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Combined Therapy Of Dietary Fish Oil And Stearoyl-Coa Desaturase 1 Inhibition Prevents The Metabolic Syndrome And Atherosclerosis, J. Mark Brown, Soonkyu Chung, Janet K. Sawyer, Chiara Degirolamo, Heather M. Alger, Tam M. Nguyen, Xuewei Zhu, My-Ngan Duong, Amanda L. Brown, Caleb Lord, Ramesh Shah, Matthew A. Davis, Kathryn Kelley, Martha D. Wilson, Jennifer Madenspacher, Michael B. Fessler, John S. Parks, Lawrence L. Rudel

Department of Nutrition and Health Sciences: Faculty Publications

Background—Stearoyl-CoA desaturase 1 (SCD1) is a critical regulator of energy metabolism and inflammation. We have previously reported that inhibition of SCD1 in hyperlipidemic mice fed a saturated fatty acid (SFA)-enriched diet prevented development of the metabolic syndrome, yet surprisingly promoted severe atherosclerosis. In this study we tested whether dietary fish oil supplementation could prevent the accelerated atherosclerosis caused by SCD1 inhibition.

Methods and Results—LDLr^-/-, ApoB^100/100 mice were fed diets enriched in saturated fat or fish oil in conjunction with antisense oligonucleotide (ASO) treatment to inhibit SCD1. As previously reported, in SFA-fed mice, SCD1 inhibition dramatically protected against …

Discovery, Steinman Laboratory

Introduction to Dendritic Cells

Introduction to Dendritic Cells: Discovery

Proteome-Wide Prediction Of Acetylation Substrates, Amrita Basu

Student Theses and Dissertations

Eukaryotic DNA is found packaged with proteins and RNA, which forms a substance called chromatin. This packaging is dynamic and regulates access to DNA for essential cellular processes such as transcription, replication, and repair. In recent years, studies have shown that regulated changes in the chemical and physical properties of chromatin often lead to dynamic changes in multiple cellular processes by affecting the accessibility of the DNA. These changes can be brought about in part through posttranslational modifications of histone proteins, which are involved in disrupting chromatin contacts or by recruiting effector proteins to chromatin. Acetylation is one of the …

Characterization Of The Functional Role Of The Intracellular Cholesterol Transporter Stard4 In Knockout Mice, And Investigation Of Epigenetic Modulation Of Apoa-I Transcription, Joshua Riegelhaupt

Student Theses and Dissertations

Cholesterol is crucial for mammalian survival by playing important roles, such as regulating membrane fluidity and as a precursor for the synthesis of steroid and sex hormones, bile acids, and Vitamin D. In addition, cellular and organismal regulation of cholesterol is important for health. For example, increased levels of plasma LDL cholesterol are a risk factor for coronary heart disease and stroke. Intracellular cholesterol levels are regulated by a variety of mechanisms, but numerous studies indicate a very important role for transcriptional regulation by Sterol Regulatory Binding Proteins (SREBPs), Liver X Receptors (LXRs) and Unfolded Protein Response (UPR) or ER …

Structural Studies On The Regulation Of Sporulation In Bacillus And Antibiotic Sulfation, Matthew J. Bick

Student Theses and Dissertations

The microbial world is rich with complexity. Bacteria have evolved several mechanisms to help them survive the harshest environments on earth. These range from complex developmental transformations to the synthesis of small molecule antibiotics that can fend off competing species. Both of these topics will be the focus of this thesis, specifically the regulation of endospore formation in Bacillus and the generation of a sulfated small molecule antibiotic from an uncultured soil bacterium. Using standard molecular biology methods and the techniques of X-ray crystallography, synthetic chemistry, and protein semisynthesis, we have: (1) Solved the X-ray crystal structure of a Geobacillus …

Mechanistic Insights Into The Stimulation Of Dot1l-Mediated Methylation Of Histone H3 By Semisynthetically Ubiquitylated Histone H2b, Robert Kendall Mcginty

Student Theses and Dissertations

Post-translational modification of histones plays an integral role in regulation of chromatin-templated processes through modulation of chromatin structure and function. One such modification, ubiquitylation of histone H2B on lysine 120 (uH2B), has been correlated with enhanced methylation of lysine 79 (K79) of histone H3 by K79-specific methyltransferase, disruptor of telomeric silencing-like (Dot1L/KMT4). However, the specific function of uH2B in this crosstalk pathway was not understood, in part due to the challenges associated with isolating or generating homogeneously ubiquitylated H2B for use in biochemical studies. As both modifications are integral to transcriptional regulation and DNA damage repair, full elucidation of their …

Synaptic Specificity In The Zebrafish Lateral Line, Aaron Nagiel

Student Theses and Dissertations

The proper wiring of the vertebrate brain represents an extraordinary developmental challenge, requiring billions of neurons to select their appropriate synaptic targets. In view of this complexity, simple vertebrate systems provide necessary models for understanding how synaptic specificity arises. The posterior lateral-line organ of larval zebrafish consists of polarized hair cells organized in discrete clusters known as neuromasts. Here I show that each afferent neuron of the posterior lateral line establishes specific contacts with hair cells of the same hair-bundle polarity. I quantify this specificity by modeling the neuron as a biased selector of hair-cell polarity and find evidence for …

Genome-Scale Genetics: Lessons From Founder Populations, Eimear Elizabeth Kenny

Student Theses and Dissertations

The potential benefits of using population isolates in genetic mapping due to reduced genetic and environmental heterogeneity are offset by the challenges posed by these populations for traditional association methods. Population isolates often contain large amounts of direct and cryptic relatedness that confound baseline assumptions of independence among genotypes and phenotypes and require specialized approaches to account for this sample structure. We examined three such approaches for association testing: (i) scoring allele transmission to offspring within families (ii) incorporating a permutation-based association score between families into the test statistic and finally (iii) incorporation of a kinship matrix to capture the …

Structural And Molecular Correlates Of Individual Differences In Anxiety Behavior And The Stress Response, Melinda Miller

Student Theses and Dissertations

Although many people are exposed to stressful experiences during their lifetime, only 5-35% will be diagnosed with Post-Traumatic Stress Disorder (PTSD). While most animal models of stress are effective at producing behavioral changes in an entire group of animals, they fail to account for individual differences in the human stress response or for the variability in baseline anxiety. Animal models of acute and chronic stress increase anxiety behavior and induce structural and neurochemical changes in brain regions necessary for learning, memory, fear responses, and executive function; the hippocampus, amygdala and medial prefrontal cortex (mPFC). In the following experiments, we measured …

Variability And Dynamics Of Apoptotic Events In Single Cells, Patrick Bhola

Student Theses and Dissertations

Apoptosis is a form of programmed cell death that is necessary for development and organism homeostasis. The early stages of apoptosis are marked by the permeabilization of the mitochondrial outer membrane, the release of cytochrome c and Smac from mitochondria, and the activation of the caspase family of proteases. The permeabilization of the mitochondria outer membrane is thought to represent the commitment of a cell to an apoptotic fate, and the subsequent release of mitochondrial proteins is an "all or none" phenomenon that is completed in an individual cell within minutes. Although the cellular decision to commit to apoptosis is …

The Impact Of Host Factors On Retroviral Evolution And The Identification Of A Novel Receptor That Was Used By An Ancient Primate Retrovirus, Steven J. Soll

Student Theses and Dissertations

The resurrection of inactive endogenous retroviruses allows us to learn about interactions between extinct pathogens and their hosts that occurred millions of years ago. Two of these paleoviruses, chimpanzee endogenous retrovirus 1 and 2 (CERV1 and CERV2), are relatives of modern murine leukemia viruses that are found in the genomes of a variety of old world primates, but are absent from the human genome. The nonexistence of human CERV1 and CERV2 homologues is peculiar given the numerous apparent cross-species transmissions that occurred between ancestors of old world monkeys, gorillas, and chimpanzees. It is possible that antiviral proteins were able to …

Regulation Of Anaphase Promoting Complex Coactivators, Jonathan Robbins

Student Theses and Dissertations

Ubiquitin-mediated proteolytic degradation is fundamental to eukaryotic cell cycle progression. From late mitosis through early G1, the Anaphase Promoting Complex (APC) is essential for cell-cycle relevant proteolytic degradation, and its activity is targeted to appropriate substrates by the evolutionarily conserved coactivators Cdc20 and Cdh1. After an initial wave of APC-Cdc20 activity, APC-Cdh1 degrades multiple mitotic proteins from mitotic exit through G1; inhibitory phosphorylation of Cdh1 by CDK and Polo kinase may allow accumulation of Cdh1 targets in the subsequent cell cycle. I demonstrate lethality of exact endogenous gene replacement of CDH1 with the CDK-unphosphorylatable CDH1-m11 allele; neither polo kinase sites …

Genome-Wide Decoding Of Mrnp And Mirna Maps, Sung Wook Chi

Student Theses and Dissertations

The limited number of primary transcripts in the genome has promoted interest in the possibility that much of the complexity in the regulation of gene expression may be determined by RNA regulation controlled by RNA-binding proteins (RNABPs) and/or microRNAs (miRNAs). However, applying biochemical methods to understand such interactions in living tissues is major challenge. Here we developed a genome-wide means of mapping messenger ribonucleoprotein (mRNP) sites in vivo, by high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). HITS-CLIP analysis of the neuron-specific splicing factor Nova provides genome-wide maps of Nova-RNA interactions in vivo and leads to a new finding …

Unzipping Amyloid Fibrils: How A Novel Calcium-Binding Protein, Nucb1, Prevents The Formation Of Amyloid Fibrils, Neeraj Kapoor

Student Theses and Dissertations

Nucleobindin 1 (NUCB1) is a widely expressed multi-domain Ca²⁺-binding protein whose precise physiological and biochemical functions are not well understood. We engineered and heterologously expressed a soluble form of NUCB1 (sNUCB1) and characterized its biophysical and biochemical properties. We show that sNUCB1 exists as a dimer in solution and that each monomer binds two divalent Ca²⁺ cations. Ca²⁺-binding causes conformational changes in sNUCB1 as judged by circular dichroism and fluorescence spectroscopy experiments. Earlier reports suggested that NUCB1 might interact with heterotrimeric G protein α-subunits. We show that dimeric Ca²⁺-free sNUCB1 binds to heterologously …

Biochemical And Structural Analysis Of The Nucleoporin Nup214 And Its Involvement In Mrna Export, Johanna Napetschnig

Student Theses and Dissertations

In order to gain a deeper understanding of the role of nups in leukemogenesis, and to make sense of the architecture and regulation of the mRNA export machinery at the NPC, I set out to biochemically and structurally characterize Nup214. In this thesis, I present the crystal structure of the human Nup214 N-terminal domain at 1.65 Å resolution. The structure reveals a sevenbladed β-propeller fold followed by a 30-residue C-terminal extended peptide segment (CTE). The CTE folds back onto the β-propeller and binds to its bottom face. Conserved surface patches on the Nup214 NTD reveal putative proteininteraction sites, one of …

Mitotic Exit Control In Budding Yeast: Regulators And Dynamics, Ying Lu

Student Theses and Dissertations

In budding yeast, the phosphatase Cdc14 is released from nucleolus to promote mitotic exit (ME). Cdc14 release and ME is controlled by mitotic cyclin-Cdk oscillation, the FEAR network including a non-proteolytic function of separase (Esp1), and the Mitotic Exit Network (MEN) indirectly activated by spindle elongation through cohesin cleavage by the proteolytic function of Esp1. The MEN contributes strongly to ME efficiency. Esp1 contributes to Cdc14 release and ME kinetics mainly through cohesin cleavage: the Esp1 requirement can be largely bypassed if cells are provided Esp1-independent means of separating sister chromatids. In the absence of Esp1 activity we observed only …

Mechanisms Of Presynaptic Cav2 Calcium Channel Localization In Caenorhabditis Elegans, Yasunori Saheki

Student Theses and Dissertations

Neurotransmitter release at nerve terminals is a fundamental aspect of communication in the nervous system. Voltage changes in the presynaptic membrane are sensed by presynaptic voltage-gated calcium channels that mediate calcium influx at the nerve terminals to execute exocytosis of various kinds of neurotransmitters. Defects in presynaptic calcium channels lead to many neurological disorders, emphasizing the importance of these channels in the regulation of neuronal activities in the brain. Previous physiological studies have focused on opening kinetics of the channels and their modulation by auxiliary subunits. Cell biological questions such as trafficking and clustering of the channels at the presynaptic …

Localization And Function Of Rna Interference In The Cerebral Cortex, John T. G. Pena

Student Theses and Dissertations

During the course of adult cortical plasticity, a number of signal transduction mechanisms are brought into play. To study regulatory genes implicated in this process, we inhibited gene expression by harnessing the machinery of RNA interference (RNAi) pathway via small hairpin RNAs (shRNA) delivered by viral vectors. Using this technology we sought to influence plasticity of the mouse vibrissal barrel cortex. In this model system, chronic whisker plucking reliably leads to the expansion of the cortical representation of the adjacent non-deprived whiskers. The mechanism underlying this process involves changes in synaptic efficacy and sprouting of axon collaterals. Initial studies describing …

Structural And Functional Characterization Of B. Anthracis Udp-Glcnac 2-Epimerase And Salmonella Secreted Effector I, Shyam Bhaskaran

Student Theses and Dissertations

Structural biology has been an exceptionally powerful tool in the study of bacterial pathogenesis, not only in elucidating the molecular basis of disease as the first step towards the design of therapeutics but also in many cases leading to insights into hostpathogen biology that would not have been possible otherwise (Remaut & Waksman 2004). In this work such an approach has been taken towards the study of the Bacillus anthracis cell-wall enzyme UDP-GlcNAc 2-Epimerase and the Salmonella secreted virulence effector SseI. UDP-GlcNAc 2-Epimerase is an essential B. anthracis enzyme required for formation of the peptidoglycan cell wall precursor UDP-ManAc. We …

Regulation Of Chromosome Segregation By The Chromosomal Passenger Complex, Boo Shan Tseng

Student Theses and Dissertations

Life depends on faithful DNA segregation. The molecular underpinnings controlling this segregation are not fully understood. Here I describe the role of the chromosomal passenger complex (CPC) in the regulation of chromosome segregation, using Xenopus egg extracts. The CPC contains the kinase Aurora B, which is well known for its phosphorylation of serine 10 on histone H3 (H3S10). While this phosphorylation is a hallmark of M-phase, its functional significance is enigmatic. In the first part of my thesis, I, with my collaborators, provide a molecular function for this phosphorylation in the chromosomal dissociation of HP1 (heterochromatin protein 1) in M-phase. …

Identification And Characterization Of Apobec1 Mrna Editing Targets: A Transcriptomics Approach, Brad Randall Rosenberg

Student Theses and Dissertations

RNA editing is generally defined as the alteration of an RNA sequence from that encoded by the genome through nucleotide insertion, deletion or modification. The Apolipoprotein B mRNA Editing Catalytic polypeptide 1 (APOBEC1) cytidine deaminase is an mRNA editing enzyme that modifies a specific cytidine in the apolipoprotein B (apoB) transcripts of small intestine enterocytes. APOBEC1-mediated cytidine to uridine editing generates an inframe stop codon and results in translation of a truncated apoB isoform with distinct functions in lipid transport. Other physiological mRNA targets of APOBEC1 editing have remained largely unknown. This thesis presents the development of an RNA-Seq method …

Lipid Chemistry And Mechanical State Of The Membrane Modulate Ion Channel Function, Daniel Schmidt

Student Theses and Dissertations

My research focused on voltage-dependent K⁺ (Kv) channels. Kv channels serve many different functions in different cells, but most notably underlie action potentials in electrically excitable cells, such as neurons and muscle (Hodgkin and Huxley, 1952, 1945). Kv channel gating is governed by the transmembrane voltage, they are therefore voltage-dependent switches for ionic current (Hille, 2001). Changes in the transmembrane voltage are sensed by the channel's voltage sensor domains, which contain charged amino acids (most often arginines) called gating charges. Shortly before I started to work on my PhD project, the crystal structure of the eukaryotic Kv1.2 channel had …

Cell Death Mechanisms In Drosophila Differentiated Photoreceptor Neurons, Alexis Robert Gambis

Student Theses and Dissertations

Apoptosis, or programmed cell death, is a form of physiological cell death that is essential for normal development and homeostasis. At the end of pupal development of the Drosophila retina, cell death terminates and photoreceptor neurons complete their differentiation process. We use these terminally differentiated photoreceptor neurons as a system to study neurodegeneration. We first adapt and develop fluorescent tools for photoreceptor visualization in vivo. These tools enable a recessive genetic screen to search for genes required for the survival of differentiated photoreceptors. Many redox and mitochondrial genes were found to protect photoreceptors from late cell death. Here, we focus …

Genome-Wide Localization And Novel Deposition Pathways Of Histone Variant H3.3 In Embryonic Stem And Neuronal Precursor Cells, Aaron David Goldberg

Student Theses and Dissertations

The eukaryotic genome is composed of chromatin, a complex polymer of genomic DNA, RNA, and closely associated proteins. Histone proteins form the core of the nucleosome, the fundamental repeating unit of chromatin. Variant histone proteins play critical roles in the epigenetic regulation of gene expression and in the development of multicellular organisms. In this thesis, I describe the first genome-wide profiles of histone H3 variants in pluripotent mammalian embryonic stem (ES) cells, and I establish the dependence and independence of these patterns on the histone chaperone Hira. To distinguish H3 variants, I use designed zinc finger nucleases (ZFNs) to rapidly …

Characterization Of Histone H2a Functional Domains Important For Regulation Of The Dna Damage Response, Elizabeta Gjoneska

Student Theses and Dissertations

DNA double strand breaks represent deleterious lesions which can either be caused by environmental or endogenous sources of DNA damage. Efficient DNA damage response which ensures repair of these lesions is therefore critical for maintenance of genomic stability. The repair happens in the context of chromatin, a three-dimensional nucleoprotein complex consisting of DNA, histones and associated proteins. As such, mechanisms that modulate chromatin structure, many of which involve the histone component of chromatin, have been shown to play a role in regulation of the DNA damage response. In my thesis work I characterize two conserved histone H2A functional domains that …

The Role Of Lysogenic Bacteriophage In Virulence And Survival Of Streptococcus Pyogenes, Chad W. Euler

Student Theses and Dissertations

This thesis investigates the interactions of lysogenic bacteriophage with Streptococcus pyogenes. We were specifically interested in elucidating novel ways in which the prophage influence GrAS virulence and survival either within the host cell or within the bacterial population. In turn, we also hoped to determine how the bacteria (and possibly other integrated phage) might influence prophage activity or gene expression. Our studies focused on two polylysogenized GrAS strains, a M6 serotype (MGAS10394) and a M1 serotype (SF370). In our M6 studies, we chose a streptococcal strain lysogenized by a chimeric bacteriophage element (Φ10394.4), which contains the erythromycin resistance gene mefA, …

Cspcr: A Computational Tool For The Simulation Of The Polymerase Chain Reaction, Sandhya Dasu, April Williams, Yuriy Fofanov, Catherine Putonti

Bioinformatics Faculty Publications

Dasu S, Williams A, Fofanov Y, Putonti C., csPCR: A computational tool for the simulation of the Polymerase Chain Reaction, Online J Bioinformatics, 11 (1): 30-33, 2010. Herein we present a computational simulation package PCR (csPCR) which models the PCR reaction, taking into consideration the issues of specificity, sensitivity, potential mishybridizations throughout the primer sequence as well as at the 3’ end, and primer interactions, including selfcomplementarity and primer-primer interactions. A single target sequence or multiple target sequences can be considered simultaneously in addition to multiple primer sequences; thus a complex community and/or a multiplex assay can be simulated in …

Law, Economics, And Evolutionary Theory: State Of The Art And Interdisciplinary Perspectives, Peer Zumbansen, Gralf-Peter Calliess

Comparative Research in Law & Political Economy

This paper is the introduction essay to an edited collection entitled “Law, Economics, and Evolutionary Theory”, forthcoming with Edward Elgar. The volume brings together work by legal scholars, economists, historians and sociologists and aims at a critical investigation of the parallel and often competing theoretical architectures of legal and economic governance from an evolutionary perspective. By reconstructing discussions in law over the relationship between legal realism, law & society, and law & economics, and in economics over the merits and prospects of institutional and neo-institutional economics from an evolutionary perspective, the introduction argues that a theory of governance must today …

Corporate Governance And First-Best Solutions: A Sociological Perspective, Aviv Pichhadze

Comparative Research in Law & Political Economy

Drawing on insights from social science methodology and systems analysis, the article adopts a holistic view of the equity markets and highlights how market forces have been driving the evolution in the equity markets towards a first-best corporate governance model. This governance model is the Market Oriented Blockholder Model (MOBM). The analysis shows that the process is not yet complete given that the legal framework has not yet updated its foundation to fully accommodate the MOBM. Such a failure by policy makers creates an opportunity for the introduction, into the financial system, of regulatory systemic risk – long-term imbalances introduced …

Body Polluted: Questions Of Scale, Gender And Remedy, Dayna Nadine Scott

Comparative Research in Law & Political Economy

This paper offers a critique of tort remedies grounded in feminist theory of the body. It demonstrates how tort law is invested in a notion of an individuated legal subject, which fails to capture the critical interconnectedness of bodies in a social, political, historical, and colonial context. Taking the ‘injury’ of endocrine disruption in a Canadian aboriginal community as an example of a contemporary pollution harm, the analysis considers various torts on a conceptual level, and what they might offer the Aamjiwnaang First Nation in the way of remedies. In each case, what the tort can do depends on how …