure to 1,4-diazabicyclo-2,2,2-octane (DABCO) with a 16-carbon chain tethered onto a mannose functionalized poly(amidoamine) (PAMAM) dendrimer (denoted as DABCOMD), a membrane disrupting multivalent QAC. Stock Escherichia coli (E. coli) (denoted as wild type) and DABCOMD mutated E. coli (denoted as mutants) were collected in the mid log and stationary phases. The same procedures were used for Bacillus cereus (B. cereus) as for E. coli samples (denoted as unchallenged), except that a DABCOMD challenged sample set was added (denoted as challenged). The challenged sample set procedures were identical to the unchallenged, except DABCOMD was included at 33 % of the MIC value in the growth media for growth curve acquisition and sample collection. The greatest differences observed between the metabolic profiles of the wild type and mutated E. coli samples and between the challenged and unchallenged B. cereus samples were in energy-associated metabolites and membrane-related pathways. The mutants in all sample types were associated with higher levels of spent energy molecules (including AMP and NAD+) and peptidoglycan related compounds (including N-acetylglucosamine). Overall, more changes were observed for B. cereus (gram-positive), especially in challenged mutant B. cereus samples, than for E. coli (gram-negative) samples. Since DABCOMD is a positively charged multivalent membrane disruptor, both B. cereus and E. coli mutated to garner protection by altering their peptidoglycan layer composition, which is energetically costly.Item Quantitative 1 H NMR analyses of immunometabolic modulation in human macrophages(Montana State University - Bozeman, College of Letters & Science, 2019) Fuchs, Amanda Lee; Chairperson, Graduate Committee: Valerie Copie; Sage M. Schiller was an author and Wyatt J. Keegan, Mary Cloud B. Ammons, Brian Eilers, Brian Tripet and Valerie Copie were co-authors of the article, 'Quantitative 1 H NMR metabolomics reveals distinct metabolic adaptations in human macrophages following differential activation' in the journal 'Metabolites' which is contained within this dissertation.; Sage M. Schiller was an author and Isaac R. Miller, Mary Cloud B. Ammons, Brian Eilers, Brian Tripet and Valerie Copie were co-authors of the article, 'Pseudomonas aeruginosa planktonic- and biofilm-conditioned media elicit divergent responses in human macrophages' submitted to the journal 'PLoS pathogens' which is contained within this dissertation.Macrophages are innate immune cells that are found ubiquitously in nearly all human tissues, where they support host innate and adaptive immune responses in an effort to maintain systemic homeostasis. They are inherently plastic in nature and can dramatically modulate their functional phenotype according to pathogen and microenvironmental stimuli. Previous studies have shown that macrophages are particularly important for the resolution of inflammation in acute wound healing, which is marked by a phenotypic transition of wound macrophages from pro-inflammatory to anti-inflammatory. Chronic, or non-healing, wounds, such as diabetic, pressure, and venous leg ulcers, feature a prolonged host inflammatory response due in part to aberrant wound macrophage behavior. Non-healing in chronic wounds has also been shown to be dependent upon the establishment of pathogenic biofilms, which are more resistant to host defense mechanisms than planktonic, or free-floating, bacteria. Therefore, investigating macrophage dysregulation in the presence of bacterial biofilms has gained considerable interest. Here, 1D 1 H NMR-based metabolomics was utilized to identify metabolic pathways that are differentially modulated following primary human monocyte-derived macrophage activation with pro-inflammatory or anti-inflammatory stimuli relative to resting macrophages. Metabolic profiling of inflammatory macrophages indicated a substantial increase in oxidative stress as well as a decrease in mitochondrial respiration. These metabolic profiles also provided evidence that inflammatory macrophages divert metabolites from de novo glycerophospholipid synthesis to inhibit oxidative phosphorylation. In addition, we investigated which metabolic pathways are differentially modulated following primary human monocyte-derived macrophage exposure to Pseudomonas aeruginosa planktonic- and biofilm-conditioned media. Metabolic profiling of PCM- and BCM-exposed macrophages indicated a significant depletion of intracellular glucose without elevation of downstream glycolytic products. These metabolic patterns suggest that PCM- and BCM-exposed macrophages potentially divert glycolytic intermediates towards inositol phosphate metabolism. Overall, our studies provide additional support to previous findings, generate novel results regarding metabolic modulation of human macrophages following activation and exposure to planktonic- vs. biofilm-conditioned media, and contribute new insight to the field of immunometabolism.Item Structural investigation of IsdB, the hemoglobin receptor of Staphylococcus aureus(Montana State University - Bozeman, College of Letters & Science, 2014) Fonner, Brittany Anne; Chairperson, Graduate Committee: Valerie CopieStaphylococcus aureus is an opportunistic pathogen which when left unchallenged can cause severe toxicity and death in mammals. Critical to S. aureus growth is the ability to scavenge iron from hemoglobin (Hb). To acquire iron S. aureus has evolved a sophisticated protein-mediated heme acquisition pathway, which comprises nine iron-regulated surface determinant (Isd) proteins involved in heme capture, transport and degradation. A key protein of the acquisition pathway is the hemoglobin receptor protein IsdB, which comprises two NEAr transporter (NEAT) domains that act in concert to bind Hb and extract heme for subsequent transfer to downstream acquisition pathway proteins. Despite significant advances in the structural knowledge of other Isd proteins, the mechanisms and molecular basis of the IsdB-mediated heme acquisition process is not well understood. In order to provide more insights into the mode of function of IsdB, structural studies via nuclear magnetic resonance (NMR) spectroscopy were employed on different domains of IsdB. The three-dimensional solution structure of IsdBN1 revealed an immunoglobulin-like fold that is consistent with other NEAT domain proteins. Site directed mutagenesis studies revealed two key aromatic residues, F164 and Y167, involved in methemoglobin (metHb) interactions with IsdB. The protein variant F164D did not bind to metHb under NMR conditions. In heme transfer studies between metHb and IsdB constructs containing the two NEAT domains and the linker region, the amino acid substitution of F164D diminished but did not knock out the ability of IsdB to remove heme from metHb. A double amino acid substation of F164D and Y167D did abolish heme transfer from metHb to IsdB, therefore identifying key residues of IsdBN1 interaction with metHb. Studies of the linker region revealed an overall alpha-helical propensity and an interaction between the linker region and the second NEAT domain, IsdBN2. Solving the apo-IsdBN2 structure revealed slight differences in the heme-binding pocket, specifically in beta-strands 7 and 8 that interact with the heme moiety, when compared to the published crystal structure of holo-IsdBN2. The findings in this thesis provide a structural role for IsdBN1 enhancing the rate of extraction of heme from metHb by IsdBN2 and interactions between the domains of IsdB.Item Solid state and solution NMR studies of model systems for parts of membrane proteins(Montana State University - Bozeman, College of Letters & Science, 1994) Adams, Earle RaymondItem Synthesis, characterization, and mechanistic studies on a group of platinum complexes using 195PT nuclear magnetic resonance(Montana State University - Bozeman, College of Letters & Science, 1991) Bedgood, Danny RichardItem Structure determination of a retinal rod G protein peptide segment bound to rhodopsin by nuclear magnetic resonance spectroscopy(Montana State University - Bozeman, College of Letters & Science, 1994) Furstenau, Julie EgeItem Steric effects on o-(N,N-disubstitutedamino)-thiophenols [pt. I] Synthesis of 1,1-dimethyl-2,3-dimethylenecyclopropane from delta-3-carene [pt. II](Montana State University - Bozeman, College of Letters & Science, 1972) Miller, Steven WayneItem NMR investigation of non-local effects in a temperature sensitive mutant of the 25 kD tryptophan repressor protein(Montana State University - Bozeman, College of Letters & Science, 2003) Tyler, Robert Charles; Chairperson, Graduate Committee: Valerie CopieItem Structural investigations of the cancer-associated laminin binding protein and Nos L : a novel copper binding protein(Montana State University - Bozeman, College of Letters & Science, 2005) Taubner, Lara Marie; Chairperson, Graduate Committee: Valerie CopieThis thesis consists of two distinct projects, one on the metastasis-associated laminin binding protein and the other on the putative copper chaperone NosL, both related by the common aim of investigation of the relationship between protein structure and function using nuclear magnetic resonance techniques. In the first part of this dissertation, the role that the metastasis-associated laminin binding protein or LBP plays in the spread and development of cancer was investigated. Functional domains of LBP were delineated by limited proteolysis, overexpressed, and then assayed for their ability to bind to the previously identified in vivo binding partner laminin. These assays demonstrated that, at least under the conditions used in this assay, binding to laminin was localized to domain 137-230, a region that encompasses a previously identified binding site known as Peptide G. This protein, like the full-length recombinant laminin binding protein, aggregated under conditions used for nuclear magnetic resonance experiments and therefore could not be analyzed with this technique. Contrary to previous findings on a synthetic peptide corresponding to residues 205-229, this sequence within the context of the 200-295 construct demonstrated no laminin binding activity. Furthermore, the peptide lacked the predicted alpha-helical content and tertiary structure as ascertained by nuclear magnetic resonance and by circular dichroism spectroscopy. A potential role for the disorder exhibited by this region of LBP is proposed, and suggests possible new functions for the laminin binding protein in angiogenesis. NosL, the subject of the second part of this thesis, is a highly conserved copper(I) binding lipoprotein encoded by the nitrous oxide reductase (nos) gene cluster of denitrifying bacteria. To identify functional features and structural homologues of this protein, the structure of apo NosL Was solved using nuclear magnetic resonance techniques. The high-resolution structure of NosL consists of one four-strand antiparallel beta sheet, one three strand antiparallel beta sheet and two alpha-helices organized in a twisted butterfly-like fold that is structurally homologous to MerB, an alkyl mercury lyase. Chemical perturbation mapping performed on the copper(I)=protein defined regions of NosL potentially involved in copper binding, and thus allowed preliminary identification of the copper-binding ligand Met 109.