Biochemistry, Biophysics, and Structural Biology Commons^™

Modulation Of Kras Structure And Dynamics By Kras Ubiquitination And Membrane Depolarization, Vinay Nair

Dissertations & Theses (Open Access)

KRAS, a 21 kDa small GTPase protein, functions as a molecular switch playing a key role in regulating cell proliferation. Dysregulation of KRAS signaling by oncogenic mutations leads to uncontrolled cell proliferation, a hallmark of cancer cells. Attempts to therapeutically target oncogenic KRAS have led to limited success resulting in a need to identify new mechanisms to targeting KRAS. The interaction of KRAS with its regulators, effectors, and the membrane present one such avenue. In this study, we investigated how post-translational covalent and environmental modifications could modulate these interactions of KRAS. Using computational molecular dynamics simulations, nuclear magnetic resonance spectroscopy …

Development Of Computational Tools To Target Microrna, Luo Song

Dissertations & Theses (Open Access)

MicroRNAs (a.k.a, miRNAs) play an important role in disease development. However, few of their structures have been determined and structure-based computational methods remain challenging in accurately predicting their interactions with small molecules. To address this issue, my thesis is to develop integrated approaches to screening for novel inhibitors by targeting specific structure motifs in miRNAs. The project starts with implementing a tool to find potential miRNA targets with desired motifs. I combined both sequence information of miRNAs and known RNA structure data from Protein Data Bank (PDB) to predict the miRNA structure and identify the motif to target, then I …

Ionic Mechanism Of Lysosomal Function And Cell Metabolism, Jian Xiong

Dissertations & Theses (Open Access)

Two Pore Channels (TPCs) are endolysosomal ion channels that are permeable to sodium and calcium. Defects in TPCs have been implicated to impair vesicle trafficking, autophagy and cell metabolism control; however, the detailed mechanism remains largely unknown. In this study, I show that TPCs are critical for appropriate cargo delivery to the lysosomes and deletion of either TPC1 or TPC2 leads to delayed clearance of autophagosomes, resulting in enlarged lysosomes and accumulated contents inside the lysosomes. Cells with both TPC deleted also exhibit 50% reduction in lysosomal amino acids under normal culture conditions, leading to reduced homeostatic mTORC1 activation.

Glutamine …

Methods To Investigate Hyperthermia Induced By Tumor Treating Fields, Ruchi Singh

Dissertations & Theses (Open Access)

Tumor Treating Fields (TTFields) are an antineoplastic treatment delivered via application of alternating electric fields using insulated transducer arrays placed directly on the skin in the region surrounding the tumor. TTF’s is a non-invasive application of low-intensity (1-3 V/cm), intermediate-frequency (100-500 kHz) alternating electric fields. The predominant mechanism by which TTFields are thought to kill tumor cells is the disruption of mitosis through the depolymerization of microtubules and interruption of the spindle structure leading to mitotic catastrophe and the formation of non-viable daughter cells. Tumor Treating fields do not stimulate nerves and muscle because of their high frequency, and do …

Investigations Of The Structure-Function Relationship In Kainate Receptors Using FöRster Resonance Energy Transfer, Douglas Litwin

Dissertations & Theses (Open Access)

Kainate receptors belong to the family of ion channels known as the ionotropic glutamate receptors. Ionotropic glutamate receptors mediate the majority of excitatory synaptic transmission, modulate the release of presynaptic glutamate, and facilitate dendrite formation. Kainate receptors are unique among the ionotropic glutamate receptors in being modulated by sodium ions. They have also been implicated in the development of higher learning and epilepsy. In recent years a wealth of structural data has become available for the AMPA and NMDA classes; however, the structural characterization of kainate receptors has been limited. The work in this dissertation utilizes luminescence resonance energy transfer …

Nanoscale Organization Of The Small Gtpase Rac1, Kelsey Maxwell

Dissertations & Theses (Open Access)

Rac1 is a small, guanine-nucleotide binding protein that cycles between an inactive GDP-bound and active GTP-bound state to regulate actin-mediated motility, migration, and adhesion. Plasma membrane (PM) localization is essential for its biological activity. Rac1 PM targeting is directed by a C-terminal membrane anchor that encompasses a geranylgeranyl-cysteine-methyl-ester, palmitoyl, and a polybasic domain (PBD) of contiguous lysine and arginine residues. Using high-resolution imaging combined with spatial mapping analysis, I found that Rac1 forms nanoclusters on the PM. Cycling between the GTP- and GDP-bound states, Rac1 forms nanoclusters that are non-overlapping, consequently undergoing guanine nucleotide-dependent spatial segregation. I further found that …

Fret-Based Investigations Of The Structure-Function Relationships In The Nmda Receptor, Drew M. Dolino

Dissertations & Theses (Open Access)

The N-methyl-D-aspartate (NMDA) receptor is one member of a class of proteins known as the ionotropic glutamate receptors. Ionotropic glutamate receptors mediate the majority of excitatory neurotransmission in the central nervous system, with the NMDA receptor standing out among these receptors for its requirement of a co-agonist, its magnesium-block-based coincidence detection, its slow kinetics, its calcium permeability, its allosteric modulation, and its especially important functional roles in synaptic plasticity, excitotoxicity, and more. In recent years, a wealth of structural information has come about describing endpoint structures to high resolution, but such structures are unable to fully resolve the movements …

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

Dissertations & 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 …

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

Dissertations & 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 …

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

Dissertations & 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 – …

Swarna Ramaswamy_Thesis, Swarna S. Ramaswamy

Dissertations & 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 …

Determining The Genotype-Phenotype Connection In Synthetic Inducible Gene Expression Systems, Rhys M. Adams

Dissertations & Theses (Open Access)

Introduction Gene expression is an important process whereby the genotype controls an individual cell’s phenotype. However, even genetically identical cells display a variety of phenotypes, which may be attributed to differences in their environment. Yet, even after controlling for these two factors, individual phenotypes still diverge due to noisy gene expression. Synthetic gene expression systems allow investigators to isolate, control, and measure the effects of noise on cell phenotypes. I used mathematical and computational methods to design, study, and predict the behavior of synthetic gene expression systems in S. cerevisiae, which were affected by noise.

Methods I created probabilistic …

Conformational Changes In The Extracellular Domain Of Glutamate Receptors, Anu Rambhadran

Dissertations & Theses (Open Access)

The family of membrane protein called glutamate receptors play an important role in the central nervous system in mediating signaling between neurons. Glutamate receptors are involved in the elaborate game that nerve cells play with each other in order to control movement, memory, and learning.

Neurons achieve this communication by rapidly converting electrical signals into chemical signals and then converting them back into electrical signals. To propagate an electrical impulse, neurons in the brain launch bursts of neurotransmitter molecules like glutamate at the junction between neurons, called the synapse. Glutamate receptors are found lodged in the membranes of the post-synaptic …