Theses and Dissertations at Montana State University (MSU)
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Item Improving the two-photon absorption properties of fluorescent proteins for neuroscience(Montana State University - Bozeman, College of Letters & Science, 2020) Molina, Rosana Sophia; Chairperson, Graduate Committee: Thomas Hughes; Yong Qian, Jiahui Wu, Yi Shen, Robert E. Campbell, Mikhail Drobizhev and Thomas E. Hughes were co-authors of the article, 'Understanding the fluorescence change in red genetically encoded calcium ion indicators' in the journal 'Biophysical Journal' which is contained within this dissertation.; Tam M. Tran, Robert E. Campbell, Gerard G. Lambert, Anya Salih, Nathan C. Shaner, Thomas E. Hughes and Mikhail Drobizhev were co-authors of the article, 'Blue-shifted green fluorescent protein homologues are brighter than enhanced green fluorescent protein under two-photon excitation' in the journal 'The Journal of physical chemistry letters' which is contained within this dissertation.; Jonathan King, Jacob Franklin, Nathan Clack, Christopher McRaven, Vasily Goncharov, Daniel Flickinger, Karel Svoboda, Mikhail Drobizhev, Thomas E. Hughes were co-authors of the article, 'An instrument to optimize fluorescent proteins for two-photon excitation' which is contained within this dissertation.Untangling the intricacies of the brain requires innovative tools that power basic research. Fluorescent proteins, first discovered in jellyfish, provide a genetically encodable way to light up the brains of animal models such as mice and fruit flies. They have been made into biosensors that change fluorescence in response to markers of neural activity such as calcium ions (Ca 2+). To visualize them, neuroscientists take advantage of two-photon excitation microscopy, a specialized type of imaging that can reveal crisp fluorescence images deep in the brain. Fluorescent proteins behave differently under twophoton excitation compared to one-photon excitation. Their inherent two-photon properties, namely brightness and peak absorption wavelength, limit the scope of possible experiments to investigate the brain. This work aims to understand and improve these properties through three projects: characterizing a set of red fluorescent protein-based Ca 2+ indicators; finding two-photon brighter green fluorescent proteins; and developing an instrument to screen for improved fluorescent proteins for two-photon microscopy. Analyzing nine red Ca 2+ indicators shows that they can be separated into three classes based on how their properties change in a Ca 2+-dependent manner. In one of these classes, the relative changes in one-photon properties are different from the changes in two-photon properties. In addition to characterizing, identifying and directly improving fluorescent proteins for enhanced two-photon properties is important. Presented here is a physical model of the light-absorbing molecule within the green fluorescent protein (the chromophore). The model predicts that green fluorescent proteins absorbing at higher energy wavelengths will be brighter under two-photon excitation. This proves to be the case for 12 blueshifted green fluorescent proteins, which are up to 2.5 times brighter than the commonly used Enhanced Green Fluorescent Protein. A way to directly improve fluorescent proteins is through directed evolution, but screening under two-photon excitation is a challenge. An instrument, called the GIZMO, solves this challenge and can evolve fluorescent proteins expressed in E. coli colonies under two-photon excitation. These results pave the way for better two-photon fluorescent protein-based tools for neuroscience.Item Characterization and embellishment of protein cages for nanomedical and nanomaterial applications(Montana State University - Bozeman, College of Letters & Science, 2014) Servid, Amy Eloise; Chairperson, Graduate Committee: Trevor Douglas; Laura E. Richert, Ann L. Harmsen, Agnieszka Rynda-Apple, Soo Han, James A. Wiley, Trevor Douglas and Allen G. Harmsen were co-authors of the article, 'A virus-like particle vaccine platform elicits heightened and hastened local lung mucosal antibody production after a single dose' in the journal 'Vaccine' which is contained within this thesis.; Paul Jordan, Alison O'Neil, Peter Prevelige and Trevor Douglas were co-authors of the article, 'Location of the bacteriophage P22 coat protein C-terminus provides opportunities for the design of capsid-based materials' in the journal 'Biomacromolecules' which is contained within this thesis.In nature, protein cages are found within the structures of viruses, heat shock proteins, and ferritins. They assemble from subunits into spherical oligomeric structures, which serve to encapsulate, protect, and/or deliver cargo. The fundamental understanding of protein cage structure is a key component in the design of novel nanomaterials that best exploit and expand upon the natural functions of protein cage architectures. By mimicking the re-occurring design strategies employed by natural systems, the protein cages produced during virus infection and/or stress responses can be modified to yield particles that fight disease and/or serve as the building blocks for materials design. In particular, the work described here highlights the design and characterization of protein cages in an effort to develop and uncover new strategies for immunization of the lung against a variety of pathogens. A combination of genetic and chemical engineering approaches is described here in order to better understand the structure of protein cage architectures and the relationship between the structure and in vivo function. This work describes the chemical cross-linking of large antigens and immunomodulatory molecules to the surface of a protein cages, and it shows that intensified and accelerated immune responses result from the display of antigens on a protein cage surface. Genetic incorporation of point mutations within the capsid structure provided unique attachment points for chemical functionalization. In addition, genetic modifications revealed information about the location of the C-terminus of the bacteriophage P22 capsid. The knowledge that this position was displayed on the capsid exterior prompted its use to promote inter-capsid interactions and target nanoparticles to melanoma cells. This research emphasizes that both the protein cage structural design and the local in vivo environment can influence the outcomes of protein cages when administered to the lung environment. It also lays the foundation for the logical design of biomaterials that offer enhanced protection against influenza and other respiratory diseases. Finally, regions of protein cages that are amendable to chemical and genetic modifications are described herein, and these have paved the way for the continued development of protein cage platforms for nanomedical and material applications.Item Novel pharmaceutical combination confers protection from delayed cell death following transient cerebral ischemia(Montana State University - Bozeman, College of Letters & Science, 2009) Chapman, Courtney Myfanwy; Chairperson, Graduate Committee: A. Michael BabcockStroke is a leading cause of death and disability throughout the world; ischemia is the most common form of stroke. Medical procedures such as cardio-pulmonary bypass surgery can cause ischemic stroke can be caused. There are no treatments to limit neural impairment following stroke. The current research investigates neuroprotection offered by treatment with a novel drug combination consisting of Simvastatin TM, Gemfibrozil TM, Troglitazone TM, and Spironolactone TM. Animals were treated with the drug cocktail three weeks proceeding and one week subsequent to surgery. Ischemic insult was induced by clamping the carotid arteries for 5 min. Sham subjects underwent similar surgical procedures, but the carotids were not clamped. Twenty-four hrs following the surgical procedure locomotor activity was monitored in an open field for 5 min. Seven to fourteen days following ischemia or the sham procedure animals were sacrificed and sections containing the hippocampal CA1 region were mounted on slides and stained with cresyl violet. The CA1 region was rated on a 4-point scale for level of damage. Rodents generally show increased locomotor activity following transient global ischemia in an open field. In our study, ischemic animals that received vehicle demonstrated increased activity relative to the animals that received the drug treatment on all behavioral measures. Ischemic animals that received vehicle treatment had significantly more neural damage in the hippocampal CA1 region than ischemic animals receiving the drug. The appearance of neurons in the CA1 hippocampal regions of animals in the sham condition was not significantly different from ischemic animals in the drug treatment condition. It is concluded that the drug treatment is effective in offering neuroprotection during transient global ischemia. The next step is to characterize the biochemical mechanisms behind the neuroprotection conferred by the drug treatment. Contrasting the protein expression levels of animals receiving the vehicle treatment with animals receiving the drug treatment following an ischemic insult will assist in elucidating these pathways. Predictions are made regarding the biochemical mechanisms affected by the drug treatment based on previous research on the biochemical pathways affected by each pharmaceutical.