Browsing by Author "Woody, Robert, committee member"

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Open Access
Biophysical, structural, and functional studies of histone binding proteins
(Colorado State University. Libraries, 2010) Sudhoff, Keely B., author; Luger, Karolin, advisor; Chen, Chaoping, committee member; Henry, Charles, committee member; Woody, Robert, committee member; Hansen, Jeffrey C., committee member
Eukaryotic genomes are extensively compacted with an equal amount of histone proteins to form chromatin. A high level of control over chromatin structure is required to regulate critical cellular processes such as DNA replication, repair, and transcription. To achieve this feat, cells have developed a variety of means to locally or globally modulate chromatin structure. This can involve covalent modification of histones, the incorporation of histone variants, remodeling by ATP-dependent remodeling enzymes, histone chaperone-mediated assembly/disassembly, or any combination of the above activities. To understand how chromatin structure is affected by histones, it is essential to characterize the interactions between histones and their associated proteins. In Saccharomyces cerevisiae, the multi-subunit SWR1 complex mediates histone variant H2A.Z incorporation. Swc2 (Swr1 complex 2) is a key member of the SWR1 complex and is essential for binding and transfer of H2A.Z. Chz1 (Chaperone for H2A.Z/H2B) can deliver H2A.Z/H2B heterodimers to the SWR1 complex in vitro. Swc2 1-179 (a domain of Swc2 that retains histone binding and the apparent preference for variant dimers) and Chz1 are intrinsically disordered, but become more ordered upon interaction with histones. Quantitative measurements done under physiological in vitro conditions demonstrate that Chz1 and Swc2 1-179 are not histone variant-specific. They bind to histones with an affinity lower than that of previously described histone chaperones, and lack the ability to act on nucleosomes or other histone-DNA complexes. Small-angle X-ray scattering demonstrates that the intrinsic disorder of the proteins allows them to adopt a multitude of structural states, perhaps facilitating many different interactions and functions. We show that Swc2 1-179, despite its overall acidic charge, can bind double stranded DNA, in particular, 3-way and 4-way junction DNA. These junctions are thought to mimic the central intermediates found in DNA damage repair. This characteristic is unique to Swc2 1-179. Consistent with this unexpected activity, yeast phenotypic assays have revealed a role for SWC2 in DNA damage repair, as indicated by sensitivity to DNA damaging agent methane methylsulfonate. Importantly, our data has exposed a novel role for Swc2 in DNA damage repair. In an independent study, we investigated the histone chaperone Vps75, a Nap1 homolog. Rtt109 is a histone acetyltransferase that requires a histone chaperone for the acetylation of histone H3 at lysine 56 (H3K56). Rtt109 forms a complex with the chaperone Vps75 in vivo and is implicated in DNA replication and repair. We show that deletion of VPS75 results in dramatic and diverse mutant phenotypes, in contrast to the lack of effects observed for the deletion of NAP1. The flexible C-terminal domain of Vps75 is important for the in vivo functions of Vps75 and modulates Rtt109 activity in vitro. Our data highlight the functional specificity of Vps75 in Rtt109 activation.
Open Access
Contribution of ²³⁸U and ²³²TH to radiation dose and risk from fly ash effluent of coal-fired power plants
(Colorado State University. Libraries, 2010) Beckfield, Felicity Cunningham, author; Johnson, Thomas, advisor; Woody, Robert, committee member; Volckens, John, committee member
The goal of this project was to determine the activity concentrations of 238U and 232Th emitted from a coal-fired power plant that could potentially impact human health and the environment. The activity concentration of 238U and 232Th in fly ash was used to estimate effluent uranium and thorium. The estimate of effluent activity was then used to model radiation dose and evaluate any associated increase in cancer risk to employees working in the plant and individuals living near the plant. Grab samples of fly ash were obtained and manually fractionated using the soil sizing techniques of sieving and pipetting. The respective samples were counted using alpha spectroscopy to determine the activity concentrations of 238U and 232Th. Whole body dose was calculated using 10 CFR 20 Appendix B annual limits on intake (ALI). The alpha emissions from 238U and 232Th are of particular interest as they are significant contributors to dose in the lungs and other tissues due to their high relative biologic effectiveness and short range. The results of this study indicate that fly ash contains both 238U and 232Th but is not a radioactive substance as defined by the IAEA transportation safety standards and Title 49 of the Code of Federal Regulations. Although the relative concentration of radionuclides in the fly ash of this study is quite low, it is still possible for individuals to receive a measurable dose. Exceeding occupational and public dose limits would require inhalation of approximately 1-1000 kg of fly ash for 232U and approximately 50 g to 20 kg for 232Th. The highest CEDE (ICRP 30) per unit mass incurred by inhalation of fly ash was class W 232Th (1.81 mrem g-1), while class W 238U had the lowest CEDE per unit mass (3.32 prem g-1). The general relationship between activity concentration of 238U and 232Th found using data from radiochemical analysis and particle size suggest that activity concentration increases with increasing particle size. However the relationship between activity concentration and particle size found in the literature suggests that activity concentration increases with decreasing particle size. The accompanying health risk from 238U and 232Th in fly ash is predicted to be less than 10-5 percent.
Open Access
Dose profile surrounding a waste repository
(Colorado State University. Libraries, 2014) Parson, Jenelle, author; Brandl, Alexander, advisor; Johnson, Thomas, committee member; Woody, Robert, committee member
The waste repository analyzed is an interim storage facility that houses low and intermediate level conditioned radioactive waste. In total, it contains 9996 200-L waste barrels. The barrels are arranged in a crate geometry to ensure easy access to each barrel. The repository walls are 0.2 m thick with extra shielding (0.7 m) present on the west, north, and east sides of the repository. Instead of extra shielding the south side of the repository has a 5.25 m gap from the crates to the wall, allowing for crane maneuverability. The dose rate profile was analyzed using Monte Carlo N-Particle Transport eXtended (MCNPX) for the south, west, and north sides. The east side was not explicitly analyzed, because of the symmetry between the west and east sides. The dose rate was analyzed using f5 detector tallies and fluence rate to dose rate conversion factors from ICRP 21. Here, contributions due to skyshine and other wall effects are analyzed in detail. For the west and north side (where shielding was present), it was found that as distance from the source increases the dose rate initially increases logarithmically to a maximum and subsequently falls off following an exponential function. The initial increase in dose rate is significant with a peak dose rate as much as 300% of the dose rate at the wall and remaining elevated until approximately 60 m from the waste repository. A similar dose rate increase is not observed for the southern side of the repository; instead, the dose rate falls off with a power function corresponding to a function between that for an infinite plane and a point source. The dose rate profile was analyzed with and without repository structures, and the initial increase is only present with the repository structure. This indicates that the initial rise is due to the presence of walls and/or roof. The shape of the dose rate profile closely follows observed skyshine functions at accelerator facilities. Skyshine and wall effects have been analyzed extensively for medical accelerator facilities, but are generally not considered for a waste repository; the work suggests that skyshine and wall effects may be more significant than previously thought and should be considered in the design and construction of waste repositories.
Open Access
Functional characterization of nucleosome assembly proteins
(Colorado State University. Libraries, 2021) Krzizike, Daniel, author; Luger, Karolin, advisor; Kennan, Alan, committee member; Nyborg, Jennifer, committee member; Stargell, Laurie, committee member; Woody, Robert, committee member
The amount of DNA found within the human body will span from the earth to the sun ~50 times. With the DNA providing the genetic blueprint of all living things, it needs to be packaged in a way that allows accessibility. The first step in this packaging involves nucleosomes, large macromolecular complexes made up of histone proteins and DNA. Nucleosomes must remain dynamic as they are constantly assembled and disassembled for processes such as DNA replication, repair, and transcription. Both assembly and disassembly occur in a specific stepwise manner orchestrated by multiple proteins employed by the cell. Specifically, histone chaperones have been implicated in almost every aspect of nucleosome dynamics such as shuttling histones into the nucleus, histone storage, and both nucleosome assembly and disassembly in an ATP-independent manner. While the structures of many histone chaperones have been determined, the mechanism of how they regulate nucleosome dynamics is still largely unknown. I investigated the mechanism of the nucleosome assembly protein family (Nap family) through several biochemical approaches. The Nap family of proteins are implicated in histone homeostasis through interactions with core histones, histone variants, and linker histones. They are conserved among all eukaryotes from yeast to humans. Members of the Nap family contain a conserved core region flanked by highly disordered N- and C-terminal tails varying in length and charge between species. Using yNap1, we investigated how these tails impact the overall function in regards to histone binding, histone selectivity among core histones and histone variants, and in mediating histone-DNA interactions. We found that the tails are critical for overall function, with the charge of the tails being crucial in regulation. We also investigated Vps75, another member of the Nap family. Similar to Nap1, Vps75 binds core histones, but also stimulates the acetylation activity of Rtt109, a histone acetyltransferase. In light of a recent debate regarding the stoichiometry with which these Nap members bind their histone cargo, we characterized the Vps75-histone interaction using core histones H2A-H2B and H3-H4. Comparing Vps75 with yeast Nap1, we found that the mechanism of histone binding is not conserved among these Nap family members. Further expanding on Vps75, we investigated the interaction with Rtt109 in both the presence and absence of H3-H4. We discovered dimeric Vps75 is capable of binding either one histone tetramer or two units of Rtt109 with the ternary complex consisting of only one unit of Rtt109 and one H3-H4 tetramer. While characterizing Nap family members I became very familiar with Analytical Ultracentrifugation (AUC). AUC is a powerful in-solution technique that provides first-principle hydrodynamic information to determine size, shape, and molecular interactions, making it ideal for the characterization of proteins, DNA, and the interactions among them. As our lab traditionally used AUC to obtain van Holde-Weischet plots, an excellent graphical representation of homogeneity or heterogeneity, we incorporated new analysis techniques for improved accuracy in molecular mass and gross shape determination. Using the added-on fluorescence detection system, we obtained a level of sensitivity and selectivity that was otherwise not possible. Using the powerful method of analytical ultracentrifugation combined with fluorescent studies, we provide insight into the regulation mechanism of Nap family members along with establishing a framework to study other macromolecular complexes.
Open Access
I. Ground-state association between phenothiazine and tris(diimine)ruthenium(II) complexes: its role in highly efficient photoinduced charge separation. II. Ligand modifications of cobalt complexes to increase efficiency of electron-transfer mediators in dye-sensitized solar cells
(Colorado State University. Libraries, 2012) Weber, John, author; Elliott, C. Michael, advisor; Rappe, Anthony, committee member; Levinger, Nancy, committee member; Woody, Robert, committee member; Van Orden, Alan, committee member
Supramolecular triad assemblies consisting of a central trisbipyridineruthenium(II) chromophore (C2+), with one or more appended phenothiazine electron donors (D) and a diquat-type electron acceptor (A2+) have been shown to form long-lived photoinduced charge separated states (CSS) with unusually high quantum efficiency. Up to now, there has been no explanation for why such large efficiencies (often close to unity) are achieved from these systems when other, seemingly similar, systems are often much less efficient. In the present study, using a bimolecular system consisting of chromophore-acceptor diad (C2+-A2+) and an N-methylphenothiazine donor we demonstrate that a ground-state association exists between the RuL32+ and the phenothiazine prior to photoexcitation. It is this association process that is responsible for the efficient CSS formation in the bimolecular system and, by inference, also must be an essential factor in the fully intramolecular process occurring with the D-C2+-A2+ triad analogs. Alkyl-substituted bipyridine ligands in cobalt II/III complexes were modified in order to serve as efficient electron-transfer mediators in dye-sensitized solar cells. Attempts at halogen substitution reactions are described. Ultimately isopropyl groups appended to bipyridine ligands were modified by introducing a hydroxyl group at the benzylic position. The electrochemical behavior of the modified ligand is described, as well as its performance as part of a cobalt complex electron-transfer mediator in dye-sensitized solar cells.
Open Access
Poly (ADP-ribose) polymerase 1 (PARP1) and its DNA-binding characteristics
(Colorado State University. Libraries, 2011) Kramer, Michael A., author; Luger, Karolin, advisor; Woody, Robert, committee member; Bailey, Susan M., committee member
The poly(ADP-ribose) polymerase (PARP) family is evolutionarily diverse, containing 18 different protein members. Roles played by PARP1 in the cell appear to be significant in establishing cellular complexity, as a correlation exists between higher eukaryotes and prevalence of PARP family members. Each member of the PARP family contains a conserved catalytic domain, which upon activation cleaves molecules of NAD+ to form polymers of ADP-ribose, with the release of nicotinamide. Poly(ADP-ribosyl)ation reactions carried out by PARP family members have been found to function in regulation of cellular systems including DNA-damage repair, transcription, mitotic spindle formation, telomere maintenance and cell-death signaling. The most well established member of the PARP family is poly(ADP-ribose) polymerase 1 or PARP1. PARP1 has been found to associate with an assortment of DNA structures within the cell. Despite being able to complex with any DNA present in the cell, PARP1 displays a propensity to interact with sites of DNA-damage. As such, PARP1 has been found to play a major role in initiation of DNA-damage repair. Through its catalytic activity PARP1 recruits additional DNA-damage repair machinery and promotes exposure of the site of damage through chromatin relaxation. Due to its ability to regulate chromatin structure, PARP1 has also been frequently connected with transcription regulation. Variable regulation of transcription by PARP1 has been observed. Catalytically inactive PARP1 can function in a similar fashion as the protein H1 to condense chromatin. Alternatively, active PARP1 functions to relax chromatin surrounding promoter regions and recruit transcription machinery. PARP1 activity appears to be primarily regulated through its association with DNA. Little is known regarding PARP1-DNA-binding affinity. Here I present a high-throughput in-solution FRET-based assay that I utilize to better characterize PARP1's interaction with sites of DNA-damage. In addition, the PARP1-nucleosome complex was analyzed utilizing the same FRET-based assay. Discrepancies found between PARP1 binding affinities to various DNA-damage and mononucleosome constructs provide insight into a potential variable mode of interaction exhibited by PARP1.
Open Access
Relationships between hydrogen bonds and halogen bonds in biomolecular engineering
(Colorado State University. Libraries, 2019) Hartje, Rhianon Kay Rowe, author; Ho, P. Shing, advisor; Reynolds, Melissa, committee member; Snow, Christopher, committee member; Stasevich, Tim, committee member; Woody, Robert, committee member
In this dissertation, we will explore the interconnectedness between halogen bonds (X-bonds) and hydrogen bonds in rational biomolecular engineering efforts. As X-bonds are not readily designed into biomolecules, we aim to show how they can be advantageous for molecular design. We will begin by considering how X-bonds compare to H-bonds and show how the two can work in harmony to provide enhanced stabilizing potential. In two unique protein engineering efforts we will show 1) how the X-bond can be just as specifying in terms of molecular assembly as compared to the H-bond, and 2) how it can coordinate with the H-bond to increase protein stability. One study shows the specifying potential the X-bond possesses in terms of coiled-coil assembly. While the study points to a direct application of a sensing probe, the scope of the work will aid others using coiled-coils for materials purpose, designing protein interfaces or potential ligand binding sites. In the other protein engineering study, we will survey how a protein with an intrinsically disordered region responds to hydrogen enhanced halogen bond engineering. We show how we can drastically increase the thermal stability of the protein through minimal change to its primary sequence. This study lends itself to exploring bigger structure-function questions and how the stabilizing capacity of halogen bonds fits into this. Through this work we aspire to show how useful X-bonds can be for biological engineering efforts by exhibiting their specifying and stabilizing characteristics in these settings.
Open Access
Skin tissue optical and thermal reactions to pulse sequences of thulium yttrium aluminum garnet laser irradiation
(Colorado State University. Libraries, 2010) Schaaf, David Nicholas, Jr., author; Johnson, Thomas E., advisor; Brazile, William J., committee member; Harmon, Joseph F., committee member; Woody, Robert, committee member; Zimbrick, John D., committee member
The increasing interest in new lasers operating in the mid-infrared region has produced a need for better understanding of tissue reactions for safety purposes. One of the lasers of interest for skin irradiation is the Tm:YAG which produces 2.0 micron wavelength light. However, there are many vital pieces of knowledge missing to calculate thermal effects of two micron light interactions with human skin. This work aims to fill several of these deficiencies. The first unresolved issue is the fundamental optical absorption coefficient of skin layers for 2.0 pm because current published values span two orders of magnitude. A new method for measuring optical absorption rates which avoids most of the factors introducing uncertainties in current approaches is therefore presented. The second issue unaddressed in literature is the effect of delivering the laser energy in multiple pulses which nullifies the normal assumption of thermal confinement. The third issue that published literature leaves confused is the values of thermal constants used in heat modeling. The wide ranging values of these constants is shown to allow models to drastically differ from measured temperatures. The final issue to be resolved for the first time is the effective depth of measurement of thermal imaging non-contact temperature measurement instruments.
Open Access
Spn1, a highly conserved and essential node of RNA polymerase II dependent functions
(Colorado State University. Libraries, 2011) Almeida, Adam Raymond, author; Stargell, Laurie A., advisor; Luger, Karolin, committee member; Woody, Robert, committee member; Suchman, Erica, committee member
A multitude of proteins are responsible for regulating the activity of RNA Polymerase II (Pol II) in the nucleus of a eukaryotic cell. Two types of themes are used by these proteins to control transcription: recruitment-regulation and postrecruitment-regulation. The main difference between the two is the rate-limiting step for producing transcript. This rate-limiting step for the first mechanism is the recruitment of Pol II to the promoter. For the second mechanism, Pol II constitutively occupies the promoter, is "poised", and an unknown rate-limiting postrecruitment step prevents transcription from commencing. The highly conserved and essential transcription factor Spn1 was identified as a protein that functions postrecruitment of Pol II and has been characterized for having a direct role at regulating the poised CYC1 gene in Saccharyomyces cerevisiae. This activity has been determined from mutations made within the most conserved portion of Spn1 made up of a highly folded central domain. Little is known about the functions of the N-and C-terminal regions flanking this central domain, which is the focus of the work done here. Genetic characterization indicates that these regions have physiologically relevant and important functions within the cell outside of optimum growth conditions, but do not involve significant regulation of the CYC1 gene. A broader approach of experimentation is likely required to understand all of the Spn1 protein's functions regarding transcription. This led to the observation that Spn1 is able to bind to nucleosomes in vitro and that this interaction is dependent on the N-and C-terminal regions of the protein. The possibility that Spn1 could affect nucleosome dynamics in the cell is consistent with the physical and genetic interactions observed between Spn1 and the Spt6 and Swi/Snf histone chaperone and chromatin remodeling complexes. This result will provide several new avenues for future Spn1 research. A genomic ChIP-chip experiment performed by two independent groups revealed that Spn1 is recruited to a majority of the genes in the yeast genome. Evidence indicates that there are multiple, evolutionarily conserved pathways within the cell that are responsible for determining the rate at which an organism will age that include: ribosome biogenesis, protein translation, mitochondrial activity and function, heterochromatic stability, maintenance of the genome, and apoptosis. The possibility that Spn1 regulates the genes involved in these pathways is highly suggestive that this protein could be an aging factor within the cell. Chronological aging assays revealed that the removal of the N-and C-terminal regions of the Spn1 protein dramatically increase the lifespan of the BY4741 strain of yeast. These results further verify the physiological importance of this protein and the need for further Spn1 research.
Open Access
Temperature increase measurements of multiple 10 ms pulses of 2.01 μm laser light incident on ex-vivo rabbit corneas
(Colorado State University. Libraries, 2011) Kelly, Edward, author; Johnson, Thomas, advisor; Brandl, Alexander, committee member; Woody, Robert, committee member
Current laser safety standards for multiple-pulse lasers are based primarily on modeling and the results of single-pulse studies. Previous thermal effects studies have focused on histological and visible endpoints, with only a few studies examining the actual temperatures achieved. The goal of this research was to probe the actual temperature profile produced by 2.01-micron laser pulses in the cornea. In this study the corneal temperature rise from multiple 2.01-micron Tm:YAG laser pulses was investigated using ex-vivo rabbit eyes. An infrared thermal camera employing microbolometer detectors captured surface temperature rises resulting from laser pulses. Thermal measurements were taken with single 10-ms pulses as well as two-, three-, and four-pulse sequences while holding the total energy delivered constant for the two- through four-pulse train measurements. An average temperature increase of 8.3 degrees C was observed with the single pulse at 2.8 J/cm2/pulse irradiance. For two pulses, an average temperature increase of 10.0 degrees C was observed for 2.7 J/cm2/pulse irradiance. For three pulses, an average temperature increase of 5.8 degrees C was observed for 1.9 J/cm2/pulse irradiance. For four pulses, an average temperature increase of 4.4 degrees C was observed for 1.5 J/cm2/pulse irradiance. A comparison of the data to temperatures required for denaturing proteins and the current laser safety guidelines is presented. It appears that the MPE may be overly conservative by a factor of at least 15. It is recommended that the 2.01-micron laser MPE be investigated to determine if a revision of the standard is warranted.
Open Access
The biophysical, biochemical and structural characterization of Poly(ADP-ribose) Polymerase-1 (PARP-1) and its complexes with DNA-damage models and chromatin substrates
(Colorado State University. Libraries, 2013) Clark, Nicholas James, author; Luger, Karolin, advisor; Bailey, Susan, committee member; DeLuca, Jennifer, committee member; Hansen, Jeffrey C., committee member; Woody, Robert, committee member
Eukaryotic DNA is highly dynamic and must be compacted and organized with the help of cellular machines, proteins, into 'heterochromatin' state. At its basic level, chromatin is comprised of spool-like structures of protein complexes termed histones, which bind and organize DNA into larger fibrous structures. Cellular processes like transcription and DNA-damage repair require that chromatin be at least partially stripped of its protein components, which in turn allows for complete accessibility by DNA-repair or transcription machinery. A number of protein factors contribute to chromatin structure regulation. Poly(ADP-ribose) Polymerase-1 (PARP-1) is one of these proteins that exists in all eukaryotic organisms except for yeast. In its inactive form, it compacts chromatin, but performs its chromatin-opening function by covalently modifying itself and other nuclear proteins with long polymers of ADP-ribose in response to DNA damage. Thus, it also serves as a first responder to many types of DNA damage. The highly anionic polymers serve to disrupt protein-DNA interactions and thus allow for the creation of a temporary euchromatin structure. Contained herein are investigations aimed at addressing key questions regarding key differences between the interactions of PARP-1 and chromatin and its DNA-damage substrates. Our experiments show that human PARP-1 interacts with and is enzymatically activated to a similar level by a variety of different DNA substrates. In terms of chromatin, it appears that PARP-1 fails to interact with nucleosomes that do not have linker DNA. PARP-1 most effectively interacts with chromatin by simultaneously binding two DNA strands through contacts made by its two N-terminal Zn-finger domains. Small-Angle X-ray (SAXS) and Neutron Scattering (SANS) and molecular dynamics (MD) experiments were combined with biophysical and biochemical studies to better describe the structural effects of DNA binding on PARP-1. The average solution structure of PARP-1 indicates that the enzyme is a monomeric, non-spherical, elongated molecule with a radius of gyration (Rg) of ~55Å. The DNA-bound form of PARP-1 is also monomeric and binding DNA causes the molecule to become more elongated with an average Rg of ~80Å.
Open Access
Yeast prion physiology
(Colorado State University. Libraries, 2016) Nelson, Aaron C. Gonzalez, author; Ross, Eric, advisor; Woody, Robert, committee member; Peersen, Olve, committee member; Zabel, Mark, committee member
Prions, or proteinaceous infections, are caused by proteins that have the unique ability to adopt an alternative, self-replicating structure. These self-replicating structures are the causative agent of a number of mammalian diseases including Bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, and Kuru. More recently, yeast were discovered to carry at least a dozen proteins capable of making this structural conversion. Yeast prions are unique in that their prion-forming domains are intrinsically disordered domains, with unusual compositional biases. This thesis addresses two broad questions about yeast prion physiology. First, a recent mutagenic screen suggested that both aromatic and non-aromatic hydrophobic residues strongly promote prion formation. However, while aromatic residues are common in yeast prion domains, non-aromatic hydrophobics are strongly under-represented. The second chapter of this dissertation explores the effects of hydrophobic and aromatic residues on prion formation. Insertion of even a small number of hydrophobic residues is found to strongly increase prion formation. These data, combined with bioinformatics analysis of glutamine/asparagine-rich domains, suggest a limit on the number of strongly prion-promoting residues tolerated in glutamine/asparagine-rich domains. Recent studies have demonstrated that aromatic residues play a key role in the maintenance of yeast prions during cell division. Taken together, these results imply that non-aromatic hydrophobic residues are excluded from prion domains not because they inhibit prion formation, but instead because they too strongly promote aggregation, without promoting prion propagation. Despite more than 20 years of research, we still don’t know why yeast carry so many prion and prion-like domains. It has been proposed that prions may serve some biological function. Chapter Three presents progress on two lines of investigation designed to resolve this issue First, a novel bioinformatics algorithm (GARRF) is used to screen a wide range of proteomes to find examples of Q/N rich domains outside of Saccharomyces cerevisiae. Identifying other species that carry these unusual regions provides insight into their role in cellular biology. We find a wide range species carry prion-like domains at levels comparable to Saccharomyces cerevisiae, and a small number carry up to an order of magnitude more. Second, currently researchers rely primarily on yeast genetic methods to discover and monitor prions. These methods have a number of drawbacks, including a glacially slow readout time. Chapter Three reports on progress towards the development of a novel fluorescence based prion assay. This assay takes advantage of bi-molecular fluorescence complementation, a technique that uses complementary fragments of a fluorescent protein to indicate when two interacting domains are in proximity to one another. When completed, this assay will provide a means to monitor protein aggregations that is both faster and more sensitive than any existing assay.