Biochemistry, Biophysics, and Structural Biology Commons^™

Developing And Using Methyl-Specific Antibodies To Study The Biological Roles Of Arginine Methylation, Vidyasiri Vemulapalli

Dissertations and Theses (Open Access)

Arginine residues can be modified in three different ways to produce asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and monomethylarginine (MMA). These modifications are catalyzed by a family of nine protein arginine methyltransferases (PRMT1-9), which are of three types (I, II, and III). The majority of Type I enzymes asymmetrically dimethylate Glycine- and Arginine-rich (GAR) motifs, except for PRMT4, which methylates Proline-, Glycine-, and Methionine-rich (PGM) motifs. The same substrates (GAR or PGM motifs) can also be dimethylated by PRMT5 in a symmetric fashion. However, it is not clear whether there are dedicated residues within these motifs for ADMA and SDMA, …

Mechanisms Of Regulation Of Tau Ires Mediated Translation, Niza Nemkul

Dissertations and Theses (Open Access)

The translation of most eukaryotic mRNAs occurs in a cap-dependent manner. However, a subset of mRNAs are capable of initiating translation in a cap-independent manner by utilizing sequences in their 5’ UTR called IRES. It was previously shown that the 5’ UTR of the tau mRNA contains an IRES. In this study I show that IRES dependent translation of tau IRES is regulated at multiple levels in order to regulate the expression of the tau protein.

Tau protein is ubiquitously expressed but is concentrated in the brain. In this study, I utilized neural and non-neural cell lines to show that …

The Roles Of Malt1 In Nf-Κb Activation And Solid Tumor Progression, Deng Pan

Dissertations and Theses (Open Access)

The transcription factor NF-κB plays a central role in many aspects of biological processes and diseases, such as inflammation and cancer. Although it has been suggested thatNF-κB is critical in tumorigenesis and tumor progression, the molecular mechanism by which NF-κB is activated in solid tumor remains largely unknown. In the current work, we focus on growth factor receptor-induced NF-κB activation and tumor progression, including epidermal growth factor receptor (EGFR)-induced NF-κB in lung cancer and heregulin receptor (HER2)-induced NF-κB in breast cancer. We found that Mucosa-associated lymphoma translocation protein 1 (MALT1), also known as paracaspase, is required for EGFR-induced NF-κB activation …

Cll Metabolism Is Regulated By Prognostic Factors, Modulated By Stroma And Abrogated By Pi3k Inhibition, Hima Vangapandu

Dissertations and Theses (Open Access)

Metabolism of chronic lymphocytic leukemia (CLL), a disease characterized by the relentless accumulation of mature B cells has been little explored. Bone marrow stromal cells provide a survival benefit to CLL cells, in part through PI3K/AKT pathway. Compared with proliferative B-cell lines, metabolic fluxes of oxygen and lactate were low in quiescent malignant B lymphocytes from CLL patients. Glycolysis (extracellular acidification rate, ECAR) was consistently low in CLL samples, but oxygen consumption (OCR) varied considerably. Higher OCR was associated with poor prognostic factors such as ZAP 70 positivity, unmutated IgVH, high β2M levels, and higher Rai stage. Co-culture with the …

Preeclampsia: The Roles Of Acute Inflammation And Intrauterine Stress, Nicholas Parchim

Dissertations and Theses (Open Access)

Preeclampsia (PE) is a severe, acute disease of pregnancy affecting approximately 8% of pregnant women after week 20 of gestation. PE is characterized by hypertension and renal damage reflected by proteinuria and has significant morbidity to both mother and fetus. Maternal symptoms range from headaches, nausea, edema, to visual changes, but once maternal symptoms present, damage to the fetus has begun. Mothers who progress untreated through the disease can also experience a condition called eclampsia characterized by seizure, coma, and, ultimately, death. PE-affected newborns experience features similar to prematurity—abnormal lung and renal development, intrauterine growth retardation (IUGR), and, possibly, fetal …

Preventing Thymus Involution In K5.Cyclin D1 Transgenic Mice Sustains The Naïve T Cell Compartment With Age, Michelle L. Bolner

Dissertations and Theses (Open Access)

The thymus maintains T cell receptor (TCR) repertoire diversity through perpetual release of self-MHC restricted naive T cells. However, thymus involution during the aging process reduces naïve T cell output, leading to defective immune responsiveness to newly encountered antigens. We have found that early thymus involution precipitates the age-associated shift favoring memory T cell dominancy in young control mice. Furthermore, we have shown that age-related thymus involution is prevented in mice expressing a keratin 5 promoter-driven Cyclin D1 (K5.D1) transgene in thymic epithelial cells (TECs). Thymopoiesis occurs normally in K5.D1 transgenic thymi and sustains T cell output to prevent the …

The Tumor Suppressor Notch Inhibits Head And Neck Squamous Cell Carcinoma (Hnscc) Tumor Growth And Progression By Modulating Proto-Oncogenes Axl And Ctnnal1 (Α-Catulin), Shhyam Moorthy, Shhyam Moorthy

Dissertations and Theses (Open Access)

Background: Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common malignancy worldwide, with roughly 300,000 cancer related deaths occurring globally each year. The survival of patients with HNSCC has not changed significantly over the past decade, leading investigators to search for promising molecular targets. To identify new treatment targets and biomarkers that could better guide therapy, we previously characterized the genomic alterations from primary HNSCC patient samples. We were among the first to discover that NOTCH1 is one of the most frequently mutated genes in this cancer type. The spectrum of inactivating NOTCH1 mutations in HNSCC suggested …

Normal Glycolytic Enzyme Activity Is Critical For Hypoxia Inducible Factor-1a Activity And Provides Novel Targets For Inhibiting Tumor Growth, Geoffrey Grandjean Phd

Dissertations and Theses (Open Access)

Normal Glycolytic Enzyme Activity is Critical for Hypoxia Inducible Factor-1α Activity and Provides Novel Targets for Inhibiting Tumor Growth

By Geoffrey Grandjean

Advisory Professor: Garth Powis, D. Phil

Unique to proliferating cancer cells is the observation that their increased need for energy is provided by a high rate of glycolysis followed by lactic acid fermentation in a process known as the Warburg Effect, a process many times less efficient than oxidative phosphorylation employed by normal cells to satisfy a similar energy demand ^[1]. This high rate of glycolysis occurs regardless of the concentration of oxygen in the cell and …

Molecular Regulation Of Vascular Calcification In Murine Models Of Atherosclerosis, Shanshan Gao

Dissertations and Theses (Open Access)

Background: Calcification occurs often in the atherosclerotic lesions of patients with coronary heart disease and animals with hypercholesterolemia, such as apolipoprotein-E deficient (ApoE-/-) mice. However, the mechanism(s) underlying the development of calcification in atherosclerosis remains unclear. ApoE acts as a lipid transporter, but also has been recognized as a potential regulator of osteogenesis. Little information is available as to whether ApoE has any direct impact on osteogenesis and calcification in vascular smooth muscle cells (VSMC). Several signal transduction pathways play a role in regulation of calcification, including the Wnt/β-catenin system and potentially GTAP, an ubiquitin-conjugating enzyme responsible for protein …

Gating Mechanisms Of The Canonical Trp Channel Isoform Trpc4, Dhananjay P. Thakur

Dissertations and Theses (Open Access)

Non-selective cation channels formed by Transient Receptor Potential Canonical (TRPC) proteins play important roles in regulatory and pathophysiological processes. These channels are known to be activated downstream from phospholipase C (PLC) signaling. However, the mechanism by which the PLC pathway activates TRPC4/C5 remains unclear. Uniquely, TRPC4 is maximally activated only when two separate G protein pathways, G_q/11 and G_i/o, are co-stimulated, making it a coincidence detector of G_q/11- and G_i/o -coupled receptor activation. Using HEK293 cells co-expressing mouse TRPC4β and selected G protein-coupled receptors, I observed that coincident stimulation of G_i/o proteins and …

Structure And Composition Of Postsynaptic Densities, Madeline Farley

Dissertations and Theses (Open Access)

Communication between neurons within the brain occurs at chemical synapses and is fundamental for all brain functions. Modulation of the strength of communication is controlled by both presynaptic and postsynaptic mechanisms and is termed synaptic plasticity. One postsynaptic structure postulated to regulate synaptic strength is the postsynaptic density (PSD), a large electron dense protein complex located just below the synaptic membrane. The PSD, which is composed of signaling, scaffold and cytoskeletal proteins, supports and organizes neurotransmitter receptors within the synaptic membrane in addition to bridging signaling with the actin cytoskeletal network. The protein composition and structure of PSDs is known …

Investigating The Interaction Of Aurka And Ube2c In Colorectal Cancer Cells, Apurva M. Hegde

Dissertations and Theses (Open Access)

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in the US. Among the many genomic aberrations previously implicated in colorectal cancer, recurrent amplification of chromosome 20q is frequently associated with liver metastasis. Previous research in our lab identified a gene signature on chromosome 20q associated with colorectal cancer progression. In this study, one of the genes in the signature, the ubiquitin conjugating enzyme UBE2C, was identified through preliminary bioinformatics analysis as a candidate for further examination of its role in CRC progression. Co-expression analysis of UBE2C in tumor-normal datasets from the public database Oncomine revealed all the …

Prophylactic Cranial Irradiation Reduces The Incidence Of Brain Metastasis In A Mouse Model Of Metastatic Breast Cancer, Daniel L. Smith

Dissertations and Theses (Open Access)

Prophylactic cranial irradiation (PCI) is a preventative whole-brain irradiation technique used to reduce the incidence of brain metastasis and improve overall survival in select patients with small cell lung cancer and acute lymphoblastic leukemia. A population of breast cancer patients – stage IV, HER2+ or triple-negative – has emerged as having a high risk of developing brain metastases. Because only 10-20% of breast cancer patients diagnosed with brain metastases survive longer than one year, in this high-risk population the benefit of PCI – potential for reduced incidence of brain metastasis and improved overall survival – may outweigh the risks – …

Direct Regulation Of Apoptosis By Linear Ubiqutin Chain Assembly Complex (Lubac) And Feedback Regulation Of Lubac Function By Caspases, Donghyun Joo

Dissertations and Theses (Open Access)

Tumor Necrosis Factor-alpha (TNF-α) is a cytokine that plays a role in various cellular processes such as proliferation, differentiation (mainly through NF-κB signaling) and death (via apoptosis signaling). Recently, linear ubiquitination by LUBAC (linear ubiquitin chain assembly complex) was reported to have a regulatory function in TNF-α mediated NF-κB activation. Although LUBAC is suggested to control not only NF-kB signaling but also the apoptosis pathway, the precise mechanism of apoptosis regulation remains unknown. Moreover, NF-κB and apoptosis pathways have opposed but fundamental functions for various cellular processes. Although these two pathways actively interplay to balance the death and survival, the …

Histone H3 K4 Methylation Regulates The Spindle Assembly Checkpoint Through Direct Binding Of Multiple Checkpoint Components And Cdc20, Andria C. Schibler

Dissertations and Theses (Open Access)

Histone H3K4 methylation is conserved across species and is associated with active transcription. By using Saccharomyces cerevisiae, we found histone H3K4 methylation has a previously unknown role in regulating mitosis through the Spindle Assembly Checkpoint. The Spindle Assembly Checkpoint ensures duplicated chromosomes are segregated correctly and each daughter cell receives one full copy of the genome. Our data show SET1 mutants and histone H3K4 mutants display a resistance to the mitotic poison, benomyl. Moreover methylated histone H3 directly binds to Spindle Assembly Checkpoint proteins Bub3 and Mad2 as well as the activator of the Anaphase Promoting Complex (APC) protein …

Computational Modeling Of Rna-Small Molecule And Rna-Protein Interactions, Lu Chen

Dissertations and Theses (Open Access)

The past decade has witnessed an era of RNA biology; despite the considerable discoveries nowadays, challenges still remain when one aims to screen RNA-interacting small molecule or RNA-interacting protein. These challenges imply an immediate need for cost-efficient while predictive computational tools capable of generating insightful hypotheses to discover novel RNA-interacting small molecule or RNA-interacting protein. Thus, we implemented novel computational models in this dissertation to predict RNA-ligand interactions (Chapter 1) and RNA-protein interactions (Chapter 2).

Targeting RNA has not garnered comparable interest as protein, and is restricted by lack of computational tools for structure-based drug design. To test the potential …

The Effect Of Activation Induced Cytidine Deaminase Phosphorylation And Herpes Virus Uracil Dna Glycosylase On Antibody Diversification, Marc Macaluso

Dissertations and Theses (Open Access)

Activation-induced cytidine deaminase (AID) is a mutagenic enzyme that is expressed in mammalian B-cells and initiates the antibody diversification processes of somatic hypermuntation (SHM) and isotype class switch recombination (CSR). AID is targeted to the immunoglobulin gene locus where it deaminates cytosines to generate uracil residues in DNA. This generates guanine-uracil (U:G) mismatch lesion which are recognized by uracil DNA glycosylase (UNG), a DNA repair enzyme that removes uracil from DNA and triggers downstream repair of the lesion. While UNG is a ubiquitously expressed DNA repair enzyme, its recognition and removal of AID introduced uracils is essential in both SHM …

Investigation Of The Role Of The Scaffold Protein Shc In Erk Signaling, Kin Man Suen

Dissertations and Theses (Open Access)

Cells respond to environmental changes by converting extracellular signals into intracellular events. Scaffolding proteins play important roles in generating specificity in intracellular signaling through the combinatorial use of protein domains and posttranslational modifications. Using the scaffold Shc (Src-homology collagen-like) as a model system, we explored novel mechanisms whereby this class of protein shapes signaling output. In the present work, we focused on the role of Shc in the MAP kinase Erk signaling both prior to and post-growth factor stimulation. Prior to growth factor stimulation, we found Erk to be a direct interacting partner of Shc. The two proteins associate through …

Dna Polymerase Θ (Polq) And The Cellular Defense Against Dna Damage, Matthew J. Yousefzadeh

Dissertations and Theses (Open Access)

In mammalian cells, DNA polymerase θ (POLQ) is an unusual specialized DNA polymerase whose in vivo function is under active investigation. The protein is comprised of an N-terminal helicase-like domain, a C-terminal DNA polymerase domain, and a large central domain that spans between the two. This arrangement is also found in the Drosophila Mus308 protein, which helps confer resistance to DNA interstrand crosslinking agents. Homologs of POLQ and Mus308 are found in eukaryotes, including plants, but a comparison of phenotypes suggests that not all of these genes are functional orthologs. Flies with defective Mus308 are sensitive to DNA interstrand crosslinking …

Developmental Origins Of Renal Connecting Tubule And Collecting Duct: Role Of Aqp2+ Progenitor Cells, Lihe Chen

Dissertations and Theses (Open Access)

The connecting tubule interconnects the nephron and collecting duct, which arise from kidney mesenchyme and the ureteric bud, respectively, to generate the functional tubular networks. The collecting duct is comprised of principal cells and intercalated cells, which bear different molecular signatures and regulate sodium/water and acid/base balance, respectively. The progenitor cells of the connecting tubule and the collecting duct remain virtually unknown.

We generated two Aqp2 lineage tracing mouse models. In these models, Aqp2Cre transgene drives Cre expression by the Aqp2 promoter to exclusively either inactivate histone H3 K79 methyltransferase Dot1l (Dot1l^f/f Aqp2Cre) or activate RFP in …

The Structural Mechanism Of Allosteric Modulation Of The Nmda Receptor: A Balance Of Tensions, Rita E. Sirrieh

Dissertations and Theses (Open Access)

N-methyl-D-aspartate (NMDA) receptors are one of the three main types of ionotropic glutamate receptors in the central nervous system. NMDA receptors mediate the rapid excitatory neurotransmission that underlies learning and memory formation. Conversely, NMDA receptors are implicated in a variety of neurological disorders. Studies targeting the mechanism of allosteric modulation, such as this study, hope to contribute to the understanding of how NMDA receptors are modulated to allow for better drug development.

NMDA receptors are obligate heterotetramers, typically composed of glycine-binding GluN1 subunits and glutamate-binding GluN2 subunits. The GluN2 subunits can be one of four subtypes (A-D). Each subunit is …

Selection Methods For Genetically-Modified T Cells: In Support Of Translational Therapy, David Rushworth

Dissertations and Theses (Open Access)

T cells are blood cells which organize the immune system of the host. These cells are necessary for the host to respond appropriately to threats from foreign organisms and cancerous growth. However, in the case of certain infections and cancer, T cells are unable to respond appropriately to a threat and establish immunity. This leads to disease when the infection or cancer is not sufficiently eliminated. On the other hand, T cells can lack tolerance for healthy tissue and perceive healthy tissue as infected. The ensuing over-reactive immune response also leads to disease. A delicate balance must exist between immunity …

Investigating The Roles Of P63 And P73 Isoforms To Therapeutically Treat P53-Altered Cancers, Avinashnarayan Venkatanarayan

Dissertations and Theses (Open Access)

Investigating the roles of p63 & p73 isoforms to therapeutically treat

p53-altered cancers

Avinashnarayan Venkatanarayan, M.S.

Supervisory Professor: Elsa R. Flores, Ph.D.

The TP53 tumor suppressor is mutated in approximately 50% of human cancers rendering cancer therapies ineffective. p53 reactivation suppresses tumor formation in mice. However, this strategy has proven difficult to implement therapeutically. An alternate approach to overcome p53 loss is to manipulate the p53-family members, p63 and p73, which interact and share structural similarities to p53. p63 and p73, unlike p53 are less frequently mutated and have two major isoforms with distinct functions …

Measuring Single Cell Responses To Lapatinib In A Heterogeneous Population, Preety Priya

Dissertations and Theses (Open Access)

Cancer is notonedisease butasaga of diseases and is the outcome of disturbed homeostasis in the normal cells due to the deregulation of its genetic makeup. With advent of technologies thatallowdetailed molecular characterizationoftumors, targeted therapies have emerged as a more promising and specific mode of treatment. However, a major challenge with targeted therapy is the acquired resistance in the cancer cells to these therapies, quite often very rapidly in the course of a few months. One of the major targets in cancer has been the EGFR/ErbB2 network in breast and other cancer types. Prior work from our lab and others have …

Regulation Of Cell Adhesion By The Ferm Proteins, Ptpn14 And Merlin, Patty Dimarco Hewitt

Dissertations and Theses (Open Access)

Cell-cell adhesion is critical for the control of tissue organization and homeostasis. A family of proteins that regulate cell-cell adhesions is the FERM (4.1 protein, Ezrin, Radixin, Moesin) domain-containing proteins.One FERM domain protein, the non-receptor tyrosine phosphatase PTPN14, is mutated or deleted in several human cancers suggesting that it may be involved in tumor development and/or progression. Additionally, the loss of the FERM domain protein Merlin is associated with tumor development and metastasis.Both PTPN14 and Merlin have been shown to localize and possibly regulate adherens junction (AJ) functions. This work sought to determine if …

Novel Posttranslational Modification In Lkb1 Activation And Function, Szu-Wei Lee

Dissertations and Theses (Open Access)

Cancer cells display dramatic alterations in cellular metabolism to meet their needs of increased growth and proliferation. In the last decade, cancer research has brought these pathways into focus, and one emerging issue that has come to attention is that many oncogenes and tumor-suppressors are intimately linked to metabolic regulation (Jones and Thompson, 2009). One of the key tumor-suppressors involved in metabolism is Liver Kinase B1 (LKB1). LKB1 is the major upstream kinase of the evolutionarily conserved metabolic sensor—AMP-activated protein kinase (AMPK). Activation of the LKB1/AMPK pathway provides a survival advantage for cells under energy stress. LKB1 forms a heterotrimeric …

Swarna Ramaswamy_Thesis, Swarna S. Ramaswamy

Dissertations and Theses (Open Access)

STRUCTURAL INVESTIGATIONS OF LIGAND GATED ION CHANNELS

Swarna Ramaswamy, B.S

Advisor: Vasanthi Jayaraman, Ph.D.

Ion channels form an integral part of membrane proteins. In the nervous system including the central and the peripheral nervous system, ligand gated ion channels form a very important part of intercellular communications. They receive chemical signals and convert them to electrical signal, mainly by allowing ion passage across the cell membrane. Ion passage also translates into downstream signaling events. Faithful translation of these signals and transmittance is crucial for several physiological functions, implying that irregular ion channel function could lead to serious consequences.

This thesis …

P120-Catenin Regulates Rest And Corest, And Modulates Mouse Embryonic Stem Cell Differentiation, Moonsup Lee

Dissertations and Theses (Open Access)

The canonical-Wnt pathway and beta-catenin have been extensively studied to determine their contributions to stem cell biology, but less is known about p120-catenin in the nuclear compartment. P120 is developmentally required as a consequence of its biochemical and functional interactions with cadherins, small-GTPases and transcriptional regulators. We report here that p120-catenin binds to and negatively regulates REST and CoREST, that others have indicated form a repressive complex having diverse key roles in developmental and pathologic gene regulation. We thus provide the first evidence for a direct upstream modulator of REST/CoREST function. Using mouse embryonic stem cells (mESCs), mammalian cell lines, …

Jab1 Negatively Regulates Pten And Promotes Resistance To Trastuzumab In Her2-Positive Breast Cancer, Thuy T. Vu

Dissertations and Theses (Open Access)

HER2-positive breast cancer, which is characterized by the over-expression of the HER2 onco-protein, accounts for approximately 20% of all breast cancer cases. Trastuzumab (Herceptin), the first targeted therapy approved for HER2-positive disease, potently prevents the activation of signaling pathways downstream of HER2 and significantly improves patients’ outcomes. However, resistance to trastuzumab is inevitable; such resistance can occur through reduced expression of PTEN protein.

Jab1 is over-expressed in 50% of primary cancers and 90% of metastatic tumors. Our lab previously showed that depletion of Jab1 in combination with trastuzumab treatment up-regulated PTEN in mouse xenografts refractory to trastuzumab. PTEN was not …

Mapping The Human Vasculature By In Vivo Phage Display, Julianna Bronk

Dissertations and Theses (Open Access)

In vivo phage display screenings by intravenous injection of a random phage-displayed peptide library allow for the selection of peptides that localize to specific vascular beds. At the University of Texas MD Anderson Cancer Center, we have had the opportunity to perform phage display screenings in cancer patients in order to select for cancer specific targets directly in humans. These targets serve to define biochemical diversity of endothelial cell surfaces and can be validated and explored towards the design of vascular-targeted pharmacology. In the most recent patient screen, samples were recovered from hepatocellular carcinoma (HCC) as well as 26 additional …