Mountain Scholar :: Browsing by Author "Zabel, Mark, committee member"

Browsing by Author "Zabel, Mark, committee member"

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Open Access
A cellular prion protein-dependent signaling pathway for proinflammatory cytokine- and β-amyloid-induced cofilin-actin rod formation
(Colorado State University. Libraries, 2014) Walsh, Keifer P., author; Bamburg, James, advisor; Zabel, Mark, committee member; Tjalkens, Ron, committee member
Stimulus of oxidative stress in neurodegeneration leads to synaptic dysfunction and the eventual loss of neurons in the central nervous system. The actin cytoskeleton of neurons under acute or chronic stress experiences dynamic remodeling due to functional alterations in the actin depolymerizing factor (ADF)/cofilin family of actin-binding proteins. Once oxidized, disulfide cross-linked cofilin incorporates into the formation of tandem arrays of 1:1 cofilin:actin rod-like bundles (rods). Rods sequester cofilin, which is required for synaptic remodeling associated with learning and memory, and interrupt vesicular transport by occluding the neurite within which they form. Different rod-inducing stimuli target distinct neuronal populations within the hippocampus. Rods form rapidly (5-30 min) in >80% of cultured hippocampal neurons which are treated with excitotoxic levels of glutamate or energy depleted (hypoxia/ischemia or mitochondrial inhibitors). In contrast, slow rod formation (50% maximum response in ~6 h) occurs in ~20% of neurons upon exposure to soluble beta-amyoid dimer/trimer (Aβd/t), a physiologically relevant species in Alzheimer disease (AD). Here we show that proinflammatory cytokines (TNFα, IL-1β, IL-6) induce rods at the same rate and in the same subpopulation of hippocampal neurons that respond to Aβd/t. Rod formation by proinflammatory cytokines may link the neuroinflammatory hypothesis for AD with the Aβ hypothesis by providing a common target. Neurons from PrPC-null mice form rods in response to glutamate or antimycin A, but not in response to Aβd/t or proinflammatory cytokines. Prion-dependent rod inducers require the activation of NADPH oxidase (NOX) to generate reactive oxygen species (ROS), but NOX activity is not required for rods induced by glutamate or energy depletion. Aβd/t and TNFα stimulate cofilin dephosphorylation and increased ROS production in a subpopulation of neurites at levels that exceed a minimum threshold to maintain stable rods. Removing inducers or inhibiting NOX activity in cells containing prion-dependent rods causes rod disappearance with a half-life of ~36 minutes. Interestingly, the overexpression of PrPC alone is sufficient to induce rods in >40% of hippocampal neurons, nearly twice the number that respond to Aβd/t or TNFα. This suggests that membrane microdomains containing PrPC recruit the oxidizing machinery necessary to initiate and sustain rod formation. Our hypothesis is supported by the inhibition and reversal of prion-dependent rods by the naturally occurring plant triterpene, ursolic acid (UA), and the pharmacological peptide RAP310. UA and related compounds to RAP310 have been proposed to inhibit changes in the membrane lipid profile that permit LR coalescence. The vast majority of neurodegenerative disorders are considered sporadic in incidence and multifactorial in cause, making treatment at an early stage a significant challenge. If cofilin-actin rods indeed bridge multiple disease initiating mechanisms into a common pathway leading to synapse loss, they provide a valuable target for therapeutic intervention.
Open Access
An exploration of viral RNA-mediated strategies to stall and repress the cellular exoribonuclease XRN1
(Colorado State University. Libraries, 2018) Charley, Phillida A., author; Wilusz, Jeffrey, advisor; Zabel, Mark, committee member; Perera, Rushika, committee member; Reddy, Anireddy, committee member
The regulation of mRNA decay plays a vital role in determining both the level and quality control of cellular gene expression in eukaryotes. Since they are likely recognized as foreign/unwanted transcripts, viral RNAs must also successfully navigate around the cellular host RNA decay machinery to establish a productive infection. This bypass of the cellular RNA decay machinery can be accomplished in many ways, including the sequestering of regulatory proteins or inactivating enzymatic components. One attractive way for RNA viruses to undermine the cellular RNA decay machinery is to target the cellular exoribonuclease XRN1 since this enzyme plays a major role in mRNA decay, appears to coordinate transcription rates with RNA decay rates, and is localized to the cytoplasm and thus readily accessible to cytoplasmic RNA viruses. We have previously shown that many members of Flaviviridae (e.g. Dengue, West Nile, Hepatitis C and Bovine Viral Diarrhea viruses) use RNA structures in their 5' or 3' untranslated regions (UTRs) to stall and repress XRN1. This results in the stabilization of viral RNAs while also causing significant dysregulation of cellular RNA stability (and thus dysregulation of overall cellular gene expression). In this dissertation we first extend this observation to another member of the Flaviviridae, Zika virus, by demonstrating that structures in the 3' UTR of the viral genomic RNA can stall and repress XRN1. Significantly, we also demonstrate that the 3' UTR of the N mRNA of the ambisense segment of Rift Valley Fever virus, as well as two other phleboviruses of the Phenuiviridae, also can effectively stall and repress XRN1. This observation establishes XRN1 stalling in an additional family of RNA viruses, in this case in the order Bunyavirales. We have mapped the region responsible for XRN1 stalling to a G-rich core of ~50 nucleotides and provide evidence that the formation of a G-quadruplex is contributing to stalling of XRN1. In addition to phleboviruses, we also detected RNA regions that stall XRN1 in the non-coding regions of two other virus families. The 3' UTRs of all four ambisense transcripts of Junin virus, an arenavirus, stall and repress XRN1. This observation was extended to two additional arenaviruses, suggesting that XRN1 stalling may be a conserved property of the 3' UTRs in the Arenaviridae. Finally, we demonstrate that the non-coding RNA from beet necrotic yellow vein virus RNA segment 3 is produced by XRN1 stalling and requires a conserved sequence called the coremin motif. Collectively, these observations establish XRN1 stalling and repression as a major strategy used by many virus families to effectively interface with the cellular RNA decay machinery during infection. We performed two proof of principle studies to extend the significance of the observation of XRN1 stalling during RNA virus infections. First, since XRN1 stalling may be associated with successful viral gene expression as well as cytopathology, we explored whether we could identify a small molecule compound that could interfere with the knot-like three helix RNA junction structure that stalls XRN1 in the 3' UTR of flaviviruses. We tested several triptycene-based molecules, compounds that have been previously shown to intercalate into three helix junctions and identified four triptycene derivatives that interfere with XRN1 stalling. Lastly, we explored whether there might be a cellular exoribonuclease that could navigate through the well-characterized flavivirus structure that effectively stalls XRN1. Our efforts focused on the mammalian Dom3z/DXO enzyme which contains both 5' decapping and 5'-3' exoribonuclease activity. Interestingly, recombinant Dom3z/DXO enzyme did not stall on RNAs containing the 3' UTR of either Dengue virus or the Rift Valley Fever Virus N mRNA. This may suggest that there is a molecular arms race of sorts between the cell and the virus for supremacy of regulating the 5'-3' decay of RNA during infection.
Open Access
Analytical spectroscopy method development to study mechanisms of Alzheimer's and tuberculosis diseases
(Colorado State University. Libraries, 2020) Beuning, Cheryle Nicole, author; Crans, Debbie C., advisor; Levinger, Nancy E., committee member; Barisas, George, committee member; Fisher, Ellen R., committee member; Zabel, Mark, committee member
This dissertation covers the analytical method development created to study and enhance the knowledge of two specific disease mechanisms important to Alzheimer's disease and Mycobacterium tuberculosis. There are two parts in this dissertation where Part 1 is entitled Measurement of The Kinetic Rate Constants of Interpeptidic Divalent Transition Metal Ion Exchange in Neurodegenerative Disease. Part 2 is entitled The Electrochemistry of Truncated Menaquinone Electron Transporters with Saturated Isoprene Side Chains Important in Tuberculosis. These diseases appear on the World Health Organization's top 10 leading causes of death worldwide. The amyloid-beta (Aβ) peptides are associated with Alzheimer's disease, where neurodegeneration is caused by the aggregation of the peptide into senile plaques within neuronal synaptic cleft spaces. Alzheimer's disease currently has no cure due to its multi-causative pathology. One disease mechanism is the coordination of divalent metal ions to the peptide. Specifically, Aβ coordinates Cu(II) and Zn(II) ions that can enhance the aggregation of Aβ into plaques. These metal ions are highly regulated within the human body and are usually found bound to peptides and not as free ions. Therefore, the Aβ must sequester the metals from other proteins and peptides. The primary research in this dissertation advances fluorescence method development to measure interpeptidic Cu(II) exchange kinetics to be able to measure this type of disease mechanism. The small peptides GHK (Gly – His – Lys) and DAHK (Asp – Ala – His – Lys) both chelate Cu(II) with nM affinity, have biological relevance as they are motifs found in human blood like Aβ, and chelate Cu(II) with similar nitrogen-rich binding ligands as Aβ. By substituting non-coordinating lysine residues with fluorescent tryptophan, the interpeptidic exchange rates can be measured since tryptophan fluorescence is statically quenched when within 14 angstroms of a paramagnetic bound Cu(II). Thus Cu(II) transfer from Cu(H-1GHW) to either GHK or DAHK can be monitored by recovered GHW fluorescence as the Cu(II) is exchanged and second-order kinetic rate constants were determined. This methodology was then used to monitor the Cu(II) exchange from truncated Cu(Aβ1-16) and Cu(Aβ1-28) complexes to GHW and DAHW, where second-order reaction kinetic rate constants were determined. While in the exchanges between Cu(H-1GHW) with GHK/DAHK the second-order rate constants were on the magnitude of 102 or 101 M-1s-1, respectively, the exchanges from Cu(Aβ) complexes were 2-3 orders of magnitude larger, 104 M-1s-1 (to GHW and DAHW). These differences in rate constant magnitude arise from the fact that the affinity of GHW (KA = 1013 M-1) for Cu(II) is larger than Aβ (KA =1010 M-1). This method development is an important step to an accurate measurement of the interpeptidic exchange between Aβ peptides, including in their fibril and plaque formations. Since senile plaques are found in synaptic cleft spaces with nanometer distances between neurons, a model system was generated to study coordination reactions at the nanoscale. In order to do this, the metal ion would need to be released in a controlled manner. Studies of metal ion burst reactions through the use of photocages can simulate bursts of ions like those seen in the synaptic cleft. Zn(II) is often released in its ionic form within the synapse in its function as a neurotransmitter, so we simulated a burst of Zn(II) ions by using a photocage, [Zn(NTAdeCage)]- which releases Zn(II) when irradiated with light. The photocage was irradiated to release Zn(II) then we followed its reaction progress with an in situ chelator, Zincon, in reverse micelles and in bulk aqueous buffer. The coordination reaction was 1.4 times faster in an aqueous buffer than in reverse micelles, despite the Zn(II) and Zincon being closer in the nanoparticle. These observations suggested that there is an impact on coordination reactivity within a highly heterogeneous environment with a cell-like membrane, which is due to the partitioning of each ligand. We observe that the photocage stays in the water pool of the reverse micelle and the Zincon partitions into the membrane interface. Thus, the coordination reactivity is diminished, likely due to the need for Zn(II) to diffuse to the Zincon, crossing a highly organized Stern layer to encounter the Zincon. Whereas in aqueous buffer, these are free to encounter each other despite being hundreds of nanometers apart. These proof of concept studies are integral to studying initial binding dynamics of metal ions with peptides at the nanoscale present in cells and neuronal synapses. Tuberculosis is a pathogenic bacterium which despite having a curable medication, can be drug-resistant. Menaquinone (MK) analogs with regiospecific partial saturation in their isoprenyl side chain, such as MK-9(II-H2), are found in many types of bacteria, including pathogenic Mycobacterium tuberculosis and function as electron transport lipids cycling between quinone and quinol forms within the electron transport system. While the function of MK is well established, the role of regiospecific partial saturation in the isoprenyl side chain on MK remains unclear and may be related to the redox function. Recently, an enzyme in M. tuberculosis called MenJ was shown to selectively saturate the second isoprene unit of MK-9 to MK-9(II-H2). The knockout expression of this enzyme was shown to be essential to the survival of the bacterium. A series of synthesized truncated MK-n analogs were investigated using a systematic statistical approach to test the effects of regiospecific saturation on the redox potentials. Using principal component analysis on the experimental redox potentials, the effects of saturation of the isoprene tail on the redox potentials were identified. The partial saturation of the second isoprene unit resulted in more positive redox potentials, requiring less energy to reduce the quinone. While full saturation of the isoprene tail resulted in the most negative potentials measured, requiring more energy to reduce the quinone. These observations give insight into why these partially saturated menaquinones are conserved in nature.
Open Access
Clinical and molecular characterization of canine small cell B-cell lymphocytosis disorders
(Colorado State University. Libraries, 2020) Rout, Emily, author; Avery, Anne, advisor; Avery, Paul, committee member; Zabel, Mark, committee member; Weil, Michael, committee member
To view the abstract, please see the full text of the document.
Open Access
Cytokines, antibodies and plasma viremia of cats infected with feline immunodeficiency virus
(Colorado State University. Libraries, 2013) Wood, Britta Ann, author; VandeWoude, Sue, advisor; Avery, Paul, committee member; Zabel, Mark, committee member; Hussey, Gisela, committee member
Feline immunodeficiency viruses (FIVs) are naturally occurring lentiviruses (family Retroviridae) of felid species, including domestic and wild cats. Studies on FIVs are beneficial for understanding the host immune response associated with disease progression (e.g., domestic cat FIV) or the viral kinetics and molecular ecology associated with naturally occurring infections in wildlife (e.g., bobcat and mountain lion FIVs). Here we describe the development and validation of the following microsphere immunoassays (MIAs) for evaluating the cytokine and antibody response of domestic cats: i) the quantification of cytokines (interferon gamma (IFNγ), interleukin (IL)-10, and IL-12/IL-23) in cell culture supernatant, and ii) the quantification of these cytokines in plasma; iii) the quantification of total IgG and IgA in plasma, and iv) the detection of IgG and IgA antibodies to feline CD134 (the primary cell receptor for FIV), and FIV capsid (CA) and surface (SU) proteins in plasma. These assays were used to evaluate temporal cytokine and antibody responses of domestic cats experimentally infected with various FIV strains. To analyze viral RNA loads associated with naturally occurring FIV infections in bobcats or mountain lions, we are adapting existing quantitative PCR assays for use with plasma samples. The eight assays described here are/will be beneficial for addressing questions related to lentiviral immune response and viral kinetics.
Open Access
Defining interactions between Mycobacterium leprae and Langerhans cells
(Colorado State University. Libraries, 2021) Fletcher, Darcy, author; Belisle, John, advisor; Henao-Tamayo, Marcela, committee member; Hess, Ann, committee member; Zabel, Mark, committee member
Leprosy is a chronic infection that affects the skin and peripheral nerves. Written accounts of the disease date back to at least 600 BC. Mycobacterium leprae, the causative agent of leprosy was first discovered by Dr. Gerhard Armauer Hansen in 1873. Leprosy remains a major health problem in several low- and middle-income countries including Brazil, India, and Indonesia. There are numerous clinical presentations of the disease which presents many challenges for controlling the disease including diagnosis, treatment regimen and duration, and occasional instances of drug resistant cases. Further challenges exist in studying the disease, knowledge of the intricate interactions with innate immune cells has made advances in some cell subsets but is limited in others leaving an incomplete picture of the disease. These gaps limit advances in disease management. M. leprae is an obligate, intracellular pathogen that grows preferentially between 33-35° C and selectively invades peripheral nerves and skin-resident innate immune cells including macrophages. Numerous host cells including macrophages and Schwann cells have been studied to understand their interaction with M. leprae, but other skin-resident immune cells like dendritic cells, specifically Langerhans cells, have not been studied as extensively. The findings that M. leprae antigens can be presented via CD1a on Langerhans cells has spurred interest in understanding how Langerhans cells interact and uptake M. leprae leading to downstream effects on T cell activation and overall immune responses. The hypothesis of this study is that M. leprae interacts with Langerhans cells via various cell surface receptors that influence a Th1 or Th2 immune response. This study interrogates the complex interactions between Mycobacterium leprae and Langerhans cells via multiple cell surface receptors. In Chapter 2, an ex vivo optical tissue clearing method was modified for fragile skin samples to analyze innate cell recruitment to the site of infection. Colocalization between Langerhans cells and a closely related mycobacterial spp. to M. leprae, M. haemophilum, was observed in a 3D optically cleared tissue. These observations indicate that wholistic insight of bacteria/innate immune cell interactions can be gleaned using experimentally infected tissues or human skin biopsies. Chapter 3 presents the contributions from multiple cell surface receptors present on Langerhans cells in recognizing and binding M. leprae. Langerin was found to play a role in binding M. leprae, however, was not the only cell surface receptor involved in recognition of M. leprae. CD5+ Langerhans cells can be separated into CD5high and CD5low LCs that have differences in binding capacity for M. leprae. This study builds the foundation to explore the wholistic contributions of Langerhans cells interactions and uptake of M. leprae. Further work should be conducted to identify M. leprae ligand(s) for CD5 and downstream effects on cytokine secretion and T cell activation.
Open Access
Detecting the temporal status of blood-borne prions in transmissible spongiform encephalopathy-infected hosts
(Colorado State University. Libraries, 2015) Elder, Alan Michael, author; Mathiason, Candace, advisor; Hoover, Edward, committee member; Zabel, Mark, committee member; Bartz, Jason, committee member; Bamburg, Jim, committee member
Transmissible spongiform encephalopathies (TSEs), or prion diseases, are infectious, fatal neurodegenerative diseases with a protracted subclinical disease state spanning months to years. Prion diseases develop when the normal cellular prion protein (PrPC) undergoes a conformational change into an aberrant, disease-causing, isoform (PrPSc/PrPres/PrPD), which aggregates into amyloid fibrils. Prions are unique from all other infectious diseases in that they lack nucleic acid. Prion diseases are known to naturally occur in cattle, sheep, mink, cervids, and humans; however, the exact mechanisms of transmission are unknown. Sufficient infectious prions to transmit and cause disease are known to be present in tissues and bodily fluids of all TSE-infected mammals during clinical and subclinical stages of disease. Extensive extraneural PrPSc-deposition has been observed in chronic wasting disease (CWD)-infected cervids and transmissible mink encephalopathy (TME)-infected hamsters and is very similar to what has been described for variant Creutzfeldt-Jacob disease (vCJD)-infected humans. Importantly, blood taken from humans and animals lacking overt clinical symptoms is capable of transmitting disease through transfusion. In this thesis we set out to answer questions regarding hematogenous prions: 1) How long does it take for prions to enter the blood after initial TSE exposure? 2) Are hematogenous prions present in all animals infected with CWD and TME? and 3) Does the route of prion entry affect the temporal status of hematogenous prions. To answer these questions, we analyzed longitudinally-collected whole blood samples from TSE-exposed animals by a modified version of the highly sensitive in vitro amyloid-amplification assay "real-time quaking-induced conversion" (RT-QuIC) we termed whole blood (wb) RT-QuIC. Longitudinal whole blood samples (15 minutes post exposure-terminal disease) were collected from experimental CWD-exposed (oral, aerosol, and intravenous inoculation) white-tailed and Reeves' muntjac deer and TME-exposed (extranasal inoculation) Syrian hamsters. We detected PrP conversion-competent amyloid in the blood of 100% of infected animals as early as 15 minutes post inoculation throughout terminal clinical TSE disease. These results were observed for all inoculation routes. Furthermore, we observed the presence of prions in the blood in two phases--a primary and secondary prionemia. The results of this work suggest that: 1) inoculated prions traverse mucosal barriers and enter the blood within 15 minutes of exposure; 2) the route of inoculation has little effect on the temporal status of prions in the blood; 3) there are two distinct phases of prionemia representing the initial inoculum (primary prionemia) and de novo host-generated prions (secondary prionemia); and 4) the observed characteristics of prionemia can be recapitulated in various TSE-host combinations and may recapitulated the extraneural pathogenesis of human TSEs.
Open Access
Development and application of new diagnostic assays for the detection of prion proteins in transmissible spongiform encephalopathies
(Colorado State University. Libraries, 2018) Ishii, Toru, author; Telling, Glenn, advisor; Liber, Howard, committee member; McGrath, Stephanie, committee member; Ross, Eric, committee member; Zabel, Mark, committee member
Transmissible spongiform encephalopathies (TSEs), well known as prion diseases, are fatal neurodegenerative disorders in humans and animals, which a prion protein (PrP) mainly implicates with the TSE pathogenesis. The normal cellular PrP isoform, referred to as PrPC, predominantly forms an α-helical structure. A structural alteration of the PrPC isoform can misfold into the infectious and pathogenic PrP isoform, referred to as PrPSc (or PrPD as disease-associated PrP). The PrPSc isoform consists of a β-sheet rich structure and accumulates in the central nervous system (CNS). The structural change and accumulation of this abnormal conformer alters physiochemical properties of the PrPC isoform. However, the conversional mechanism from PrPC to PrPSc isoforms is not clearly known. In general, the PrPC structure has two conserved Asparagine (N)-glycosylation sites that generate four various glycosidic forms (unglycosyl, two differing monoglycosyl and diglycosyl). In preliminary studies, TSE-disease mice exhibited the increased detection of under-glycosylated PrP forms, compared to controls. Although protein glycosylation plays various structural and functional roles, the importance of these glycans is not clarified in TSE pathogeneses. Recently, novel monoclonal antibodies (mAbs) against PrP molecules were reported. Of these mAbs, PRC7 mAb can recognize an unglycosyl form and one monoglycosyl form (mono-1) of PrP molecules specifically. In addition, PRC7 mAb has a unique feature to require denaturation and renaturation of PrP molecules to recognize PrPSc isoforms. Since PRC7 mAb cannot react with a diglycosyl PrP form that an abundance of normal PrPC isoforms express, PRC7 mAb preferentially detects PrPSc isoforms. Thus, these features of PRC7 mAb were applied to develop a sensitive enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of under-glycosylated PrP forms in TSE-infected samples. My central hypothesis is that, the detection of under-glycosylated PrP forms is the hallmark of TSEs as diagnostic biomarkers for the disease progression. Here, I propose that loss of full glycosylation is implicated in the pathological mechanisms of TSEs. For instance, glycosylation is involved in the maintenance of protein structure. Thus, its modulation can initiate unstable conditions for maintaining proper PrPC conformations, which induce pathologic alterations of the PrPC structure. These aberrant formations could lead functional impairments of normal PrPC isoforms. In another aspect, the PrPSc isoform may lose glycans during the disease development of TSEs. Therefore, I assume that under-glycosylated PrP forms can be preferentially generated during the disease progression. To accomplish the proposed studies, I have developed two sensitive ELISA methods for the detection and quantification of PrP molecules in TSE-infected samples. Using the PRC7 and PRC5 mAbs as capture and detecting antibodies respectively, the 7-5 ELISA method specifically recognizes the certain under-glycosylated PrP forms that are significantly detected in TSE-infected materials. In addition, the D-5 ELISA method uses D18 anti-prion antibody as a capture antibody and has an exceptional capability to determine levels of only PrPC or total PrP molecules by different sample preparations. Using the D-5 ELISA method, I have detected the reduction of PrPC levels in TSE-infected materials at terminal stages, whereas total PrP levels were increased. These results were similar observations to a recent article using the conformation-dependent immunoassay. Since my protocols do not require a proteinase K (PK) reagent, these ELISA methods will be ultimately beneficial for TSE diagnoses, especially for detecting PK-sensitive PrPSc forms. Furthermore, the ELISA approaches would contribute to understand the TSE pathogenesis under the specific detections of PrPSc isoforms, based on glycosylated forms for distinguishing PrPC from PrPSc isoforms. This dissertation study will provide an innovative framework of the proposed projects that will achieve beneficial impacts into the fields of veterinary medicine, human medicine, and public health.
Open Access
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.
Embargo
Furthering the development of the Jamaican fruit bat as an animal model for immunology
(Colorado State University. Libraries, 2023) Burke, Bradly E., author; Schountz, Tony, advisor; Henao-Tamayo, Marcela, advisor; Zabel, Mark, committee member; Bosco-Lauth, Angela, committee member; Hansen, Tod, committee member
Bats are the only flying mammals capable of powered sustained flight and encompass over 1,400 species. Bats interconnect important ecological services, agricultural crop services, agricultural husbandry health, and human health. The order of chiroptera is largely understudied for immunological research even though bats are reservoirs for viruses that are transmissible to livestock and humans including, henipaviruses, filoviruses, coronaviruses, and lyssaviruses. In 2006 Colorado State University established a breeding colony of Jamaican fruit bats (Artibeus jamaicensis) for use in the study of bat-borne viral infections. Establishment of a bat colony is only the first step to the development of an animal model. The next step to develop an animal model is to elucidate the immunological systems and functionality of them. To elucidate the immunological systems, bat specific reagents are needed to characterize and perform hypothesis driven research. However, the paucity of bat specific reagents largely limits hypothesis driven research. Antibodies and cell lines are foundational reagents to immunology and virology. Antibodies are integral reagents used for many immunological and biochemical assays: ELISAs, ELISPOTs, western blots, cytometric bead assays, magnetic bead pull-down, surface plasmon resonance, microscopy, in vivo cellular depletion, and flow cytometry. Cell lines allow for in vitro assays and the propagation of viruses to be performed. The production and validation of Jamaican fruit bat specific antibodies targeting CD3γ, CD4, and CD8α epitopes for the investigation of cytotoxic CD8 T cells, and CD4 T helper cells are described in this study. Furthermore, the identification and validation of commercially available cross-reactive antibodies targeted to epitopes for various proteins and glycolipids: asialo GM1, CD3ε, CD8α, CD19, CD34, CD40, CD44, CD45, CD80, CD104, CD154, CD161, and MHC-II. This work has described their use in highly quantitative flow cytometric analysis, enrichment of cell populations by fluorescently activated cell sorting, and highly qualitative anatomical data by microscopy. This study has identified a novel CD19+CD3+ T cell population under homeostatic conditions of the Jamaican fruit bat immune system that has not been identified in humans, mice, or other bat species under a homeostatic state. The use of anti-asialo GM1 in vivo treatment of Jamaican fruit bats to target natural killer cells is also described in this work. Lastly the construction of plasmids for the production of Jamaican fruit bat growth factors: epidermal growth factor, wnt3a, R-Spondin-2, noggin, and gastrin are described for their use in culturing Jamaican fruit bat primary cells – especially gastrointestinal crypt stem cells. Validation of cellular markers and reagents is a crucial first step in the investigative process that allows for the generation of informed conclusions – in any study. Furthermore, validation of cellular markers holds a higher level of imminent need and accuracy in underdeveloped animal models. This work provides a framework for other researchers in the advancement of underdeveloped animal models for immunology to more rigorously test antibody cross-reactivity. Furthermore, this work highlights the need to build a robust body of literature of cross-reactive antibodies for underdeveloped animal models.
Open Access
HIV prophylaxis: an essential role for T cells and adjuvants in recombinant mucosal Lactobacillus acidophilus vaccines
(Colorado State University. Libraries, 2016) LeCureux, Jonathan Spicer, author; Dean, Gregg, advisor; Aboellail, Tawfik, committee member; Chen, Chaoping, committee member; Zabel, Mark, committee member
Current HIV vaccines have poor efficacy, with inconsistent levels of protection following mucosal HIV exposure. Lactic acid bacteria offer an alternative vaccine vector targeting the primary site of HIV infection, the mucosa. In these studies we evaluated the immunogenicity of several strains of Lactobacillus acidophilus expressing HIV membrane proximal external region (MPER), a portion of HIV envelope that contains broadly neutralizing antibody binding sites. We evaluated MPER-only expressing strains along with strains expressing adjuvants (interleukin-1β or flagellin) to improve immunogenicity against the HIV MPER. We compared the adjuvant strains to the MPER-only strain by oral administration in BALB/c mice to observe these improvements, and in CD40L-/- mice to observe if T cell help was necessary. Some BALB/c animals were also placed on a modified diet supplemented with prebiotic rice bran to observe any influence on vaccine immunogenicity. Resulting antibody responses and interleukin-17 levels were measured by ELISA, and T and B cell levels were measured by flow cytometry. Here we show that the addition of adjuvants, including dietary rice bran, to L. acidophilus vaccine strains improves their immunogenicity against HIV MPER. Our results indicate that anti-MPER IgG and IgA levels, as well as the number of anti-MPER antibody secreting cells, are improved with adjuvants, and that T cell help is required for an effective immune response. These results, combined with the many advantages offered by this lactic acid bacteria vaccine system make L. acidophilus an attractive vaccine vector for primate and human trials.
Open Access
Immune modulatory and antimicrobial properties of canine and human mesenchymal stem cells
(Colorado State University. Libraries, 2018) Chow, Lyndah, author; Dow, Steven, advisor; Koch, Peter, committee member; Zabel, Mark, committee member; Tjalkens, Ronald, committee member
To view the abstract, please see the full text of the document.
Open Access
Interruption of neuron-microglia bidirectional communication to modulate cofilin:actin rod formation
(Colorado State University. Libraries, 2022) Zoller, Maia, author; Bamburg, James, advisor; Chanda, Soham, committee member; Zabel, Mark, committee member
Immune responses in the central nervous system are mediated by microglia, whose responses to CNS threats can be replicated in vitro to study the role of microglia in the onset, progression, and treatment of neurodegenerative diseases. Previous work has identified a pathway common to neurodegenerative diseases such as Alzheimer's Disease, Parkinson's Disease, and HIV-Associated Neurocognitive Dementia in which the actin-severing protein, cofilin, forms a 1:1 bundle with actin making rod-shaped inclusions (rods) that can be found in the dendrites and axons of neuronal cells. This thesis focuses on developing methods for examining the role of primary microglia, activated by different factors, to secrete rod-inducing chemokines/cytokines or directly attacking neurons leading to neuronal death. Understanding both of these mechanisms is important in study of neuroinflammation and disease progression. Hemin, a hemoglobin metabolite, and alarmin, S100B, a astrocyte secreted, calcium binding protein, protein, were used to model the environment of intracerebral hemorrhage and general neuroinflammation respectively. Preliminary experimental results suggest blockage of actin-rod inducing signaling pathways via CXCR4/CCR5 receptor antagonist improves neuronal survival to both microglia conditioned medium and direct exposure the microglia-activating hemin or S100B. Further studies are in progress to obtain sufficient statistical data to verify these results.
Open Access
Investigation of the sequence features controlling aggregation or degradation of prion-like proteins
(Colorado State University. Libraries, 2017) Cascarina, Sean Micheal, author; Ross, Eric, advisor; Ho, P. Shing, committee member; Di Pietro, Santiago, committee member; Zabel, Mark, committee member
Protein aggregates result from the conversion of soluble proteins to an insoluble form. In some cases, protein aggregates are capable of catalyzing the conversion of their soluble protein counterparts to the insoluble form, resulting in a mode of molecular self-replication. Many of these infectious proteins, or "prions", have been identified and characterized in yeast. This has led to the development of prediction algorithms designed to identify protein domains capable of forming prions. Recently, a number human proteins with aggregation-prone prion-like domains (PrLDs) have been identified, and mutations within PrLDs have been linked to muscular and neurodegenerative disorders. However, the number and diversity of PrLD mutations linked to disease are currently limited. Therefore, the extent to which a broad assortment of PrLD mutations affect intrinsic aggregation propensity, and how well this correlates with aggregation in a cellular context, has not been systematically examined. In Chapter 2, I present evidence suggesting that our prion aggregation prediction algorithm (PAPA) is capable of predicting the effects of a diverse range of mutations on the aggregation propensity of PrLDs in vitro and in yeast. PAPA was also able to predict the effects of many but not all PrLD mutations when the protein was expressed in Drosophila, but with slightly. Therefore, while great strides have been made in predicting intrinsic aggregation propensity, a more complete understanding of the cellular factors that influence aggregation in vivo may lead to further improvement of prion prediction methods. Many intracellular protein quality control factors specialize in recognizing and degrading aggregation-prone proteins. Therefore, prions must evade or outcompete these quality control systems in order to form and propagate in a cellular context. However, the sequence features that promote degradation versus aggregation of prion domains and PrLDs have not been systematically defined. In Chapter 3, I present evidence that aggregation propensity and degradation propensity can be uncoupled in multiple ways. First, we find that only a subset of classically aggregation-promoting amino acids elicit a strong degradation response in PrLDs. Second, the amino acids that promoted degradation of the PrLDs did not induce degradation of a glutamine/asparagine (Q/N)-rich prion domain, and instead led to a dose-dependent increase in the frequency of spontaneous prion formation, suggesting that protein features surrounding aggregation-prone amino acids can modulate their ultimate effects. Furthermore, degradation suppression correlated with Q/N content of the surrounding prion domain, potentially indicating an underappreciated role for these residues in yeast prion domains. The protein features that foster susceptibility or resistance to degradation are further explored in Chapter 4. We find that Q/N-rich domains resist degradation in a primary sequence-independent manner, and can even exert a dominant degradation-inhibiting effect when coupled to a degradation-prone PrLD. Furthermore, susceptibility to degradation was a relatively de-centralized feature of the PrLD, requiring a large portion of the domain surrounding degradation-promoting amino acids to permit efficient protein turnover. Collectively, these results provide key insights into the relationship between intrinsically aggregation-prone protein features and the ability to aggregate in the context of intracellular protein quality control factors.
Open Access
Molecular basis of [PSI+] yeast prion nucleation
(Colorado State University. Libraries, 2013) Ben Musa, Zobaida A., author; Ross, Eric, advisor; Crans, Debbie, committee member; Zabel, Mark, committee member; Di Pietro, Santiago, committee member
Many fatal diseases arise from the conversion of soluble, functional proteins to insoluble misfolded amyloid aggregates. Amyloid fibers are characterized by filamentous morphology, protease resistance and cross]beta structure. Prions (infectious amyloids) are a specific subset of amyloid fibers, differing from other classes of amyloids by their infectivity. Prions are found in both mammals and yeasts, but there are differences between these two groups. Most yeast prions are characterized by the presence of large numbers of glutamine and asparagine (Q/N) residues, and some other common characteristics have been noted, including the presence of few hydrophobic and charged residues. Although, several attempts have been made with limited success to develop valuable systems to predict prion activity, there is no accurate algorithm that has the ability to predict the prion-forming proteins among the Q/N-rich protein group. In the yeast, it has been shown that amino acid composition, not primary sequence, drives prion activity. Recently, preliminary efforts to define the role of amino acid composition in prion formation have been examined. The fundamental question of this project is how, in yeast Q/N-rich prions, the sequence requirements for nucleation versus propagation differ, and how this information can be used in order to develop a precise prion prediction system. By answering this question we will be able to more accurately identify additional prions in both yeast and other organisms. Our long-term goal in the comprehensive studies of prion formation and propagation mechanisms is to apply this information to mammalian prion diseases. Consequently, we will be able to identify targets for therapeutic intervention to avoid, slow-down, or reverse the development of related diseases. The study determined that the amino acids required for prion formation differ from those required for prion propagation. Identifying the sequence feature for both activities is the first step towards mechanistic studies to examine how these sequences perform their function.
Open Access
Neuroinflammation and the two-hit hypothesis of Parkinson's disease
(Colorado State University. Libraries, 2019) Bantle, Collin M., author; Tjalkens, Ronald B., advisor; Zabel, Mark, committee member; Moreno, Julie, committee member; Kato, Takamitsu, committee member; Randall, Elissa K., committee member
The ever-increasing prevalence of neurodegenerative diseases, Alzheimer's Disease (AD) and Parkinson's disease (PD), impose one the most significant medical and public health threats throughout the world. Characteristic PD symptoms include loss of voluntary motor control due to α-synuclein protein-aggregation, neuroinflammatory glial activation, mitochondrial dysfunction, oxidative stress, and progressive neuronal loss. There are currently no disease-modifying therapies for the disease nor has the etiology of PD been elucidated. Epidemiologic and experimental evidence suggests that genetic susceptibility, environmental pesticide exposure, and viral infections are possible risk-factors for PD, but a clear understanding of the environmental links to PD and how these factors can act in concert remains extremely limited. Research is beginning to shed light on neuroinflammation as a converging and coalescing pathway in the pathogenesis and pathophysiology of genetic, sporadic, and postencephalitic PD. While it has been appreciated since the late 1980s that brain inflammation is a hallmark of PD and other age-related neurodegenerative diseases, the immunological role of glia and the key trophic and inflammatory factors and pathways responsible for neurotoxicity and neuronal death in PD have not been clearly elucidated. Understanding how these pathways are regulated in glia during genetic, sporadic, and postencephalitic PD, and how they can directly or secondarily affect the onset and progression of PD is of keen interest. Therefore, the subject of this work will be to explore mechanisms by which glial cells modulate neuronal injury in genetic, sporadic, and postencephalitic cases of PD, with an emphasis on the role of neuroinflammatory activation of glia in single and two-hit models of PD.
Open Access
Nitric oxide-releasing or generating surfaces for blood-contacting medical devices
(Colorado State University. Libraries, 2020) Zang, Yanyi, author; Reynolds, Melissa, advisor; Kipper, Matt, committee member; Li, Yan Vivian, committee member; Zabel, Mark, committee member
Medical device-induced thrombosis is a major complication that impairs the expected performance of blood-contacting medical devices. Traditional anticoagulation therapies are used to reduce thrombus formation; however, systemic anticoagulants such as heparin increase the risk of thrombocytopenia or even bleeding, which are detrimental to patients who already have injuries. To address these issues, surface modification has been widely studied to improve the performance of blood-contacting medical devices, ranging from biopassive surfaces to biomimetic surfaces. To date, such modifications are not sufficient to prevent blood clotting alone. Supplementary anticoagulation remains necessary to maintain clot-free surfaces. Nitric oxide (NO) is a well-known signaling molecule that has antiplatelet properties. Our approach is to use surfaces that can either release NO via NO donors or promote NO production via an NO catalyst. In this work, a NO-releasing polyelectrolyte multilayer coating effectively reduces platelet adhesion, platelet activation and delay blood clotting on titania nanotube array surfaces. In addition, NO-releasing polymeric surfaces mediate blood serum protein deposition in a manner that prevents platelet adhesion and platelet activation. However, the NO donors used in these two coatings are photo- and thermo- sensitive, and the NO release is limited by the amount of NO donor added to the coating. To overcome these shortcomings, a copper-based metal organic framework (MOF) was used to infinitely promote NO production from NO donors in the blood. The copper-based MOF polymer coating was successfully applied to the surfaces of extracorporeal life support catheters and circulation tubing via custom coating systems. These copper-based MOF-coating also exhibited inherent antibacterial properties under both static and dynamic flow conditions.
Open Access
Novel in vitro approaches to delineate prion strain conformational variation
(Colorado State University. Libraries, 2019) Selwyn, Vanessa Villegas, author; Telling, Glenn, advisor; Zabel, Mark, committee member; Ross, Eric, committee member; Dobos, Karen, committee member
Prions cause in invariably lethal, transmissible neurodegenerative diseases. There are no effective treatments or cures for prion diseases. Unlike other known pathogens, prions replicate in the absence of nucleic acids. Prion diseases stem from the conformational corruption of the cellular prion protein (PrPC) by the pathogenic form (PrPSc) (Prusiner, 1982). The prion phenomenon, protein-templated misfolding, is no longer limited to the prion protein (PrP). Other neurodegenerative disorders, including but not limited to Alzheimer's, Parkinson's, Huntington's are now being recognized as prion-like disorders (Soto, 2012). By exploring the intricacies of prion protein- misfolding, therapeutic approaches might emerge that will be useful in treating other neurodegenerative protein-misfolding disorders. Although the structure of PrPC has been solved (Riek et al 1997, Zahn et al 2000, Garcia et al 2000, Donne et al 2007, Antonyuk et al 2009), the three-dimensional structure of PrPSc has yet to be resolved. A confounding issue to identifying PrPSc structure is the existence of prion strains (Bett et al 2012). In the absence of nucleic acids, prion strain properties are propagated though variations in the conformational structure of PrPSc (Telling et al 1996). As such, prion strains can be defined as an infectious prion protein particle with a specific tertiary conformation that produces a specific neurodegenerative phenotype (Colby et al., 2009). Specifically, a prion strain can be considered to have a strain-specific (Peretz et al 2001) disease phenotype (Collinge et al 1996) based on the prion's ability to be stably propagated, fidelity to neuropathology, disease length, glycosylation profile, molecular weight of PK-resistant PrPSc, resistance to denaturation, amyloid seeding potential and other molecular characteristics. Ultimately, revealing PrPSc structure will provide better understanding of the basis of strains, species adaption and ultimately the species barrier. The traditional methodologies to examine prion strains are costly, time consuming, and do not provide adequate resolution of the PrPSc structure. The overarching aim of my research is to better understand how prions encrypt strain information. In Chapter 1, I outline essential background regarding prions and prion strains. In Chapter 2 and 3, I address the creation of the expanded Cell-Based Conformational Stability Assay, Epitope Stability Assay, and use of a new 7-5 ELISA Conformational Stability Assay. These represent novel tools that use chaotropic agents to probe epitope-mapped regions to identify subtle differences in prion strain structure. The prion strains evaluated were cervid (deer and elk) chronic wasting disease, murine- adapted scrapie (RML, 22L, 139A), murine-adapted chronic wasting disease (mD10) and cervid-adapted (deer and elk) RML. These techniques revealed subtle but significant prion strain structural variations within and between these strains. In Chapter 4, the techniques were used to better understand drug-induced prion evolution and strain evolution in cell culture. Drug-induced prion evolution of PrPSc structure was subtle but detectable within 24 hours of treatment. Additionally, the structural changes were not stable, but in flux. Prion strains evolve in cell culture through serial passaging, they do not recapitulate molecular characteristics of a biological prion infection. Moreover, the prion structure is not stably passaged into naïve cells, or transgenic mice. This makes reliance on chronically infected cells as a basis for anti- prion therapeutic testing inadvisable. In conclusion, the subtle variations encoded in prion strain structure can be detected with the three new techniques in this dissertation: C-CSA, ESA, and 7-5 ELISA-CSA.
Open Access
Reframing viral infections as acute metabolic disorders: dengue viruses and their dependency on host metabolic pathways
(Colorado State University. Libraries, 2022) St. Clair, Laura A., author; Perera, Rushika, advisor; Belisle, John, committee member; Nachappa, Punya, committee member; Wilusz, Jeff, committee member; Zabel, Mark, committee member
Dengue viruses (DENVs) are the etiological agent of the world's most aggressive arthropod-borne disease. At present, there are no available antivirals against DENVs. This fact underscores a dire need to examine host-virus interactions to identify and develop novel therapeutic approaches. As obligate intracellular parasites, DENVs are reliant upon and hijack several host metabolic pathways both to fulfill their replicative needs, and to evade the host immune response. We and others have previously established that infection with DENVs causes significant perturbation to host lipid metabolism, including elevations in sphingolipids in both the human and mosquito host. In addition, we and others previously discovered that the DENV NS1 protein increases sialidase activity in both in vitro and in vivo models leading to increased endothelial hyperpermeability and vascular leakage which are hallmarks of severe dengue. To further clarify and characterize these previous works, we have performed siRNA-mediated loss of function studies using human hepatoma cells (Huh7 cells) on several metabolic pathways altered during DENV2 infection. First, we examined the role of acyl-CoA thioesterases, enzymes responsible for controlling the intracellular balance of activated fatty acids and free fatty acids, on the DENV2 lifecycle. In these analyses, we determined that the cytosolic ACOT1 enzyme had an inhibitory effect on DENV2 replication and release, while mitochondrial ACOT (ACOTs 2 and 7) functionality was critical for viral replication and release. Moreover, we identified several enzymes within the ACOT family whose expression was dependent on ACOT2 and ACOT7 expression. These results highlighted complex relationships between ACOTs and DENVs, as well as identified yet unknown functional interdependence between ACOT enzymes. Next, we expanded our previous understanding of the relationship between DENVs and the human sialidase enzymes (NEU1-4). While previously studies linked upregulation of these enzymes with DENV2 pathology, we provide the first evidence showing that NEU1-4 functionality is vital for DENV2 genome replication and viral egress. Moreover, our analyses also revealed previously unknown functionality of NEU4 or its downstream products as transcriptional regulators for NEU1-3. Finally, we provide the first profile of the effect of loss of function of enzymes within the entire sphingolipid metabolic pathway (as identified through KEGG pathway database) on the DENV2 life cycle. In this study, we identified that enzymes involved the sphingomyelinase and salvage pathways of ceramide synthesis as opposed to de novo ceramide synthesis were critical to DENV2 release from Huh7 cells. In addition, we determined that enzymes involved in the synthesis and degradation of glycosphingolipids were vital for DENV2 release. An especially intriguing result within this arm of sphingolipid metabolism was that the two enzymes which hydrolyze GluCer had differential effects on DENV2 replication and release. GBA1 (lysosomal) had an antiviral effect on DENV2, while GBA2 (non-lysosomal) was required for DENV2 replication and release. This prompted us to profile the changes that occur to glycosphingolipids (GSLs) during infection, and we uncovered several species of GSLs that are elevated during infection. Moreover, we identified that Ambroxol HCl, a pharmaceutical GBA1 chaperone/GBA2 inhibitor, was able to abrogate these elevations in GSLs. Combined, our results allowed us to propose a novel function for GBA2 as a GluCer recycling enzyme during DENV2 infection. In conclusion, together, the work in this dissertation highlights critical metabolic nodes that impact virus replication and provides new directions for investigating viral infections as acute metabolic diseases.
Open Access
The influence of H2AX and γH2AX on chromatin condensation
(Colorado State University. Libraries, 2010) Norskog, Sarah Samaya, author; Hansen, Jeffrey, advisor; Luger, Karolin, committee member; Zabel, Mark, committee member
Chromatin composition and structure are essential for the condensation of the genome and the regulation of a wide range of cellular activities. Chromatin condensation is thought to be controlled predominantly through interactions mediated by the unstructured amino terminal domains of the core histones H4, H3, H2A and H2B. In addition to the amino terminal domain, histone H2A contains an unstructured carboxyl terminal domain. Multiple H2A variants, many differing from major type H2A in this C-terminal domain sequence, have been identified. The most studied of variant is H2AX, which contains a conserved serine residue that becomes phosphorylated following double strand DNA breakage (yH2AX). Although the phosphorylation of the H2AX has been identified as a key step in major genomic activities, the basic mechanism by which it functions remains controversial. Here, I have determined the structural role of H2AX and yH2AX using in vitro assays which utilize defined nucleosomal arrays. H2AX and yH2AX alter chromatin folding under high salt concentrations but show no discernable differences in low concentrations of salt or under conditions which favor oligomerization. The phosphorylation of H2AX does not alter the folding or oligomerization relative to the unphosphorylated form, indicating yH2AX more likely functions as a signaling and recruitment motif rather than as a chromatin secondary structure remodeling factor.