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Browsing by Author "Suchman, Erica, committee member"

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    Elucidating mother to offspring transmission of chronic wasting disease using a transgenic mouse model
    (Colorado State University. Libraries, 2016) Willingham, Kassandra, author; Mathiason, Candace, advisor; Zabel, Mark, committee member; Suchman, Erica, committee member; Winger, Quinton, committee member
    Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE), or prion disease, of free-ranging and farmed cervids (deer, elk and moose). CWD is the only TSE in a wildlife population, which was initially discovered in a captive mule deer herd in a study shared between Colorado State University and University of Wyoming in 1967. CWD is the most readily transmitted of all the prion diseases and since its discovery has been identified in cervid populations in 24 states, 2 Canadian provinces, and the Republic of Korea. Horizontal transmission of prion diseases is thought to account for its exceptional transmission efficiency [2-10]. Recent studies published by our group provide evidence that transmission from mother to offspring may also be a contributing factor. In the work of this thesis, we employed a transgenic mouse system that expresses the cervid prion protein Tg(CerPrP-E226) to help elucidate the role of mother to offspring CWD transmission via hemochorial placentation. Females were inoculated with known CWD-positive material and subsequently bred with CWD-naïve males at various timepoints post inoculation to investigate if maternal/vertical transmission occurs in this host, as well as to further understand how this might occur. We examined the likelihood of prion trafficking in utero by analysis of mother: offspring pairs at different timepoints in CWD-infection and gestation, in addition to looking for infectious prions in milk collected from CWD-positive dams. We have demonstrated that CWD-infected Tg(CerPrP-E226) females successfully breed and bear offspring irrespective to TSE disease stage. Offspring born to CWD- infected females did not exhibit signs of TSE disease and lacked detectible PrPres via conventional methodologies. Interestingly, conversion competent prions were identified in the brains and spleens of offspring by highly sensitive amyloid seeding assays. The lack of symptoms in these offspring indicates covert prion transmission from mother to offspring, resulting in a potential silent-carrier status. As for our studies to further the understanding of the mechanisms behind this transmission, we identified CWD-prions in reproductive and mammary tissue, and spleen of Tg(CerPrP-E226) mouse mothers as early as 72 days post inoculation. In addition, we found minute quantities of amyloid conversion material in placenta and fetal tissues from mother:offspring pairs at varying timepoints in CWD-infection. We were unable to detect prions in milk collected from CWD-positive transgenic dams, leading us to hypothesize that the route of TSE transmission to offspring is likely a combination of environmental exposure, and/or very low concentrations of prions breaching the feto-maternal interface.
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    Investigation of mechanisms of mitotic recombination in yeast
    (Colorado State University. Libraries, 2016) Harcy, Lisa Victoria, author; Argueso, Lucas, advisor; Di Pietro, Santiago, committee member; Liber, Howard, committee member; Suchman, Erica, committee member
    At the submicroscopic level within all living cells the workings of a dynamic molecular world attempt to preserve the integrity of DNA - the blueprint of life. This dissertation describes in detail the experimental systems and results from two of our studies conducted in which DNA lesions compromised genomic integrity. The unifying theme of the following chapters revolves around the mechanisms responsible for structural genomic variation, that is, what happens to chromosomes when they break. In the first phase of research, we examined outcomes associated with double-strand breaks (DSBs) at G-quadruplex DNA sequences. Here, we show that G4 DNA motifs, capable of initiating double-strand breaks, result in the formation of chromosomal aberrations. Our results provide structural context and support to a putative mechanism of homology-directed repair revealed by molecular analysis of these complex rearrangements. In the second project, we investigated chromosomal translocations that developed from spontaneously occurring DSBs in diploid yeast to ascertain the means by which the DSBs had been repaired. The objective of this study was to examine the intricate interplay between the non-allelic DSB repair (DSBR) processes: canonical reciprocal homologous recombination (CRHR) vs. break-induced replication (BIR). While numerous assays have previously measured repair pathway efficiency by isolating one of the two pathways from the other, no prior studies to our knowledge, have prospectively examined the contribution of BIR to overall double-strand break repair in diploids, using a non-inducible experimental system where either mechanism can be used freely by the cells. I designed and constructed a new assay system to study chromosomal translocations in the yeast eukaryotic model to investigate the balance between these two DSB repair processes. I characterized genotypic and phenotypic alterations in wild type background and in mutant yeast strains defective for BIR. The data obtained from this work provides an additional perspective to the field of DNA repair biology with broad relevance to DSBR regulation in eukaryotes. It provides further understanding about the role of DNA repair to undesired genetic outcomes, thus, leading the way to the design of new and more effective treatments for diseases in which these molecular actions are the instigators of pathogenesis.
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    Oral and nasal mucosal pathways of prion infection in chronic wasting disease
    (Colorado State University. Libraries, 2010) Denkers, Nathaniel David, author; Hoover, Edward Arthur, 1942-, advisor; Zabel, Mark Douglas, committee member; Suchman, Erica, committee member; Ross, Eric D., committee member
    Chronic wasting disease (CWD) is a fatal neurodegenerative prion disease of deer, elk, and moose. The unique feature of CWD as a prion disease is its efficient transmission among cervids in nature. As with other prion infections, CWD disease inception relies on the conversion of the normal host cellular prion protein (PrPC) to the abnormal, protease-resistant isoform (PrPCWD)--the diagnostic hallmark of prion diseases. Since its detection in Colorado in 1967, CWD has spread to captive and free-ranging cervid species in 16 additional states, 3 Canadian provinces, and one Asian country. CWD is also exceptional as the only prion disease to afflict a free-ranging, wildlife population. Understanding the facile means by which CWD is transmitted from animal to animal is important not only in understanding prion transmission overall but also in elucidating the potential public health consequences of cross species prion transmission. This dissertation work asks whether and how CWD prions are able to cross the oral and nasal mucosa to induce infection and disease. The above questions were addressed through the use of two strains of transgenic mice that express the normal cervid prion protein [Tg(CerPrP)1536, Tg(CerPrP-E226)5037+/-] and prion protein knockout mice [FVB PrP0/0] exposed by either aerosol, nasal, or oral route to CWD prions. In the first series of studies, cohorts of Tg(CerPrP)1536 mice were exposed to brain homogenates from either CWD-infected or CWD-naïve deer via either aerosolization or direct nasal installation. In the second series of studies, cohorts of Tg(CerPrP-E226)5037+/- mice were exposed to the same inocula via installation onto the lingual mucosal surface which had or had not been previously subjected to superficial abrasions. Mice were then observed and tissues from each cohort, at time points ranging from weeks to as long as 2 years post inoculation, were examined for the presence of the abnormal prion protein of CWD (PrPCWD) using western blotting and immunohistochemistry assays. The final studies employed the same inoculation techniques used in the first two studies to seek to identify early (less than 4 hours) sites of prion entry via the mucous membranes. The results of these dissertation studies demonstrated; 1) that CWD could be transmitted by aerosol exposure with high efficiency compared with direct inoculation onto the nasal mucosa; and 2) that micro-abrasions to the lingual surface greatly facilitated CWD prion transmission. Finally and perhaps surprisingly, we were unable to detect PrPCWD in either the nasal or oral mucosa shortly after inoculation or at any time, even after the onset of clinical symptoms of CWD. The results from these studies suggest that; 1) CWD prions can be transmitted by aerosol exposure; thus exposure to the respiratory system merits increased consideration in prion transmission and biosafety; 2) minor oral mucosal injury does greatly facilitate prion infection--a potentially significant co-factor in CWD transmission of foraging cervids; and 3) these mucosal pathways may explain how and why CWD is transmitted with high efficiency in animals exposed to low concentrations of prions in nature.
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    Role of basic and hydrophobic residues in the poliovirus polymerase elongation complex and the structure of a coxsackievirus polymerase elongation complex
    (Colorado State University. Libraries, 2011) Kortus, Matt, author; Peersen, Olve, advisor; Ho, P. Shing, committee member; Suchman, Erica, committee member
    Picornaviruses encode for and require a viral RNA-dependent RNA polymerase (RdRP) for genome replication. This enzyme synthesizes negative-sense RNA from the infecting positive sense genome producing a replicative intermediate. The negative sense RNA then serves as a template for synthesis of additional positive-sense RNA. To efficiently replicate the genome, RdRPs must form a stable and processive elongation complex (EC) by binding RNA, incorporating the first templating nucleotide, and undergoing a necessary conformational. Upon completion of these steps that comprise initiation, the newly formed EC is capable of rapidly replicating the viral genome. The work presented in this thesis 1) investigates the role that several basic and hydrophobic residues serve in forming and maintaining the poliovirus (PV) EC and 2) presents the crystal structure of a coxsackievirus (CV) EC. To determine the role of that several arginines, lysines, and tyrosines play in the PV polymerase, we assessed whether mutations to these residues affect initiation, elongation, or stability of the EC. The data indicates the basic residues within the fingers domain of the PV polymerase have a major role in binding RNA. In addition, data shows two tyrosine residues in particular are critical for formation and maintenance of the EC. Overall, the data provides evidence the fingers domain interacts with the template RNA in a manner not captured by crystal structures. Finally, we have solved the structure of a CVEC stalled after incorporation of four nucleotides. The CVEC structure closely matches the previously solved PVEC structure. In addition, one crystal form produced an elongation complex trapped in a translocation intermediate state.
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    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.