Theses and Dissertations at Montana State University (MSU)
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Item Unveiling the photophysics in solid-state organic materials: a study on BODIPY, porphyrin, and PBI based materials(Montana State University - Bozeman, College of Letters & Science, 2024) King, Alexander James; Chairperson, Graduate Committee: Erik Grumstrup; This is a manuscript style paper that includes co-authored chapters.Organic semiconductors have applications in optoelectronics, light harvesting, and sensing as soft matter materials. One of the biggest challenges to overcome with organic-based materials is structural heterogeneity that arises from the self-assembly of monomers upon solid-state deposition. In this work we have investigated solid-state organic semiconductors with three levels of solution-phase processing: i) materials prepared from drop casting with no solution-phase processing on BODIPY systems ii) films prepared from pre-aggregation of the monomers with porphyrin systems iii) films prepared from aggregated monomers that were covalently stapled with perylene bisimide systems. In the BODIPY systems, we found that: i) the electronic states are highly coupled with a major redshift from 583 nm in the solution to 614 nm in the solid. ii) Through interpretation of the broadband transient absorption spectrum, the initial excited state is delocalized and localizes within the first 10 femtoseconds. iii) Using two color pump probe, we measured ultrafast diffusion at 14.37 + or - 2.79 cm 2 s -1 that abruptly halts after 10 ps. In the porphyrin systems with level 2 solution-phase processing, we have also shown that the lifetime of the excited state is correlated with the degree of structural order. The monomer exhibits the longest lifetime with an average lifetime of 1.26 ns, the aggregate is much shorter with a lifetime of 349 ps, and the films show substantially faster relaxation, with the film fabricated from the monomer having a 72.56 ps average lifetime, and the film composed of the aggregate having a 26 ps average lifetime. These results suggest that the lifetime decreases as the order and electronic coupling of the system increases, so much so that the lifetime is two orders of magnitude different. In the perylene bisimide systems, we did a direct spectroscopic comparison between thin films formed from noncovalent assemblies and from covalently tethered molecular assemblies. This indicates that interchromophore coupling is enhanced in the covalently tethered film. We saw a 73% increase in excited state transport compared to the control film, as well as a shorter and more homogenous excited state lifetime. Covalent tethering proves to be the best strategy for generating homogeneous materials.Item Versatility of cryo-electron microscopy as a structural technique informs iron mineral nucleation and growth in a mini-ferritin(Montana State University - Bozeman, College of Letters & Science, 2024) Gauvin, Colin Charles; Chairperson, Graduate Committee: C. Martin LawrenceIron is an enigmatic element. While necessary for life, it also contributes to the generation of reactive oxygen species via the Fenton reaction. To mitigate this, cellular life has evolved the ferritin family of proteins, including the 24 subunit ferritins and bacterioferritins, and the 12 subunit DPSL "mini-ferritins". Each of these catalyze the controlled oxidation and sequestration of iron as a hydrous ferric oxyhydroxide within their hollow protein cores. While there is a wealth of structural information on the unmineralized ferritins, little is known about the structures of the biomineralized forms, and the mechanism of ferric oxyhydroxide nucleation and growth. Here we report structural and biochemical characterization of a DPS-Like protein from Pyrococcus furiosus. This "thioferritin" utilizes a bacterioferritin-like ferroxidase center, but adopts the mini-ferritin quaternary structure, and is thus thought to sit at the evolutionary boundary between mini- and maxi-ferritins. In addition to the unmineralized structure, we report the 1.91 angstrom structure of P. furiosus thioferritin as it nucleates iron-oxyhydroxide distal to the ferroxidase site. In this very low iron form, a pair of conserved glutamate residues and unsaturated carbonyls at the 3-fold axis serve to template initial nucleation. We also determine structures of higher iron forms with a biomineralized ferrihydrite core, where C-terminal residues 170-176 interact directly with the initial mineral surface, which then grows towards the particle center. These studies provide important new insight into biological mechanisms for the controlled nucleation, growth and storage of ferric oxyhydroxide in this thioferritin specifically, and the ferritin superfamily as a whole.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 Illuminating dynamic phenomena within organic microstructures with time resolved broadband microscopies(Montana State University - Bozeman, College of Letters & Science, 2024) Hollinbeck, Skyler Robert; Chairperson, Graduate Committee: Erik Grumstrup; This is a manuscript style paper that includes co-authored chapters.Materials derived from organic chromophore subunits are currently at the forefront of academic and industrial interest. This strong interest is driven in part by the tunability of their extant properties through engineering of both the intra-molecular and inter-molecular structure. The structure of organic materials affects optoelectronic properties because organic chromophores are sensitive to dipole-dipole and charge-transfer coupling interactions. This sensitivity presents both opportunities for tuning functional properties through designing specific packing geometries, and liabilities arising from the disruptive effects of structural disorder. Many organic materials are built from weak noncovalent interactions between chromophores, leading solid-state deposition, and crystallization to be susceptible to microscopic variations in environmental conditions. Structural heterogeneity is regularly intrinsic to organic materials, and even self-assembled systems of covalently linked chromophores exhibit defects. Ergo, in order to disentangle the effects of structural heterogeneity from the inherent properties of the material, the study of organic materials must be anchored with techniques that are capable of correlating optoelectronic properties and excited state evolution with microscale morphological characteristics. We have employed broadband pump-probe microscopies, in conjunction with steady-state and time resolved fluorescence techniques, to examine the effects of structure and coupling on excited state dynamics in solid-state organic microstructures. The study of perylene diimide (PDI) materials revealed that kinetically trapping PDI (KT-PDI) enhanced long-range ordering and led to distinct excited state evolution, delocalized charge-transfer states and long-lived charge separated species. In the MOF PCN-222, excitation energy dependent excited state behavior was observed. Pumping the first excited state (Q-band) led to immobile excited states that were relatively unaffected by local defect densities, whereas pumping the second excited state (Soret-band) led to mobile subdiffusive excited state species whose lifetimes are significantly impacted by morphologically correlated defect sites. Finally, we present progress made toward the construction and utilization of a frequency modulated-femtosecond stimulated Raman microscope, yielding spectra that resolve the location of photoinduced anion formation in KT-PDI. The work presented herein highlights broadband time-resolved microscopy as a potent tool for studying the structure-function relationship and photophysical behavior in molecular solids, deepening our understanding of how structural characteristics influence excited state evolution.Item Synthetic and mechanistic strategies to achieve unconventional site-selectivity in cross-couplings of dihalo-heteroarenes(Montana State University - Bozeman, College of Letters & Science, 2024) Norman, Jacob Patrick; Chairperson, Graduate Committee: Sharon Neufeldt; This is a manuscript style paper that includes co-authored chapters.Pd-catalyzed cross-couplings rank among the most powerful methods for constructing substituted biaryls, polyaryls, and heteroarenes. Frequently, di- or polyhalogenated (hetero)arenes are employed as starting materials in cross-couplings to access products with increased structural complexity via multiple cross-coupling or substitution steps. N-heteroarenes bearing multiple reactive handles--such as halides, are of particular interest as starting materials since their cross- coupled products can be medicinally relevant. Non-symmetrical dihalogenated N-heteroarenes typically exhibit a site-selectivity bias for C-X bonds which are adjacent to at least one heteroatom in Pd-catalyzed cross-couplings. However, some Pd catalysts--particularly those with hindered ligands, promote atypical selectivity at distal C-X bonds of 2,X-dichloropyridines and related heterocycles during the selectivity-determining oxidative addition step. This dissertation explores the mechanistic origins of these ligand trends and emphasizes the critical importance of Pd's ligation state--either mono (PdL) or bis (PdL 2), in controlling the site of oxidative addition. Ligation state is also relevant when selecting for the products of mono- vs difunctionalization in cross-couplings of dihalogenated substrates, since bisligated 14 e - Pd dissociates quickly from the monofunctionalized intermediate after an initial cross-coupling cycle, whereas monoligated 12 e - Pd is slow to dissociate and may "ring-walk" to the remaining reactive site(s). Additionally, this dissertation explores alternative methods to access minor regioisomers in cross-couplings of dichloro-azines. One approach involves ligand-free conditions where atypical site-selectivity at dichloropyridines and dichloropyrimidines arises from a change in Pd's speciation from mono- to multinuclearity. Another approach employs a thiolation/Liebeskind-Srogl arylation sequence to achieve site-selectivity which is orthogonal to that of Suzuki-Miyaura couplings.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 Characterization of osteoarthritis metabolism: a mass spectrometry based-approach(Montana State University - Bozeman, College of Letters & Science, 2024) Welhaven, Hope Diane Aloha; Co-chairs, Graduate Committee: Brian Bothner and Ronald K. June II; This is a manuscript style paper that includes co-authored chapters.Osteoarthritis (OA) effects 7% of the global population, equating to more than 500 million people worldwide, and is the leading cause of disability. Its multifaceted etiology includes risk factors ranging from genetics, to aging, obesity, sex, race, and joint injury. OA manifests differently across the patient population where symptom severity, rate of progression, response to treatment, pain perception, as well as others vary person to person posing significant challenges for effective management and prevention. At the cellular level, imbalanced matrix catabolism and anabolism contribute to the breakdown of cartilage, underlying bone, and other tissues affected by OA. Leveraging mass spectrometry-based techniques, particularly metabolomics, offers a promising avenue to dissect OA metabolism across musculoskeletal tissues, while considering individual patient-specific risk factors. Therefore, the goals of this research were to: (1) comprehensively characterize OA phenotypes and endotypes and (2) explore OA pathogenesis within the context of disease-associated risk factors. The first area of research focuses on profiling OA phenotypes and endotypes across disease development. These results provide clear evidence of OA-induced metabolic perturbations in OA cartilage and bone and elucidate mechanisms that shift as disease progresses. Several metabolites and pathways associated with lipid, amino acid, matrix, and vitamin metabolism were differentially regulated between healthy and OA tissues and within OA endotypes. The second area of research focuses on the impact of OA risk factors -- sex, injury, obesity, loading -- on the metabolism of circulatory fluids (i.e., serum, synovial fluid) and chondrocytes. Identification of metabolic indicators of disease, such as cervonyl carnitine, and metabolic pathways associated with these risk factors holds potential for improving screening, monitoring disease progression, and guiding preventative strategies. Overall, this work contributes to our current understanding of OA, its diverse metabolic landscape, risk factors and their interactions. Moreover, it lays the groundwork for personalized medicine by providing detailed insights into individualized phenotypic profiles, thereby advancing the prospect of tailored treatment strategies for OA individuals.Item The synthesis and characterization of fluorescently labeled, lactose-functionalized poly(amidoamine) (PAMAM) dendrimers(Montana State University - Bozeman, College of Letters & Science, 2024) Frometa, Magalee Rose; Chairperson, Graduate Committee: Mary J. CloningerCellular uptake of lactose-functionalized poly(amidoamine) dendrimers (PAMAM) has yet to be fully understood and deeply studied. Before sufficient cellular uptake studies can be made, optimization of the synthesis of the lactoside, and the coupling and purification of dye-tagged lactose-functionalized PAMAM had to be completed, as reported here. The synthesis of the requisite lactoside derivative for dendrimer functionalization was optimized. The coupling of the dye, Alexa Fluor 647, to the lactoside-functionalized PAMAM was performed in the presence of a sodium acetate buffer and utilized size separation methods to ensure purity. The structures of the lactoside derivatives and of lactose functionalized PAMAMs were confirmed via nuclear magnetic resonance (NMR) spectroscopy. The purity and degree of labeling (DOL) of the dye labeled, lactose-functionalized PAMAMs were determined with UV-vis. Results show high success of yield and purity resulting from the optimized procedure described in this study.Item Structural characterization of the Csa3/cA4 complex - a nexus for class 1 CRISPR-Cas immune response coordination & establishing a cure for highly efficient galectin expression(Montana State University - Bozeman, College of Letters & Science, 2024) Charbonneau, Alexander Anthony; Chairperson, Graduate Committee: C. Martin Lawrence; This is a manuscript style paper that includes co-authored chapters.Though Class I CRISPR-Cas systems, primarily Type I and Type III, are the most abundant CRISPR systems in archaea and bacteria, mechanisms driving their immune response regulation are not well understood. Csa3 family transcription factors, composed of N-terminal CARF and C-terminal winged helix-turn-helix domains, are frequently encoded within Type I CRISPR-Cas systems. Csa3 transcription factors are hypothesized to bind cyclic oligoadenylate (cOA) second messengers produced by Type III interference complexes, likely modulating their DNA-binding activity. Therefore, we investigated the interaction between Csa3a and cyclic tetra-adenylate (cA4). Isothermal titration microcalorimetry showed S. solfataricus Csa3a binds cA4 at biologically relevant concentrations in an entropically driven interaction. Ring nuclease assays revealed Csa3a lacks self-regulatory phosphodiesterase activity exhibited by other CARF domain proteins. We crystallized and solved the structure of the Csa3/cA4 complex, which revealed conserved motifs are responsible for cA4 binding and illuminated significant conformational changes induced by the interaction. We also identified an 18-bp palindromic motif, which we designated CAPPa, that is conserved in the 27 sequenced members of the order Sulfolobales, and shows synteny with Csa3a and acquisition genes in these genomes. We found Csa3a binds CAPPa in a nonspecific, cooperative, and cA4-independent manner. These characteristics suggest a more complex method of transcriptional regulation than previously hypothesized. However, the interaction between Csa3a and cA4 confirmed here signifies a nexus between Type I and Type III systems; we thus propose a model in which this interaction coordinates the two arms of an integrated immune system to mount a synergistic, highly orchestrated, adaptive immune response. We applied the workflow designed to produce significant protein quantities for crystallographic studies of Csa3a to the study of Homo sapiens galectin proteins, a family of beta-galactoside-binding proteins. Here, we identified a putative autoinhibitory mechanism affecting traditional IPTG-induction methods by characterizing IPTG-binding capabilities of galectins and quantifying basal protein expression over various IPTG concentrations. To bypass this predicted feedback loop, we employed a highly efficient and approachable autoinduction method, resulting in a 7-fold increase in protein expression. Much of this work was done in the context of a course-based undergraduate research experience with great success.Item Elucidating the impacts of structural heterogeneity on excited state dynamics in solution-processed materials(Montana State University - Bozeman, College of Letters & Science, 2024) Afrin, Sajia; Chairperson, Graduate Committee: Erik Grumstrup; This is a manuscript style paper that includes co-authored chapters.Solution-processed inorganic and organic semiconductors hold enormous promises due to their low manufacturing cost, scalability, and compatibility with flexible substrates. However, solution processing techniques do not require control over crystal growth, which can lead to structural defects within the crystal structure. The defects within solution-processed semiconductors can create significant challenges in optimizing device functionality; therefore, it is crucial to understand the impact of structural defects on photophysical properties. Traditional ensemble measurement techniques can conceal the effects of microscale structural defects on functional properties in the structure-averaged observation of solution-processed materials. The work presented in this dissertation employs time-resolved and spectrally resolved microscopy techniques to investigate the influence of structural heterogeneity on the photophysical properties of microscale solution-processed materials. Measurements collected across multiple discrete and highly crystalline domains of multiple classes of solution-processed materials have helped establish a relationship between the functionality and the local structure of these materials. Initially, the focus was on elucidating anisotropic carrier transport in lead halide perovskites by investigating lattice strain and energetic distribution in microcrystals. Later, the focus shifted towards characterizing and understanding the impact of structural defects on the excited state dynamics in another class of solution-processed material called metal-organic frameworks (MOFs). PCN-222 exhibited rapid exciton transport with time-averaged diffusion coefficients ranging from 0.27 to 1.0 cm2/s and subdiffusive behavior, showing transport slowing on the tens of ps time scale. Subdiffusivity indicated that excited states were rapidly transported through the porphyrin network of PCN-222 before being trapped. Moreover, the first transport measurements and transient absorption microscopic measurements in PCN-222 are reported here. Photoluminescence quenching and heterogeneous relaxation pathways were noted in regions with higher structural heterogeneity. Furthermore, the spectral evolution of porphyrinic PCN-222 MOF was investigated, which revealed excitation-dependent chromophore coupling in the MOF structure. Soret band excitation with enhanced coupling can create more mobile excited states, whereas Q band excitation with reduced coupling will generate fewer mobile excited states. Excitation-dependent chromophore coupling strongly dictates the transport and relaxation properties in MOF microstructures that also illustrate the impact of structural defects on the excited state transport and relaxation dynamics. A significant spectral shift has also been observed in microrods stemming from structural heterogeneity. These findings contribute to a deeper understanding of the impact of structural defects on the photophysical properties of solution-processed materials, facilitating the development of optimized semiconductor devices for various applications. The results reported in this dissertation will not only continue to aid in the characterization of MOFs but will also advance our understanding of excited state dynamics in a variety of solution-processed materials.