Life Sciences Commons^™

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 …

Obscurin Mediates Ankyrin Complex Formation In The Heart, Janani Subramaniam

Dissertations & Theses (Open Access)

Distinctly organized domains of receptors, ion channels, transporters, signaling molecules, cell adhesion molecules, and contractile proteins are crucial to cardiac function. Interactions between adaptor proteins such as ankyrins and cytoskeletal proteins such as obscurin play a pivotal role in organizing these functional domains in cardiomyocytes. Therefore, dysfunction of both ankyrin as well as obscurin lead to a host of cardiovascular diseases such as arrhythmias and cardiomyopathies. Alternative splicing of ankyrin yields numerous isoforms that interact with obscurin at various sub-cellular domains. And while some of these obscurin-ankyrin complexes have been studied, many others have not been characterized. Further, previous studies …

Deubiquitinating Enzymes Promote Cancer Progression And Metastasis Via Regulating Protein Stability, Zhenna Xiao

Dissertations & Theses (Open Access)

Deubiquitinating enzymes (DUBs, also called deubiquitinases) are enzymes that remove monoubiquitin or polyubiquitin chains from target proteins. DUBs have critical roles in cell homeostasis and signal transduction, as they regulate protein degradation, subcellular localization, and protein-protein interaction. Deregulation of DUBs contributes substantially to tumor formation and progression, and therefore targeting DUBs may be a promising cancer therapy strategy. My dissertation focuses on identifying the DUBs of EZH2 and SNAI1, two proteins critical for cancer progression and metastasis, and establishing these DUBs as promising anti-cancer targets.

EZH2, the catalytic component of the PRC2 complex, silences gene transcription by histone methylation. High …

Dissecting The Roles Of Human Smc Complexes In Transcription Regulation And Chromatin Organization, Ruoyu Wang

Dissertations & Theses (Open Access)

Metazoans utilize a constellation of distal regulatory elements to control gene transcription, and therefore they have to forge highly complex chromatin loops to spatially bridge these regulatory elements and genes in the three-dimensional (3D) genome. However, the hierarchy of chromatin contacts and their underlying mechanisms are not well-understood. SMC complexes including Cohesin complex and Condensin complex has been widely proposed to organize 3D genome structure, and further regulate metazoans’ gene transcription. Here, we aim to dissect the direct functions of SMC complexes (both Cohesin and Condensin) in transcriptional regulation and 3D genome organization, by utilizing an inducible protein degradation system. …

Ipsc Based Gene Correction And Disease Model Of A New Class Of Lgmd Due To Poglut1 Mutation, Jose Ortiz-Vitali

Dissertations & Theses (Open Access)

Recently, a novel class of muscular dystrophy has been discovered in a family due to autosomal recessive missense mutation in POGLUT1. Mutation of this enzyme leads to decreased O-glucosyltransferase activity and impaired Notch signaling, the pathways important for skeletal muscle stem cell (satellite cells) quiescence and activation. We hypothesize that reduced POGLUT1 activity and impaired Notch signaling is causative of this limb girdle muscular dystrophy through dysfunction of muscle stem cells and myogenic progenitors.

To test this, we have used iPSCs for disease modeling and rescue experiments. Using a CRISPR based gene targeting method, we aimed to correct the point …

The Role Of Gene Expression Noise In Mammalian Cell Survival, Kevin Farquhar

Dissertations & Theses (Open Access)

Drug resistance and metastasis remain obstacles to effective cancer treatment. A major challenge contributing to this problem is cellular heterogeneity. Even in the same environment, cells with identical genomes can display cell-to-cell differences in gene expression, also known as gene expression noise. Gene expression noise can vary in magnitude in a population or in fluctuation time scales, which is influenced by gene regulatory networks.

Currently, it is unclear how gene expression noise from gene regulatory networks contributes to drug survival outcomes in mammalian cells. An isogenic cell line with a noise-modulating genetic system tuned to the same mean is required. …

Hsp70-Mediated Regulation Of Hsf1 Transcriptional Activity In Saccharomyces Cerevisiae, Sara Peffer

Dissertations & Theses (Open Access)

In eukaryotic cells, protein homeostasis and cellular fitness is promoted by the transcription factor heat shock factor 1 (HSF1) during exposure to proteotoxic stress. HSF1 controls the basal and stress-induced expression of molecular chaperones and other protective targets. Dynamic regulation of HSF1 involves the major heat shock proteins Hsp70 and Hsp90. Recent advances in the understanding of this regulatory circuit in Saccharomyces cerevisiae have shown that the Hsp70 Ssa1 acts as a sensor for some proteotoxic stresses and is capable of a direct interaction with Hsf1. This work continues to explore the complex regulatory interaction between Hsf1 and Ssa1. I …

Development Of A High-Throughput System For Screening Of Anti-Prion Molecules, Katherine Do

Dissertations & Theses (Open Access)

The misfolded prion protein causes and transmits disease in both humans and animals. As other infectious agents, prions display strain variation, which can generate different pathological outcomes in affected individuals. Unfortunately, there are no known therapies for these diseases, which at present are invariably fatal. In this work, the Protein Misfolding Cyclic Amplification technology (PMCA, an in vitro test that replicates minimum quantities of infectious prions) has been modified to screen for small molecules inhibiting prion protein misfolding in a strain-specific manner. In order to approach a high-throughput PMCA system, technical aspects in PMCA has been optimized for application of …

Thiol-Based Misfolding: Linking Redox Balance To Cytosolic Proteostasis, Ford Amy

Dissertations & Theses (Open Access)

The eukaryotic cytosolic proteome is vulnerable to changes in proteostatic and redox balance caused by temperature, pH, oxidants and xenobiotics. Cysteine-containing proteins are especially at risk as the thiol side chain is subject to oxidation, adduction and chelation by thiol-reactive compounds. All of these thiol-modifiers have been demonstrated to induce the heat shock response and recruit protein chaperones to sites of presumed protein aggregation in the budding yeast Saccharomyces cerevisiae. However, endogenous targets of thiol stress toxicity responsible for these outcomes are largely unknown. Furthermore, I hypothesize proteins identified as redox-active are prone to misfolding and aggregation by thiol-specific …

Role Of P300 Zz Domain In Chromatin Association And Histone Acetylation, Yongming Xue

Dissertations & Theses (Open Access)

Transcription is strictly regulated by numerous factors including transcription coactivators. The p300 protein and its close paralogue CREB-binding protein (CREBBP, aka CBP) are well-known transcriptional coactivators that have intrinsic lysine acetyltransferase activity. The functions of p300/CBP largely rely on their capabilities to bind to chromatin and to acetylate the histone substrates. However, the molecular mechanisms underlying the regulation of these processes are not fully understood.

Through combination of various biochemical, biophysical and molecular approaches, we show that the ZZ-type zinc finger (ZZ) domain of p300 functions as a histone reader that specifically binds the N-terminal tail of histone H3. Crystal …

Characterizing The Recognition Motif And Novel Substrates Of Carm1, Sitaram Gayatri

Dissertations & Theses (Open Access)

A limited pool of proteins attains vast functional repertoire due to posttranslational modifications (PTMs). Arginine methylation is a common posttranslational modification, which is catalyzed by a family of nine protein arginine methyltransferases or PRMTs. These enzymes deposit one or two methyl groups to the nitrogen atoms of arginine side-chains. Elucidating the substrate specificity of each PRMT will promote a better understanding of which signaling networks these enzymes contribute to. Although many PRMT substrates have been identified, and their methylation sites mapped, the optimal target motif for each of the nine PRMTs has not been systematically addressed. Here we describe the …

Functional Similarity Of Prd-Containing Virulence Regulators In Bacillus Anthracis, Malik Raynor

Dissertations & Theses (Open Access)

Bacillus anthracis produces three regulators, AtxA, AcpA, and AcpB, that control virulence gene expression and are members of an emerging class of regulators termed “PCVRs” (Phosphoenolpyruvate-dependent phosphotransferase regulation Domain-Containing Virulence Regulators). AtxA controls expression of the toxin genes; lef, cya, and pag, and is the master virulence regulator and archetype PCVR. AcpA and AcpB are less well studied. AcpA and AcpB independently positively control transcription of the capsule biosynthetic operon capBCADE, and culture conditions that enhance AtxA activity result in capBCADE transcription in strains lacking acpA and acpB. RNA-Seq was used to assess the regulons of the …

The Regulation Of Dna Methylation In Mammalian Development And Cancer, Nicolas Veland

Dissertations & Theses (Open Access)

DNA methylation is an essential epigenetic modification in mammals, as it plays important regulatory roles in multiple biological processes, such as gene transcription, maintenance of chromosomal structure and genomic stability, genomic imprinting, retrotransposon silencing, and X-chromosome inactivation. Dysregulation of DNA methylation is associated with various human diseases. For example, cancer cells usually show global hypomethylation and regional hypermenthylation, which have been implicated in genomic instability and tumor suppressor silencing, respectively. Although great progress has been made in elucidating the biological functions of DNA methylation over the last several decades, how DNA methylation patterns and levels are regulated and dysregulated is …

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 …

Deciphering The Roles Of Δnp63 In Regulating Epithelial To Mesenchymal Transition, Cancer Progression And Metastasis, Ngoc Bui

Dissertations & Theses (Open Access)

p63 is a member of the p53 family, a well-known tumor suppressor which is considered the guardian of the genome. The TP63 gene encodes multiple isoforms that can be categorized into two main isoforms, TAp63 and ΔNp63, which are expressed in different cellular compartments and have distinct functions in many biological processes. While the Flores laboratory identified TAp63 as a tumor and metastasis suppressor, the precise roles of ΔNp63 isoforms in tumorigenesis and metastasis remain elusive. ΔNp63 is the predominant p63 isoform expressed in the epidermis and plays essential roles in regulating epidermal development and homeostasis. Utilizing a ΔNp63-conditional …

Biological Clocks, Inflammation, And Multiorgan Damage In Sickle Cell Disease, Morayo Adebiyi

Dissertations & Theses (Open Access)

Sickle cell disease (SCD) is a dangerous condition caused by a genetic mutation on the human beta-globin gene that contributes to erythrocyte sickling, the hallmark of the disease. Previous metabolomics studies have confirmed that elevated sphingosine kinase 1 (SphK1) mediates sphingosine-1-phosphate (S1P) production to promote erythrocyte sickling. S1P signals via five S1P receptors (S1PR) regulates several pathophysiological functions.

In the first chapter of this dissertation, I explored the role of S1PRs in SCD by utilizing pharmacologic and genetic tools. To determine the role of S1P-S1PRs signaling in SCD, I treated humanized Berkeley sickle mice (Berkeley HBS mice), with FTY720, a …

Investigating The Impact Of Intragenic Dna Methylation On Gene Expression, And The Clinical Implications On Tumor Cells And Associated Stroma, Michael Mcguire

Dissertations & Theses (Open Access)

Investigations into the function of non-promoter DNA methylation have yielded new insights into epigenetic regulation of gene expression. Previous studies have highlighted the importance of distinguishing between DNA methylation in discrete functional regions; however, integrated non-promoter DNA methylation and gene expression analyses across a wide number of tumor types and corresponding normal tissues have not been performed. Through integrated analysis of gene expression and DNA methylation profiles, we uncovered an enrichment of DNA methylation sites within the gene body and 3’UTR in which DNA methylation is strongly positively correlated with gene expression. We examined 32 tumor types and identified 57 …

Evolution Via Gene Duplication And Alternative Splicing In The Eukaryotic Ski7 And Hbs1 Genes, Alexandra Marshall

Dissertations & Theses (Open Access)

Gene duplication and alternative splicing are both recognized as important drivers of proteomic diversity and innovation during evolution, but the evolutionary changes over long periods of time or the interrelations of the two processes has not been extensively studied. Here I study these phenomena for the SKI7 and HBS1 gene pair. These Saccharomyces cerevisiae genes were created as part of a whole genome duplication (WGD) event and have since functionally diverged. Although both genes function in mRNA surveillance pathways, the two genes act on different RNAs and have different effects on the target mRNAs. Ski7 brings the Ski complex and …

The Functions Of Setd5 And Mir-221 In Embryonic Stem Cell Differentiation, Tsai-Yu Chen

Dissertations & Theses (Open Access)

Embryonic stem cells (ESCs) are a widely used model system to study cellular differentiation because of their pluripotent characteristics, and ESC differentiation is an epigenetic process. In an effort to identify a new epigenetic factor that is required for ESC differentiation, the function of SETD5 in ESCs was studied for this thesis. Results show that SETD5 is essential for retinoic acid (RA)-induced differentiation of mouse ESCs and for RA-induced expression of critical developmental genes (e.g., Hoxa1 and Hoxa2) and neuron-related genes (e.g., Nestin and Pax6). SETD5 was upregulated during ESC differentiation. Additional results demonstrated that SETD5 bound to …

Characterization Of Notch1 And Pi3k-Pten-Akt/Mtor Pathway Interaction In Head And Neck Squamous Cell Carcinoma, Kyriante' Henry

Dissertations & Theses (Open Access)

Head and neck squamous cell carcinoma (HNSCC) affects various mucosal sites of the upper aerodigestive tract, including the nasal and oral cavities, the nasopharynx, and the oropharynx. More than five hundred thousand new cases of HNSCC occurred in 2011 alone, with 50,000 reported cases in the United States. This trend made HNSCC the seventh most common non-skin cancer worldwide (Ferlay et al., 2015). Although significant epidemiological and pathological advancements have been made, survival rates have not improved much over the last 40 years, leaving a mortality rate that remains at approximately 50%. An unbiased drug screen demonstrated that HNSCC cell …

Endocytic Trafficking Of The Amyloid Precursor Protein In Rat Cortical Neurons, Sahily Reyes

Dissertations & Theses (Open Access)

Amyloid-beta (Aβ) aggregation and deposition into extracellular plaques is a hallmark of the most common forms of dementia, including Alzheimer’s disease. The Aβ-containing plaques result from pathogenic cleavage of amyloid precursor protein (APP) by secretases resulting in intracellular production of Aβ peptides that are secreted and accumulate extracellularly. Despite considerable progress towards understanding APP processing and Aβ aggregation, the mechanisms underlying endosomal production of Aβ peptides and their secretion remain unclear. Using endosomes isolated from cultured primary neurons, we determined that the trafficking of APP from the endosomal membrane into internal vesicles of late endosome/multivesicular bodies (MVB) is dependent on …

Insights Into The Therapeutic Potential Of Salt Inducible Kinase 1: A Novel Mechanism Of Metabolic Control, Randi Fitzgibbon

Dissertations & Theses (Open Access)

Salt inducible kinase 1 (SIK1) has been considered a stress-inducible kinase since it was first cloned in 1999. Continued efforts since this time have been dedicated to characterizing the structure and function of SIK1. Such research has laid the ground work for our understanding of SIK1 action and regulation in tissue and stimuli dependent manners. The fundamental findings of this dissertation continue in this tradition and include investigations of SIK1 regulatory mechanisms in skeletal muscle cells, the cellular and physiological effects of SIK1 loss of function in vitro and in vivo, and intracellular metabolic and mitochondrial regulation by this …

Gcn5 Impacts Fgf Signaling At Multiple Levels And Activates C-Myc Target Genes During Early Differentiation Of Embryoid Bodies, Li Wang

Dissertations & Theses (Open Access)

Precise control of gene expression during development is orchestrated by transcription factors, signaling pathways and co-regulators, with complex cross-regulatory events often occurring. Growing evidence has identified chromatin modifiers as important regulators for development as well, yet how particular chromatin modifying enzymes affect specific developmental processes remains largely unclear. Embryonic stem cells (ESCs) are self-renewing, pluripotent, and have the abilities to generate almost all cell types in adult tissues. The dual capacity of ESCs to self-renew and differentiate offers unlimited potential for studying gene regulation events at specific developmental stages in vitro that parallel developmental events during embryogenesis in vivo. …

Preclinical Development Of Therapeutic Strategies Against Triple-Negative And Inflammatory Breast Cancer, Angie M. Torres-Adorno

Dissertations & Theses (Open Access)

Triple-negative (TNBC) and inflammatory (IBC) breast cancer are the most aggressive forms of breast cancer, accounting for 20% and 10% of cancer-related deaths, respectively. Among IBC cases, 30% are additionally classified with TNBC molecular pathology, a diagnosis that significantly worsens patient’s prognosis. The current lack of TNBC and IBC molecular understanding prevents the development of effective therapeutic strategies. To identify effective treatments, we explored aberrant apoptosis pathways and cell membrane fluidity as novel therapeutic targets.

We first identified an effective therapeutic strategy against TNBC and IBC by pro-apoptotic protein NOXA-mediated inhibition of the anti-apoptotic protein MCL1 following inhibition of histone …

Investigating The Role Of Prmt1 And Arginine Methylation Of Hsp70 In Human Pancreatic Cancer, Liang Wang

Dissertations & Theses (Open Access)

Protein arginine methyltransferase 1 (PRMT1) is the major arginine methyltransferase, which catalyzes the addition of one or two methyl groups to the arginine residues of its substrate proteins. The best-known substrate for PRMT1 is histone, while more and more non-histone proteins are now found to be methylated by PRMT1. Dysregulation of PRMT1 is reported in several human cancer types. However, its biological roles in human pancreatic cancer initiation and development are still unclear. In the first part of this study, I found that the expression level of PRMT1 was elevated in both human and mouse pancreatic cancer tissues in immunohistochemistry …

Phopsphorylation And Ubiquitin Modification At Dna Damage Sites In Response To Double-Strand Breaks, Atanu Paul

Dissertations & Theses (Open Access)

Genomes of all organisms are continuously damaged by numerous exogenous and endogenous sources leading to different kinds of DNA lesions, which if not repaired efficiently may trigger wide-scale genomic instability, a hallmark of cancer development. To overcome this, cells have evolved a sophisticated sensory network called the DNA damage response (DDR) comprised of a large number of distinct protein complexes categorized as sensor, mediator, transducer and effector proteins that amplify the DNA damage signal and activate cell cycle checkpoint to initiate DNA repair or trigger apoptosis where the defect is beyond repair. This intricate signaling pathway is tightly regulated by …

Non-Coding Rnas Identify The Intrinsic Molecular Subtypes Of Muscle-Invasive Bladder Cancer, Andrea E. Ochoa

Dissertations & Theses (Open Access)

NON-CODING RNAS IDENTIFY THE INTRINSIC MOLECULAR SUBTYPES OF MUSCLE-INVASIVE BLADDER CANCER

Andrea Elizabeth Ochoa, B.S.

Advisory Professors: David J. McConkey, Ph.D. and Joya Chandra, Ph.D.

There has been a recent explosion of genomics data in muscle-invasive bladder cancer (MIBC) to better understand the underlying biology of the disease that leads to the high amount of heterogeneity that is seen clinically. These studies have identified relatively stable intrinsic molecular subtypes of MIBC that show similarities to the basal and luminal subtypes of breast cancer. However, previous studies have primarily focused on protein-coding genes or DNA mutations/alterations.

There is emerging evidence implicating …

Understanding The Mechanism Of Genomic Instability During Replicative Aging In Budding Yeast, Sangita Pal

Dissertations & Theses (Open Access)

Aging brings a gradual decline in molecular fidelity and biological functionality, resulting in age related phenotypes and diseases. Despite continued efforts to uncover the conserved aging pathways among eukaryotes, exact molecular causes of aging are still poorly understood. One of the most important hallmarks of aging is increased genomic instability. However, there remains much ambiguity as to the cause. I am studying the replicative life span (RLS) of the genetically tractable model organism Saccharomyces cerevisiae, or budding yeast using the innovative “mother enrichment program” as the method to isolate unparalleled numbers of aged yeast cells to investigate the molecular changes …

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 …

Analysis Of The Biochemical And Cellular Activities Of Substrate Binding By The Molecular Chaperone Hsp110/Sse1, Veronica M. Garcia

Dissertations & Theses (Open Access)

Molecular chaperones ensure protein quality during protein synthesis, delivery, damage repair, and degradation. The ubiquitous and highly conserved molecular chaperone 70-kDa heat-shock proteins (Hsp70s) are essential in maintaining protein homeostasis by cycling through high and low affinity binding of unfolded protein clients to facilitate folding. The Hsp110 class of chaperones are divergent relatives of Hsp70 that are extremely effective in preventing protein aggregation but lack the hallmark folding activity seen in Hsp70s. Hsp110s serve as Hsp70 nucleotide exchange factors (NEF) that facilitate the Hsp70 folding cycle by inducing release of protein substrate from Hsp70, thus recycling the chaperone for a …