Life Sciences Commons^™

Characterization And Functional Analysis Of Astrotactin 2 Reveals A Role For Endocytosis In Glial-Guided Neuronal Migration, Perrin Megan Wilson

Student Theses and Dissertations

The cerebellum is essential for learning coordinated movements, balance, eye movements, and aspects of sensory cognition. The remarkably uniform array of the principal neurons of the cerebellar circuitry, granule cells and Purkinje cells, develops by glial-guided migrations that position these cell types in specific neuronal layers. Classical EM studies and real time imaging of the migration of granule neurons along glial fibers established the mode of movement of migrating neurons, including the formation of an interstitial junction beneath the cell soma. Biochemical and molecular biological experiments demonstrated that neuronal protein astrotactin (ASTN1) functions as a neuron-glial ligand during migration. This …

The Multifunctional Capsid Gene Of Hepatitis C Virus, Laura Katherine Mcmullan

Student Theses and Dissertations

In populations worldwide, the Hepatitis C Virus displays great diversity. The capsid gene, however, is remarkably conserved and this has led to predictions of RNA secondary structure and an overlapping, alternative reading frame (ARF). We investigated a role of the ARF in HCV infection, by introducing four stop codons into the ARF of a genotype 1a H77 molecular clone. These changes did not alter the capsid protein sequence, but were predicted to disrupt the RNA secondary structures, SLV and SLVI. An infection was launched after inoculation of the mutant HCV RNA into an HCV naïve chimpanzee. The acute infection was …

Role Of Laminins In The Development Of The Peripheral Nervous System, Wei-Ming Yu

Student Theses and Dissertations

To investigate the function of laminins in peripheral nerve development, the laminin γ1 gene was specifically disrupted in Schwann cells. Disruption of laminin γ1 gene expression resulted in depletion of all other laminin chains known to be expressed in Schwann cells. Schwann cells lacking laminins fail to differentiate to myelinating and non-myelinating Schwann cells and do not extend processes required for initiating axonal sorting and mediating axon-Schwann cell interactions. These cells also fail to down-regulate Oct-6 and they arrest at the premyelinating stage. Impaired axon-Schwann cell interactions prevent phosphorylation of β-neuregulin-1 receptors, which results in decreased cell proliferation. Postnatally, laminin-null …

A Genetic-Proteomic Approach To Identify Cellular Components That Interact With Hiv-1, Yang Luo

Student Theses and Dissertations

Given the limited genetic coding capacity of HIV-1, it is reasonable to expect that the virus must interact with an extensive set of cellular factors and their complexes to complete its passage through the cell. Indeed, it is remarkable that the viral genome, comprising only about 0.0003% of the entire genetic capacity of the cell, commandeers the cellular environment to its own advantage. However, to date, only a small group of cellular proteins have been shown to be required for viral propagation. In an effort to recover and identify those host proteins that interact in complex with the viral machinery, …

Genetic And Molecular Characterization Of Programmed Cell Death In The C.Elegans Tail-Spike Cell, Carine Waase-Maurer

Student Theses and Dissertations

Work in Caenorhabditis elegans has been instrumental in deciphering the molecular basis of programmed cell death. However, despite extensive characterization of broadacting cell death genes, the molecular events triggering cell-specific activation of the cell death machinery remain, for the most part, unknown. In some C. elegans somatic cells, transcription of the egl-1 /BH3-only gene is believed to promoted cell-specific death. EGL-1 protein inhibits the CED-9/Bcl-2 protein, resulting in release of the caspase activator CED-4/Apaf-1. Subsequent activation of CED-3 caspase by CED-4 leads to cell death. But despite the important role of egl-1 transcription in promoting CED-3 activity in cells destined …

Autophagy Delivers Viral Antigens For Mhc Class Ii Presentation And Is Regulated By Viral Infection, Dorothee Schmid

Student Theses and Dissertations

MHC class II molecules generally present peptides derived from exogenous antigens after endocytosis. However, biochemical studies have revealed that MHC class II ligands are frequently derived from intracellular proteins after endogenous processing. Endogenous MHC class II antigen presentation has been described for viral and model antigens and might represent an important mechanism to initiate CD4+ T cell responses to intracellular pathogens. We studied this unusual MHC class II presentation pathway using the Epstein- Barr virus nuclear antigen 1 (EBNA1) as a model antigen with relevance for human disease. We found that EBNA1 was degraded by lysosomal proteases and detected EBNA1 …

A Search For The Counter-Immune Mechanisms Of Mycobacterium Tuberculosis, Meghan Alida Kirksey

Student Theses and Dissertations

Tuberculosis is characterized by dynamic interactions between M. tuberculosis (Mtb) and the human immune response. The cytokine IFN-γ triggers macrophage production of bactericidal nitric oxide by inducible nitric oxide synthase (NOS2) and is essential for mammalian control of Mtb infection. Mice lacking NOS2 are unable to control replication of Mtb and rapidly succumb to infection. The persistent nature of TB infection suggests that Mtb has evolved counter-immune mechanisms to survive in the face of NOS2 and other pathways downstream of IFN-γ. A differential signature-tagged transposon mutagenesis screen was conducted to identify mutants attenuated in NOS2-/- mice, but retaining virulence in …

Chronic Stress Effects On Prefrontal Cortical Structure And Function, Conor Liston

Student Theses and Dissertations

Stressful life events have been implicated clinically in the pathogenesis of major depression, but the neural substrates that may account for this observation remain poorly understood. Attentional impairments symptomatic of depression are associated with structural and functional abnormalities in the prefrontal cortex. In three parallel rodent and human neuroimaging studies, this project assessed the effects of chronic stress on prefrontal cortical structure and function and the behavioral correlates of these changes. The first study used fMRI to elucidate the precise computational contributions of frontoparietal circuitry to attentional control in human subjects, using a task that could be adapted for rats. …

The Role Of Microglia In The Effects Of Steroid Hormones On Brain Inflammation, Andres C. Gottfried-Blackmore

Student Theses and Dissertations

The conditions defining whether microglial activation is detrimental or beneficial to neuronal survival are still poorly understood. Better understanding of the factors regulating microglia activation may lead to improved therapies for neurodegenerative diseases. Clinical and animal studies point to the neuroprotective and anti-inflammatory effects of steroid hormones. However, our comprehension of the cellular targets and mechanisms of action of these hormones in the CNS is still unresolved. In view of these limitations, the main question addressed in this dissertation was the role that microglia play in the anti-inflammatory effects of steroid hormones in the brain, with particular emphasis on the …

Comparative Studies Of Processivity Clamp Loader Atp Site Function., Aaron M. Johnson

Student Theses and Dissertations

DNA replicases utilize ring-shaped sliding clamps to ensure polymerase processivity. An ATP-dependent clamp loader topologically links the clamp around DNA in a multi-step mechanism. Clamp loaders are ring-shaped pentamers of AAA+ subunits. The pentamer complex contains 3-4 ATP sites, each located at the interface of two subunits. In each ATP site, an arginine residue from one subunit is located near the γ-phosphate of ATP bound to the adjacent subunit. These arginines act as “arginine fingers” that can potentially perform two functions: sensing that ATP is bound and catalyzing ATP hydrolysis. This thesis utilizes mutations in the arginine fingers of the …

Targeted Disruption Of Lynx2 Reveals Distinct Functions For Lynx Homologues In Learning And Behavior, Ayse Begum Tekinay

Student Theses and Dissertations

Endogenous short peptide modulators of ion channels provide a new level of regulation of nervous function. Lynx1 was identified as an endogenous mammalian homologue of snake venom peptide neurotoxins capable of binding to and functionally modulating nicotinic cetylcholine receptors (nAChR). Lynx1 is a member of the Ly6-α−neurotoxin superfamily (Ly6SF) of genes. Through extensive database searches, I identified 85 members of this superfamily including previously unidentified vertebrate and invertebrate family members. I show that these proteins are very divergent in their sequences, and identify two conserved subfamilies, snake toxins and immune system expressed Ly6 genes through phylogenetic inference. I also discovered …

From Naturally Occurring Tumor Immunity To Supernatural T Cells: Isolation And Characterization Of A Murine T Cell Receptor Specific For Human Breast And Ovarian Tumor Antigen Cdr2, Bianca D. Santomasso

Student Theses and Dissertations

Patients with paraneoplastic cerebellar degeneration (PCD), a form of neuronal autoimmunity, have a co-occurring natural immune response against a protein called cdr2 in their breast and ovarian carcinomas, and thus provide an innovative starting point for understanding how to harness the immune system to fight cancer. We previously demonstrated cdr2-specific cytotoxic T lymphocytes (CTL) in the peripheral blood of HLA-A2.1+ PCD patients, suggesting that CTLs mediate tumor immunity in these patients. Cdr2 is expressed by a large proportion of breast and ovarian tumors from individuals who do not develop neurological disease, suggesting that immune responses to this antigen may develop …

Genetic And Biochemical Analyses Of The Flaviviridae Capsid Proteins, Catherine Lucy Murray

Student Theses and Dissertations

The small, enveloped viruses of the family Flaviviridae are etiological agents of numerous important human and agricultural diseases including hepatitis C, yellow fever, and bovine viral diarrhea. Efficient dissemination of these viruses is dependent on the production of infectious particles, thought to arise by budding of the capsid protein and associated genomic RNA through a host cell-derived lipid membrane outfitted with envelope glycoproteins. The process of virion morphogenesis is not well understood, but the presumed involvement of numerous viral and cellular components makes it an attractive target for novel therapeutic drug design. To investigate the early events of Flaviviridae particle …

Haplotype-Based Association Studies: Approaches To Current Challenges, Mark A. Levenstien

Student Theses and Dissertations

Haplotype-based association studies have greatly aided researchers in their attempts to map genes. However, current designs of haplotype-based association studies lead to several challenges from a statistical perspective. To reduce the number of variants, some researchers have employed hierarchical clustering. This thesis starts by addressing the multiple testing problem that results from applying a hierarchical clustering procedure to haplotypes and then performing a statistical test for association at each of the steps in the resulting hierarchy. Applying our method to a haplotype case-control dataset, we find a global p-value. Relative to the minimum p-value over all steps in the hierarchy, …

A Structural Basis For Host Cytoskeletal Disruption And Virulence By Yersinia Protein Kinase A, Gerd Prehna

Student Theses and Dissertations

Yersinia spp. cause gastroenteritis and the plague, representing historically devastating pathogens that are currently an important biodefense and antibiotic resistance concern. Although several antibiotic therapies exist, the emergence of strains that have garnered multiple drug resistances in combination with the weaponization of Yersinia, make understanding the biology of this pathogen a high priority. Yersinia, along with other pathogenic bacteria such as Salmonella, utilize a macromolecular complex, called a type III secretion apparatus, to deliver virulence proteins directly into cells. These factors commandeer several signaling pathways, often targeting the Rho family of small GTPases which regulate actin cytoskeletal dynamics. A critical …

Insect Host Seeking: Investigations Into The Molecular Mechanisms Of Chemosensation, Walton D. Jones

Student Theses and Dissertations

Among other functions, chemosensory systems play a crucial role in the host-seeking behaviors of insects that allow them to find their preferred food sources. Quite often, however, these host-seeking behaviors have a negative impact on either human health or livelihood. The following details investigations into the evolution and molecular mechanisms of two distinct pathways involved in insect chemosensation. The atypical odorant receptor gene, Or83b, is co-expressed with other ORs in most olfactory sensory neurons (OSNs) of the Drosophila antenna. OR83b acts as a generic heterodimeric partner for other ORs coupling them to the ciliary trafficking machinery, which is responsible for …

A Map Of Transcription Factor Expression In Neuronal Precursors Of The Chicken And Mouse Embryonic Cerebellum, Daniver Morales

Student Theses and Dissertations

The cerebellum is comprised of an overlying cortical structure and a basal set of cerebellar nuclei (CN). The cerebellar cortex contains two principal neurons, the Purkinje cell (PC) and the granule cell (GC), whereas the CN are formed by an heterogeneous array of cerebellar nuclei neurons. Although granule cell development is well-studied, the specification and early migratory routes of cerebellar nuclei neurons and Purkinje cells are not well understood. To map neural progenitor identity, we analyzed the expression of various members of the TALE, LIM and bHLH transcription factor families in the avian and murine cerebellar anlagen. We show here …

Regulation Of Histone Covalent Modifications During Yeast Apoptosis, Sung Hee Ahn-Upton

Student Theses and Dissertations

Chromatin compaction is a hallmark property of apoptosis, a highly coordinated suicide mechanism generally believed to be confined to vertebrates. However, invertebrates such as the budding yeast, Saccharomyces cerevisiae, display an apoptotic-like phenotypes including chromatin condensation, although its functions and mechanism are unclear. One mechanism that alters chromatin structure is the covalent modification of histones, which associates with DNA to form the nucleosome, the fundamental unit of chromatin. Phosphorylation of histone H2B at serine 14 (H2BS14ph), catalyzed by Mst1 kinase, has been linked to chromatin compaction during mammalian apoptosis. I extended these results to yeast by demonstrating that Ste20 kinase, …

Molecular Regulation Of Hair Follicle Morphogenesis, Horace Rhee

Student Theses and Dissertations

The skin epidermis develops from a single uniform layer of multipotent cells during embryogenesis. Morphogenesis of hair follicles is initiated when a series of reciprocal interactions between this undifferentiated ectoderm and its underlying mesenchyme leads to a localized invagination of epithelial cells. As hair follicle development proceeds, stem cells are specified and set aside to fuel the postnatal hair cycle and repair the epidermis after injury. Although many signaling pathways have been identified by genetic studies to be essential for the induction of hair follicle morphogenesis and differentiation, little is known about the downstream genetic targets and molecular mechanisms in …

Dissecting The Entry Pathways Of Hepatitis C And Bovine Viral Diarrhea Viruses, Donna M. Tscherne

Student Theses and Dissertations

Hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV) are closely related members of the family Flaviviridae and are important human and animal pathogens, respectively. In this work, I investigated how these viruses interact with and enter both naïve and previously infected cells, specifically, the mechanisms of superinfection exclusion, the phenomenon by which previous viral infection prevents reinfection of the same cell, and the pathways of entry into target cells for these viruses. BVDV-acutely infected cells establish two blocks to superinfection, at the levels of virus entry and RNA replication. The former is mediated by the BVDV E2 protein, …

Energy Homeostasis During Fasting And Fed States: Foxa2 Regulates Hepatic Amino Acid Uptake, Vivian M. Lee

Student Theses and Dissertations

In MODY3 patients, as well in the experimental mouse model, the Tcf1 -/- mouse, there is a defect in arginine-induced insulin secretion. In this thesis, we examined the role of arginine transporters in the insulin secreting cells in arginine-induced insulin secretion. We first characterized arginine uptake by MIN6 cells as having a Km of 102.6 μM and being partially Na-dependent and entirely Cl-dependent. We then examined Tcf1 -/- pancreatic islets, which are defective for arginine-induced insulin secretion. Using gene expression array analysis and semi-quantitative RT-PCR analysis on pancreatic islets from Tcf1 +/+ and Tcf1 -/- mice, we found eight arginine …

Death By Frustration: How Defects In Cell Differentiation Trigger Apoptosis, Ann H. Tang

Student Theses and Dissertations

Apoptosis plays a crucial role in the growth, development, and homeostasis of multicellular organisms by removing excess cells and sculpting tissues. The implications of its misregulation in diseases such as cancer, neurodegeneration, and autoimmunity bespeak of its importance. A family of proteases called caspases brings about the orderly deconstruction of a cell through an evolutionarily-conserved process. A bewildering number of molecules have been implicated to either directly or indirectly regulate these enzymes. Since the net outcome between inhibitory and activational mechanisms ultimately determines a cell’s fate, caspases and their regulation still continue to be subjects of intense study. Studies with …

The Role Of Histone H2b Ubiquitylation And Its Related Factors In Transcriptional Regulation In Mammalian Cells, Jaehoon Kim

Student Theses and Dissertations

Diverse histone modifications such as acetylation, methylation, and phosphorylation play important roles in transcriptional regulation throughout eukaryotes, and recent studies in yeast also have implicated H2B ubiquitylation in the transcription of specific genes. However, a systematic study of H2B ubiquitylation in mammalian cells has been hindered by the lack of information about mammalian homologues of the yeast enzymes responsible for H2B ubiquitylation. I report identification of a functional human homologue, the hBRE1A/B complex, of the yeast BRE1 E3 ubiquitin ligase. hBRE1A, which forms a complex with hBRE1B, specifically increases the global level of H2B ubiquitylation at lysine 120 and enhances …

The Role Of Activation-Induced Cytidine Deaminase In Antibody Diversification And B Cell Malignancies, Eleonora Market

Student Theses and Dissertations

Activation-induced cytidine deaminase (AID) is indispensable for somatic hypermutation (SHM) and class switch recombination (CSR) in B cells. SHM alters the affinity of an antibody for its corresponding antigen by introducing point mutations within the variable region of the immunoglobulin (Ig) gene heavy chain locus. CSR replaces the Ig μ constant region with one of the downstream constant region, thereby changing the physical properties of the antibody. To study the molecular mechanism of AID in these two antibody diversification events, I cloned, expressed and purified the recombinant AID protein from E. coli and showed that it can deaminate cytidines within …

Kh Domains On Brain Polyribosomes: Fmrp And Nova In Translational Regulation, Claire Elizabeth Fraser

Student Theses and Dissertations

The regulation of protein synthesis is an important aspect of the control of gene expression in neurons and is thought to contribute to neurologic diseases such as Fragile X mental retardation syndrome. We demonstrate that several neuronal RNA-binding proteins implicated in human disease are associated with brain polyribosomes, namely the Nova and Hu paraneoplastic antigens and the Fragile X mental retardation protein. We use microarray analysis of polyribosomal mRNAs in knockout mouse models of these diseases to identify target mRNAs and analyze the translational profiles of mice lacking Nova-1 or FMRP. The KH2 and RGG box RNA-binding domains of FMRP …

The Role Of Pot1 In Telomere Protection And Maintenance, Dirk Hockemeyer

Student Theses and Dissertations

POT1 is a single stranded telomeric DNA binding protein implicated in telomere length regulation in human cells. To address the role of POT1 in telomere protection we used RNAi in human cells. We confirmed that POT1 acts as a negative regulator of telomerase and showed that POT1 is required to protect telomeres. Reduced levels of POT1 elicited a strong telomere DNA damage response and a growth arrest in primary cells; the 3’ telomeric overhang shortened and the sequence of the 5’ terminus changed from its precise sequence (ATC-5’) to a randomized ending. In order to determine the phenotype of complete …

A New Type Of Programmed Cell Death In C. Elegans, Mary C. Abraham

Student Theses and Dissertations

The most intensively studied form of programmed cell death (PCD) is apoptosis,

which is characterized by stereotypical morphological features including

chromatin compaction and by a requirement for the activity of caspase proteases,

which are controlled by conserved gene pathways. Although non-apoptotic,

caspase-independent programmed cell death pathways have been postulated,

there is little evidence to convincingly prove their existence, and few insights

regarding their molecular basis or possible

in vivo functions. To investigate this

question, we have studied the developmentally regulated PCD of the

Caenorhabditis elegans

linker cell.

We have carried out transmission electron microscopy studies of dying

linker cells, which …