Full text for dissertations and theses included in this collection dates back to 2011. For older dissertations, check the library’s catalog CatalogUSMAI or Dissertations and Theses database.

Recent Submissions

  • Risk of drug overdose death following discharge from a trauma center for an injury

    Greene, Christina Reagan; Smith, Gordon S., M.B., Ch.B., M.P.H.; Albrecht, Jennifer S. (2019)
    Background: Trauma patients have a higher rate of long-term mortality due to natural and external causes, yet drug overdose (OD) death within this population has not been explored previously. Pre-existing behavioral risk factors, such as drug and alcohol use disorders (DUD/AUD), and chronic pain resulting from traumatic injury may increase trauma patient’s risk of subsequent drug overdose death. Objectives: To determine whether trauma patients are at greater risk of drug OD death than the general population and detect whether smoking status or fracture is associated with future drug OD death among surviving trauma patients. Methods: Trauma patients between 18 and 64 years of age who were discharged alive from a Level I Trauma Center between January 1999 and October 2008 were linked to the National Death Index (N=36,288). Patients who were alive at least 30 days after discharge without cancer were included in this study. Trauma patient risk of drug OD death was compared with the age, gender and race adjusted state population using a standardized mortality ratio (SMR) and 95% confidence intervals (CI). Cox proportional hazard regression was used to determine whether current smoking status or lower limb fracture injury were risk factors for drug overdose death factors. Results: Trauma patients had a significantly higher drug overdose mortality rate than the state population [SMR=6.10 (95% CI 5.35-6.93)]. Cox proportional hazard modeling revealed a significant increased risk of drug overdose for current smokers [HR = 1.66 (95% CI 1.25-2.21)]. The effect of smoking was stronger in patients with no DUD/AUD and BAC< 80mg/dL [HR=2.45, 95% CI 1.67-3.57], while smoking was not associated with drug OD death in those with DUD/AUD or BAC≥ 80mg/dL on admission. Patients with lower limb fracture were not at increased risk of drug overdose death compared to those without fracture injuries. Conclusion: Trauma patients have a higher risk of drug OD death than the general population. Smoking is a significant risk factor for drug OD following traumatic injury. Future drug overdose prevention programs should focus efforts on reducing drug overdose mortality in trauma patients, particularly those who smoke.
  • The Muscle Xenograft Model of Facioscapulohumeral Muscular Dystrophy: Development, Optimization, and Novel Applications

    Mueller, Amber; Bloch, Robert J.; 0000-0002-2842-1072 (2019)
    Aberrant expression of DUX4 in human muscle causes Facioscapulohumeral Muscular dystrophy (FSHD) which affects about 1 in 8,333 individuals worldwide, yet the mechanism by which DUX4 causes muscle wasting is unknown. The DUX4 gene is unique to humans and transgenic animal models have largely failed to exhibit the dystrophic phenotype and endogenous molecular changes characteristic of FSHD. Therefore, studies of DUX4 signaling in human muscle have been limited to patient biopsies and cultures of myogenic cells in vitro. There are no therapies that target the mechanism of disease, thus there is a pressing need for studies of DUX4 in mature human muscle. We have developed a method to xenograft human-derived muscle precursor cells, isolated from patients with FSHD and controls, into the tibialis anterior of immune-deficient mice to form xenografts of pure human muscle tissue. The FSHD xenografts are robust, mature, and well organized. Human myofibers are innervated and associate with human satellite cells, making the xenografts structurally comparable to intact human skeletal muscle. The FSHD but not control xenografts express DUX4 and DUX4 gene targets and have 4q35 methylation profiles typical of FSHD. The FSHD grafts also display a novel biomarker of FSHD, SLC34A2, measurable by immunofluorescence, which will provide a quantifiable metric for future therapeutic studies. We have described several modifications to the engraftment strategy that can be used to answer complex mechanistic and functional questions regarding the FSHD pathophysiology. Finally, we report promising data from a collaborative study with Fulcrum Therapeutics in which we were able to repress the DUX4-signaling pathway by administering a targeted small molecule therapy to FSHD grafts. Ours is the first scalable and reproducible in vivo model of FSHD muscle. Future studies will include those aimed at continuing to delineate the molecular pathogenesis of FSHD, performing functional tests to define the pathophysiology of FSHD, and testing new and exciting therapeutic strategies aimed at reducing the DUX4 program in human FSHD xenografts.
  • Molecular Mechanisms of Enzymes in Infection and Immunity

    Klontz, Erik; Sundberg, Eric J. (2019)
    Enzymes are biological catalysts that enable life by accelerating specific reactions. As new infectious diseases are discovered, the enzymes produced by these organisms require characterization. In some cases, they can be inhibited to prevent disease; in other cases, they can be coopted or altered to serve as diagnostic or therapeutic tools. In this work, we explore the mechanisms of several enzymes involved in infection and immunity. In the first major theme, we characterize the mechanisms of fosfomycin resistance proteins from Escherichia coli (FosA3) and Klebsiella pneumoniae (FosAKP), which degrade the antibiotic fosfomycin. Although the active sites of FosA enzymes are well conserved, differences in activity between enzymes may be due to the presence of an allosteric site at the dimer interface of these enzymes. We solved crystal structures with a novel small molecule FosA inhibitor, termed ANY1, which binds with high affinity to the active site and acts as a competitive inhibitor of fosfomycin binding. By inhibiting FosA, ANY1 potentiates the effects of fosfomycin in several priority Gram-negative pathogens, and may serve as a suitable lead candidate for structure-guided drug design and pre-clinical development. In the second major theme, we describe the mechanisms of enzymes with activity on the carbohydrates of IgG antibodies. EndoS and EndoS2 are enzymes produced by Streptococcus pyogenes, which remove the conserved glycan on Asn297 of IgG to evade the host immune system. We discovered that these enzymes share a conserved mechanism of substrate recognition, binding primarily to the core and α(1,3) antenna of the IgG glycan. EndoS2 recognizes a more diverse set of glycans through differences in the both the active site and a coevolved carbohydrate binding module. We then describe the structure and function of AlfC, an α(1,6)-specific fucosidase from Lactobacillus casei with activity on the core fucose of antibodies. We identified D200 and D242 as the most likely catalytic residues, and provide a structural basis for the remarkable specificity of this enzyme and transfucosidase mutants. Finally, we describe a novel application for these carbohydrate-active enzymes in the directed evolution of antibodies based on yeast display.
  • Functional characterization of Myosin Binding Protein-C slow in health and disease

    Geist Hauserman, Janelle; Kontrogianni-Konstantopoulos, Aikaterini (2019)
    Myosin Binding Protein-C (MyBP-C) comprises a family of proteins with structural and regulatory roles in muscle. There are three MyBP-C isoforms in the family, encoded by different genes. Although the isoforms share significant structural and sequence homology, slow skeletal MyBP-C (sMyBP-C), encoded by MYBPC1, is unique as it is heavily spliced in both the NH2 and COOH-termini. To study the role of sMyBP-C in healthy, adult skeletal muscles, in vivo gene transfer and CRISPR plasmids were used to knock down sMyBP-C. Decreased sMyBP-C levels resulted in significantly decreased levels of thick, but not thin, filament proteins. The reduced levels of thick filament proteins were accompanied by disorganized A- and M-bands. Moreover, examination of the contractile activity of treated muscles demonstrated that downregulation of sMyBP-C resulted in significantly decreased force production and velocities of contraction and relaxation. In addition to the extensive exon shuffling that takes place in the NH2-terminus of sMyBP-C, it also undergoes PKA and PKC mediated phosphorylation within two motifs, which flank the first Ig domain of the protein. Recombinant NH2-terminal sMyBP-C phosphomimetic peptides were tested in co-sedimentation and in vitro motility assays, indicating that phosphorylation of sMyBP-C variants regulates actomyosin binding and sliding velocity. Mutations in MYBPC1 have been implicated in the development of distal arthrogryposis, while four recently discovered mutations (Y247H, E248K, L259P, and L263R) co-segregate with the development of a new myopathy characterized by muscle weakness, hypotonia, skeletal deformities, and tremor. In vitro studies and computational modeling suggest altered myosin binding and/or protein instability for the four mutations. Further in vivo evaluation of the E248K mutation in a heterozygous knock-in mouse model revealed significant biochemical, morphological, and behavioral deficits compared to wild type littermates. Additionally, functional assessment of heterozygous E248K muscles demonstrated decreased force and power production, as well as decreased cross bridge cycling kinetics, indicating the tremor may begin at the level of the sarcomere. My studies therefore reveal that sMyBP-C has important structural and regulatory roles within the sarcomere, is modulated through phosphorylation, and that novel MYBPC1 mutations lead to the development of myopathy and tremor that is of myogenic origin.
  • An investigation into the behavioral effects of targeted memory reactivation during sleep on sensorimotor skill performance

    Johnson, Brian Philip; Westlake, Kelly P. (2019)
    Background. Memory consolidation occurs during sleep, providing an opportunity to enhance upper extremity (UE) function in people with residual impairments post-stroke. Targeted memory reactivation (TMR) has been used to enhance this process, which involves pairing auditory cues with task performance and subsequent cue replay during sleep. TMR application during sleep leads to increased task-related brain network connectivity and behavioral performance in healthy young adults. Yet it remains unknown whether TMR can enhance sensorimotor performance in individuals with stroke. Methods. Healthy younger and older adults and individuals with chronic stroke were trained on a non-dominant (or non-paretic) UE throwing task before a period of waking or sleeping consolidation, with some receiving TMR throughout the consolidation period. Study 1 involved the use of TMR throughout the first two slow wave sleep periods over a full night of sleep with young adults. Studies 2, 3, and 4 investigated whether TMR throughout a one-hour nap was sufficient to influence sensorimotor performance in young adults, older adults, and people with a history of stroke, respectively. Results. All studies found that TMR application during sleep enhanced sensorimotor performance. In addition, TMR during wake did not influence sensorimotor performance (Studies 1 and 2), and enhanced performance of a cognitive aspect of the trained task (Study 2). Additional generalization and transfer tests helped to support the hypothesis that TMR enhanced a task-specific motor program, as improvements were seen within the trained task but not un-trained, but similar tasks. Lastly, sleep alone appears to stabilize sensorimotor performance variability, but this process demonstrates an age-related decline. Conclusion. This dissertation has shown that the use of TMR during sleep is a useful method for enhancing sensorimotor performance in healthy young and old adults, as well as individuals with a history of stroke. Future research may lead to an adjunct to traditional physical rehabilitation protocols.
  • Spatiotemporal Regulation of Myosin II Dynamics during Cell Movement

    Snell, Nicole; Rizzo, Mark A. (2019)
    The goal of this research was to investigate how the phosphorylation of non-muscle myosin II (NMMII) by myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) regulates cell motility. Cellular movement is important to both biological processes such as immune response, organism development, and axon guidance and to diseases such as cancer metastasis and hypertension. Movement requires a complex series of coordinated events involving the simultaneous buildup and tear down of the actomyosin cytoskeleton. The actomyosin cytoskeleton stabilizes cellular protrusions by joining with proteins in the extracellular matrix (EM), like fibronectin, and together they produce stress fibers that allow movement. NMMII regulatory light chain (RLC) phosphorylation at Serine 19 and Threonine 18 helps drive cell movement. Removing NMMII causes cells to lose structure and lose their migratory capabilities. Subsequently, phosphorylation allows NMMII to bind to actin filaments and create actomyosin crossbridges, the structural components, of the leading and lagging edges of moving cells. The dynamic activity of NMMII and MLCK at the leading edge remains undetermined in live cells, and it is also not well understood where MLCP influences cell movement on the motile edge. I investigated the moving edge of the cell using a multiparametric imaging approach with Förster resonance energy transfer (FRET) biosensors for NMMII, MLCK, and MLCP. Transfected NIH3T3 fibroblasts were imaged using fluorescence polarization microscopy. My results suggest that NMMII and MLCK activity are compartmentalized at the leading edge during cell motility and that there are differential phases of activity on a retracting membrane. We aim to understand the spatial relationship of NMMII phosphorylation with its regulators in different areas of the cell during movement. Taken together, this thesis work advances our understanding of non-muscle myosin II phosphorylation and regulation during random cell migration.
  • Computational Modeling of Behavior and Neural Mechanisms of Decision-Making Using Reinforcement Learning Theory

    Pietras, Bradley William; Schoenbaum, Geoffrey; Dayan, Peter, 1965- (2019)
    In the study of learning and decision-making in animals and humans, the field of Reinforcement Learning (RL) offers powerful ideas and tools for exploring the control mechanisms that underlie behavior. In this dissertation, we use RL to examine the questions of (i) how rats represent information about a complex, changing, task; (ii) what are the relevant variables underlying their decision-making processes; and (iii) whether those variables are encoded in the firing rates of neurons in the orbitofrontal cortex (OFC). We addressed these questions by making inquiries across three levels of understanding: computational theory, algorithmic representation, and physical implementation. Within this tri-level framework, we hypothesize that the subjects are engaged in a form of approximately optimal adaptive control. This involves their tracking critical, task-relevant, features of their environment as these features change, and then making appropriate choices accordingly. Two classes of RL algorithms were constructed, embodying different structural assumptions. One class of so-called return-based algorithms is based on elaborations of a standard Q-learning algorithm. The other, novel, class of income-based algorithms is based on a rather weaker notion of action-outcome contingency. Both classes of algorithm were parametrized and other factors were included such as perseveration. We t the algorithms to behavioral data from subjects using complexity-controlled empirical Bayesian inference. Internal variables from our algorithms were then used to predict neural ring rates of OFC neurons that were recorded as subjects performed the task. Linear regression, randomization testing, and false discovery rate analysis were used to determine statistically significant correlations between the predictors and neural activity. We found that income-class algorithms augmented with perseveration offered the best predictions of behavior. For the least restrictive statistical test (linear regression, p < 0.05), as many as 24% of the neurons were significantly correlated with variables associated with the best-fitting algorithm. By contrast, for our most restrictive test (randomized false discovery rate < 0.05), only 3% of the neurons passed as significant for one or more of our predictor variables. Other forms of neuronal dependence were apparent, including neurons that appeared to change their computational function dynamically depending on the state of the task.
  • Neuroprotective role of nicotinamide adenine dinucleotide precursor in modulation of mitochondrial fragmentation and brain energy metabolism

    Klimova, Nina; Kristian, Tibor (2019)
    Nicotinamide adenine dinucleotide (NAD+) is a central signaling molecule and enzyme cofactor that is involved in a variety of fundamental biological processes. NAD+ levels decline with age, neurodegenerative conditions, acute brain injury, and in obesity or diabetes. Loss of NAD+ results in impaired mitochondrial and cellular functions. Administration of NAD+ precursor, nicotinamide mononucleotide (NMN), has shown to improve mitochondrial bioenergetics, reverse age associated physiological decline, and inhibit post-ischemic NAD+ degradation and cellular death. In this work we identified a novel link between NAD+ metabolism and mitochondrial dynamics. A single dose (62.5mg/kg) of NMN, administered in naïve animals and after animals are subjected to transient forebrain ischemia, increases hippocampal mitochondria NAD+ pools and drives a sirtuin 3 (SIRT3) mediated global decrease in mitochondrial protein acetylation. This results in a reduction of hippocampal reactive oxygen species (ROS) levels via SIRT3 driven deacetylation of mitochondrial manganese superoxide dismutase. Consequently, mitochondria in neurons become less fragmented due to lower interaction of phosphorylated fission protein, dynamin-related protein 1 (pDrp1 (S616)), with mitochondria. In conclusion, manipulation of mitochondrial NAD+ levels by NMN results in metabolic changes that protect mitochondria against ROS and excessive fragmentation, offering therapeutic approaches for pathophysiologic stress conditions.
  • Development of the Drude Polarizable Force Field for Molecular Dynamics Simulation

    Lin, Fang-Yu; MacKerell, Alexander D., Jr.; 0000-0003-0103-0167 (2019)
    Molecular dynamics (MD) simulations have been widely applied to study biomolecular systems. While MD simulations have been used in a variety of applications, the accuracy of the results depends strongly on the force field (FF) used. The commonly used FFs are additive or non-polarizable FFs. However, one limitation of the additive FFs is the lack of electronic polarizability to treat molecules. To overcome this, an attractive approach is to introduce the explicit treatment of electronic polarizability into the potential energy function known as polarizable FFs. In Chapter 1, an overview of the CHARMM empirical FF as well as the development and application of the polarizable CHARMM FF based on the classical Drude oscillator is presented. As noncovalent halogen bonding interactions between halogenated ligands and proteins are important in drug design, a well-parametrized polarizable FF for halogen containing compounds is required to perform more accurate modeling of halogenated molecules. During the course of this development, a significant contribution of halogen-hydrogen bond donor (X-HBD) interactions to ligand-protein complexes was uncovered in addition to halogen bond (XB) interactions (Chapter 2). In Chapter 3, the development of halogen polarizable FF to both aliphatic and aromatic systems using halogenated ethane and benzene model compounds is illustrated, with emphasis on optimizing both X-HBD and XB interactions as discussed in Chapter 2. To assure good reproduction of X-HBD interactions with proteins, further optimization of atom pair-specific Lennard-Jones (LJ) parameters is required for both the polarizable and non-polarizable halogen FFs (Chapter 4). Finally, Chapter 5 presents the current optimization of polarizable protein FF to yield a more accurate description of the polarization response in simulations. The resulting protein FF, referred to as Drude-2019, will be more applicable in the study of biomolecular systems. Overall, the advances made in this dissertation will facilitate important discoveries of a range of chemical and biological phenomena.
  • Molecular Mechanisms Underlying the Metastasis Suppressor Activity of NME1 in Malignant Melanoma

    Pamidimukkala, Nidhi Vela; Kaetzel, David M. (2019)
    Metastatic melanoma is exceedingly lethal and is responsible for the majority of skin cancer related deaths. Impactful research on melanoma metastasis is crucial to improving prognosis. The discovery of metastasis suppressor genes, genes that inhibit metastasis but do not affect tumor growth, have advanced the field of metastasis research. NME1 (also referred to as NDPK-A or NM23-H1) was the first identified metastasis suppressor gene. NME1 is a multifaceted molecule, executing numerous cellular functions which are attributable to the suppressor phenotype. Our lab previously identified a signature of NME1-regualted genes which have prognostic value in human melanoma. As such, we hypothesize that NME1 possess an understudied transcriptional activity to regulate one or more of these signature genes. In the present study, we identified Aldolase C (ALDOC) from the gene signature as an ideal candidate to assess NME1 transcriptional activity. NME1 induced mRNA and protein expression of ALDOC in multiple melanoma cell lines. Transcriptional regulation was evidenced by elevated expression of ALDOC pre-mRNA and activation of the ALDOC promoter in NME1-expressing melanoma cells. Employing chromatin immunoprecipitation, we demonstrated NME1 localizes to the ALDOC gene and enriches the presence of active transcription indicators at the ALDOC promoter. Together, we provide novel systematic evidence for NME1 transcriptional activity, which may further be explored as a mechanism for the metastasis suppressor function. Furthermore, we establish unequivocal metastasis suppressor capability of NME1 using a transgenic mouse model susceptible to developing melanoma upon exposure to ultraviolet (UV) radiation. Mice deficient in NME1 expression experienced significantly increased melanoma metastases to the lung and lymph nodes compared to wild-type mice. Taken together, our studies demonstrate NME1 as a robust metastasis suppressor of UV-induced melanoma, and provide evidence for novel transcription factor activity of NME1. We aim to enhance our understanding of melanoma metastasis through our continued research of the metastasis suppressor gene, NME1.
  • Regulation of CD8+ T cell differentiation by cytokines and T-box transcription factors

    Reiser, John Wyatt; Singh, Nevil (2019)
    A protective immune response requires naïve CD8+ T cells to differentiate into effector cells, some of which persist as a memory population to protect against future threats. This differentiation program is controlled by a transcriptional network involving the T-box transcription factors T-bet and Eomesodermin (Eomes). These factors are in turn differentially regulated by cytokines in the local milieu. The precise architecture of this regulatory network in terms of the individual, synergistic and redundant roles of T-bet, Eomes and critical cytokines is still poorly understood. Here we sought to clarify these regulatory pathways, using mice deficient in T-bet, Eomes or both. Our studies identify a key role for Eomes, but not T-bet, in regulating IL-10 expression by CD8+ T cells early after activation. We further reveal a feedback loop where Eomes and IL-10 cooperatively enhance the expression of the lymph-node homing selectin CD62L. Consistent with the importance of CD62L in promoting memory T cell homing to secondary lymphoid organs (SLOs), we identify a role for CD8+ T cell-intrinsic IL-10 in promoting the long-term persistence of memory cells in vivo. In contrast, T cells that home away from the SLOs become terminal effector T cells. Using tumor and infectious disease models, we report that these cells experience a second wave of T-bet induction in the tissue, which may contribute to a heightened effector response. We also define distinct roles for T-bet and Eomes in regulating effector and memory genes, highlighting their redundant role in repressing Tc2 and Tc17 differentiation in CD8+ T cells. Finally, we demonstrate that in the context of a tumor-specific immune response, the absence of both T-box transcription factors severely limits tumor infiltration without significant alteration in early proliferation or effector functions. Taken together, these findings offer new insights into how T-bet, Eomes and IL-10 regulate effector and memory responses in distinct tissues during the immune response. Future developments targeting individual aspects of T-box and cytokine signaling in T cells may help engineer precise outcomes in the context of tumor immunotherapies, vaccination and transplantation.
  • Pseudomonas aeruginosa adaptation, pathogenicity, and persistence in the environment and cystic fibrosis airway.

    Chandler, Courtney; Ernst, Robert K.; 0000-0003-2076-3665 (2019)
    Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium associated with airway infections of patients with cystic fibrosis (CF). Lipid A is the membrane anchor of lipopolysaccharide, the dominant component of the outer leaflet of the outer membrane of Gram-negative bacteria. Lipid A structure can be modified via a number of biosynthetic enzymes. Here, we describe two -hydroxylase enzymes, LpxO1 and LpxO2, capable of catalyzing 2-hydroxylation of lipid A in P. aeruginosa. We additionally characterize their role in persistence and infectivity. Phylogenetic analysis suggests at least one lpxO originated from lateral gene transfer and the gene duplication is a recurring feature in Pseudomonas evolution. To determine the roles of LpxO1/2 in vivo, we used a rapid extraction from lavage fluid to describe the structure of P. aeruginosa lipid A isolated from the lungs of mice after intranasal infection. Lipid A is also known to be altered during adaptation to the lungs of CF patients, where P. aeruginosa can colonize and cause infection throughout a patient’s lifetime. This is a leading contributor of morbidity and mortality in this patient population, and thus the genetic and phenotypic adaptations that P. aeruginosa undergoes during CF airway infection are of interest. We used whole-genome sequencing of 130 P. aeruginosa isolates, including 81 from young children with CF, to define early-stage genetic adaptation events, including lipid A modification. We additionally investigate the influence of patient region of residence on such adaptation. With these data, we provide the first longitudinal analysis of P. aeruginosa genetics in young patients with CF in the United States. We additionally analyzed ten sublines of laboratory-adapted strain PAO1 to determine the level of microevolution experienced by this set of isolates. In total, our analyses contribute to our understanding of lipid A structure, synthesis, and modification in P. aeruginosa, and our sequencing data will serve as a resource for the entire CF and Pseudomonas community.
  • The Role of Testisin and PAR-2 Signaling in Ovarian Cancer Metastasis

    Conway, Gregory David; Antalis, Toni M. (2019)
    Ovarian cancer is the leading cause of death among gynecological cancers in the United States. Ovarian cancer employs a unique mode of metastasis, as tumor cells disseminate within the peritoneal cavity, colonizing in several sites and driving the accumulation of ascites. Tumor recurrence and metastasis are significant factors contributing to poor prognosis. The membrane-anchored serine protease testisin is aberrantly expressed in ovarian tumors and it’s only known substrate, protease activated receptor-2 (PAR-2), is also overexpressed in ovarian tumors. In this study I have examined 1) the role of testisin and PAR-2 signaling in ovarian tumor metastasis and 2) determined whether testisin and other membrane-anchored serine proteases may be anti-cancer therapeutic targets. We generated human ovarian ES-2 tumor lines that express testisin or the catalytically inactive testisin mutant S238A and explored the role of constitutive testisin expression in late stage ovarian cancer. We show that testisin stimulates the activation and internalization of PAR-2 resulting in decreased expression of ANG2 and ANGPTL4. Using a preclinical xenograft model of late stage ovarian cancer, we find that testisin activity in ovarian cancer cells reduces intra-peritoneal tumor dissemination, tumor burden and ascites formation. Analyses of the tumors showed that testisin activity downregulates the expression of ANG2 and ANGPTL4 in vivo as well as in vitro. To examine membrane-anchored serine proteases as therapeutic targets in cancer, we utilized an engineered anthrax toxin pro-drug strategy requiring proteolytic cleavage of a protective antigen (PrAg) for activation. We explored the efficacy of these engineered PrAg toxins in several intraperitoneal models of ovarian cancer metastasis and show PrAg treatment reduced tumor burden in both an intraperitoneal NCI/ADR-Res xenograft and an ES-2 minimal residual disease model, which replicates debulking surgery in ovarian cancer patients, but had no effect in a syngeneic ID-8-Luc xenograft model of ovarian cancer. Additionally, we found that engineered PrAg toxins are capable of inducing lung and pancreatic cancer cellular death in vitro. Data presented herein provide new insight into the potential role of testisin and PAR-2 signaling in ovarian cancer metastasis and suggests membrane-anchored serine proteases as a potential therapeutic target for metastatic ovarian cancer.
  • Characterization of PfCRT F145I in piperaquine-resistant Plasmodium falciparum isolates from Cambodia through zinc-finger nuclease-mediated gene editing

    Shrestha, Biraj; Takala-Harrison, Shannon (2019)
    ACTs are the first-line treatment for clinical malaria in the malaria-endemic world and have reduced malaria-associated mortality and morbidity. However, the recent emergence of Plasmodium falciparum that is resistant to both the artemisinins and key partner drugs, piperaquine, in Cambodia and nearby countries in GMS poses a threat to the control and elimination of malaria. Identification and validation of molecular markers of antimalarial drug resistance provide surveillance tools to monitor resistance and inform drug policy decisions and insights into the molecular mechanisms underlying resistance. Previous studies have found that F145I mutation within the PfCRT and plasmepsin2/3 gene copy number are associated with resistance to piperaquine. When PfCRT F145I is introduced into Dd2 of P. falciparum, it confers piperaquine resistance. In this study, we will use gene-editing approaches to remove F145I from field isolates that contain both this mutation and amplified plasmepsin2/3, to quantify the effect on malaria parasite susceptibility to piperaquine.
  • XIAP-p47 Pairing Activates the Immune Deficiency Pathway in the Lyme Disease Tick Ixodes scapularis

    McClure Carroll, Erin; Pedra, Joao H. F. (2019)
    Globally, vector-borne diseases account for 17% of all infectious diseases. Most vectors are blood-feeding arthropods, which transmit bacterial, viral, and parasitic diseases to humans and animals. The tick Ixodes scapularis transmits seven pathogens, including Borrelia burgdorferi, the agent of Lyme disease. Lyme disease is the most important vector-borne disease in the United States and causes an estimated 329,000 infections annually. Best described in the model organism Drosophila melanogaster, the arthropod immune deficiency (IMD) pathway responds to microbial infection through activation of Relish, a nuclear factor (NF)-κB family transcription factor. In I. scapularis ticks, the E3 ubiquitin ligase X-linked inhibitor of apoptosis (XIAP) regulates the IMD pathway through ubiquitylation. Yet, the tick genome notably lacks homologs to genes encoding key IMD pathway proteins as described in Drosophila. How XIAP activates the IMD pathway in response to microbial infection is poorly characterized and targets of XIAP-mediated ubiquitylation remain unknown. In this study, we identified the XIAP enzymatic substrate p47 as a positive regulator of the I. scapularis IMD network. XIAP polyubiquitylates p47 in a lysine (K)63-dependent manner and interacts with the ubiquitin-like (UBX) domain of p47. p47 also binds to Kenny (IKKγ/NF-κB essential modulator [NEMO]), the regulatory subunit of the inhibitor of NF-κB kinase (IKK) complex. Replacement of the amino acid lysine with arginine in the p47 linker region completely abrogated molecular interactions with Kenny. Furthermore, reduction of p47 transcription levels through RNA interference in I. scapularis limited Kenny accumulation, reduced phosphorylation of IKKβ (IRD5), and impaired cleavage of the NF-κB molecule Relish. Accordingly, disruption of p47 expression increased microbial colonization of the tick-transmitted spirochete B. burgdorferi and the rickettsial agent Anaplasma phagocytophilum. In summary, we demonstrated that XIAP ubiquitylates p47 in a K63-dependent manner, culminating in Relish activation and antimicrobial responses. Manipulating immune signaling cascades in I. scapularis may lead to innovative approaches to reducing the burden of tick-borne diseases.
  • Characterizing Genetic Drift and Migration between Populations

    Harris, Daniel Nathan; O'Connor, Timothy D. (2019)
    Genetic drift and migration are two of the main mechanisms of evolution at the genetic level. These mechanisms greatly impact the genetic relatedness of populations. Therefore, by studying how genetic drift and migration impact populations, it is possible to reconstruct elements of population history throughout a geographic region. This thesis examines how genetic drift and migration rapidly impact human population structure. My first aim studies the peopling of the Peruvian region and models migration patterns. Here, I suggest that the peopling of Peru was a rapid process that occurred by 12,000 years ago. Furthermore, migration modeling suggests that the majority of migration in Peru is from populations in the high altitude Andes Mountains to the low altitude coast and Amazon. My second aim, then analyzes genetic drift and migration on the fatty acid desaturase gene region, where there is a suggestion of a public health problem due to the apparent fixation of the low efficient ancestral haplogroup in Native Americans. I confirm the near fixation of the ancestral haplogroup in Native American ancestry, and suggest that understanding the frequency of the ancestral haplogroup in the Native American founding population is crucial for determining how this apparently detrimental haplogroup reached near fixation. Furthermore, I suggest that the global distribution of the haplogroup is not indicative of Neanderthal introgression and is instead likely an ancient polymorphism that arose during the divergence of modern humans and Neanderthals. For my final aim, I use rare variant and haplotype based approaches to discern the population structure and evolutionary history of Samoans. I suggest that the early population size was small and a strong population expansion occurred approximately 35 generations ago, consistent with the Samoan archaeological record. In addition, Samoan population structure is likely strongly driven by urbanization, and not biogeography. In sum, this thesis demonstrates that migration and genetic drift can rapidly impact the genetic variation in populations. In addition, I demonstrate that rare variant and haplotype based approaches are essential for examining the impact of migration and genetic drift between populations within an extremely recently founded region with minimal geographic barriers.
  • From Cell Fate to Terminal Differentiation – Insights into Inner Ear Hair Cell Development from Cell Type-Specific Transcriptomic Analyses

    Matern, Maggie S.; Hertzano, Ronna P. (2019)
    Hair cells (HCs), which are specialized neuroepithelial sensory cells of the inner ear, are responsible for conveying information about the environment, such as sound and movement, to the brain. In adult mammals, HCs do not spontaneously regenerate. Therefore, damage that occurs to HCs after noise or ototoxic drug exposure often leads to a permanent loss of inner ear function. It is estimated that 1/3 of individuals over the age of 65 experience hearing loss that could benefit from use of a hearing aid, representing a significant healthcare burden in the United States. It is for these reasons that the NIH National Institute on Deafness and Other Communication Disorders has set understanding the normal development of the auditory and vestibular systems as the Hearing and Balance Research Priority Area 1 of their 2017-2021 Strategic Plan, so that regenerative therapies can be better formulated. Here I have utilized cell type-specific ribosomal immunoprecipitation analyses to study HC gene expression, with the goal of furthering our understanding of their normal development. First, research into control of inner ear development has indicated that early HC differentiation appears to be dependent on the expression and normal functioning of the transcription factors ATOH1, POU4F3, and GFI1. While Pou4f3 and Gfi1 expression seems to be downstream of ATOH1, and the expression of Gfi1 is dependent on POU4F3, the signaling cascades downstream of GFI1 and its role in HC development are completely unknown. Here I have identified a critical role for GFI1 in HC maturation, namely through repression of a neuronal transcriptional program that is normally present in cochlear HCs during early embryonic development. Later in this developmental program, cochlear HC precursors must further differentiate into either inner or outer HCs, however, apart from the recently described INSM1 in embryonic development, no transcription factors have been implicated in the differentiation of outer HCs. It was therefore my second goal to identify and validate candidate downstream targets of helios, a newly identified postnatal outer HC transcription factor. Finally, I have developed and validated a model for ribosome immunoprecipitation and translatome analysis to study zebrafish HC gene expression – the Tg(myo6b:RiboTag) zebrafish.
  • Functional Status and Muscle Evaluation in Older Women with Urinary Incontinence.

    Sanses, Tatiana Vyacheslavovna Deniseiko; Ryan, Alice (2019)
    Background: Urinary incontinence (UI) and mobility limitations are prevalent in older women suggesting common pathophysiology. Objectives: 1) to evaluate Short Physical and Performance Battery (SPPB) and Modified Physical Performance Test (MPPT); 2) to describe lower extremities/pelvic floor muscles among older women with UI. Methods: Women age ≥ 70 years with UI underwent functional testing, clinical, and magnetic resonance imaging evaluation. Spearman correlation coefficients were calculated between UI and mobility limitations. Results: MPPT was inversely moderately associated with UI severity impact on daily activities. SPPB was negatively associated with UI but findings were not statistically significant. Levator ani muscles were atrophic in all participants. Obturator and gluteal muscles had mild and moderate fatty infiltration, respectively. Conclusions: Lower MPPT is associated with increased UI severity impact on daily activities. Further research into shared pathophysiology between UI and mobility
  • Molecular Determinants of Pulmonary Mucormycosis and Aspergillosis

    Watkins, Tonya Nicole; Bruno, Vincent, Ph.D. (2019)
    Mucormycosis and aspergillosis are invasive fungal infections with very limited treatment options and extremely high mortality rates. During both pulmonary mucormycosis and aspergillosis, inhaled fungal spores must adhere to and invade airway epithelial cells in order to establish infection. The molecular mechanisms governing these interactions in the context of each disease are not completely understood. Mucormycoses are caused by fungi belonging to the Order Mucorales, with ~70% of all cases caused by Rhizopus species. To better understand the molecular mechanisms of fungal invasion during mucormycosis, an unbiased survey of the host transcriptional response to Mucorales infection in in vitro and in vivo murine models of pulmonary mucormycosis using RNA-seq was performed. Network analysis revealed activation of host receptor tyrosine kinase (RTK) signaling pathways and progesterone (PG) signaling pathways. By combining established models of mucormycosis, transcriptomics, cell biology, and pharmacological approaches, we demonstrated that Mucorales activate epidermal growth factor receptor (EGFR), erb-b2 tyrosine kinase 2 (ErbB2), and platelet-derived growth factor receptor (PDGFR) signaling to induce fungal uptake into airway epithelial cells. Furthermore, we demonstrated that inhibition of EGFR signaling with existing FDA-approved cancer drugs significantly increased survival following Rhizopus delemar infection in mice. We also demonstrated that inhibition of progesterone receptor (PGR) signaling decreases R. delemar invasion of airway epithelial cells in vitro. These studies enhance our understanding of how Mucorales invade host cells during the establishment of pulmonary mucormycosis and provide a proof-of concept for repurposing FDA-approved cancer drugs that target RTK function. Aspergillus fumigatus is responsible for 90% of all aspergillosis cases. To better understand how A. fumigatus senses and responds to airway epithelial cells during pulmonary aspergillosis, we used RNA-seq to analyze the transcriptomes of two commonly used clinical A. fumigatus isolates, Af293 and CEA10, during an in vitro infection model of airway epithelial cells. In this analysis, we identified 47 genes that were up-regulated in both strains and enriched for genes involved in iron acquisition, a mechanism required for A. fumigatus virulence in the mammalian host. Knowledge gained from this work could aid in the identification of therapeutic and prevention targets to combat mucormycosis and aspergillosis.
  • Association between Iron Deficiency with or without Anemia and Infants’ Cognitive, Motor, and Socio-emotional Development: Cross-sectional and Longitudinal analyses

    Yimgang Ngandjouong, Doris P.; Black, Maureen M.; 0000-0002-3909-4839 (2019)
    Background: Early child development is a major public health concern worldwide as it has long-term health and economic effects that extend beyond childhood. Iron deficiency (ID) is the most common micronutrient deficiency worldwide with the highest prevalence in low- and middle-income countries. ID may impact child development early in life. Objectives of this study were to investigate the relationship between ID and child development and to identify moderators of this association among infants. Methods: Secondary data analyses of a nutritional and early learning controlled trial were performed. Participants: 513 children age 6-14 months from rural India were enrolled and followed for 12 months. Measures: At enrollment, 6-month and 12-month follow-up, child development was assessed using Mullen Scales for Early Learning (visual reception, fine/gross motor and receptive/expressive language domains) and Behavior Observation Inventory of the Bayley Scales of Infant and Toddler Development (socio-emotional domain). Analysis included linear mixed effects models accounting for village-level clustering (all aims) and repeated measures (Aims 2 and 3.1). To assess moderating effects of child age and sex, maternal education, household assets, and home environment, interaction terms were added in models. Results: At enrollment, 47% of infants were girls, 72% had ID, 66% had anemia, and the mean age was 8.6 months. Mean maternal age was 23 years and 43% of mothers were anemic. Adjusted analyses found weak to no evidence of associations between ID and child development at enrollment or over a 12-month period. Child age and sex, maternal education, household assets, and home environment did not moderate associations of interest. Conclusion and future directions: In this rural sample, ID was not associated with child development using observational study designs. In order to properly assess the onset and duration of ID and its associations with child development, further studies need to enroll pregnant women and follow up newborns during their first years of life. It is necessary to assess iron measures and child development at multiple time points throughout the study period. Implications: Because of the pervasive nature of ID in this population, there is a need for nutritional interventions to reduce micronutrient deficiencies.

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