Molecular Biology Commons^™

Chromatin Regulation By Rb-Interacting Proteins In Cellular Immune Functions, Seung June Kim

Electronic Thesis and Dissertation Repository

The retinoblastoma protein (RB) is historically known for its function in cell cycle control. However, mice carrying targeted Rb1 mutations have revealed that RB serves various non-cell cycle control roles. Notably, RB acts as a scaffold that recruits chromatin regulatory proteins, condensin II and enhancer of zeste homolog 2 (EZH2). These complexes protect the genome integrity through maintaining proper chromosome condensation, long range contacts, and transcriptionally repressive histone modification. This thesis explores the mechanistic links that regulate such RB-condensin II complex or that are leveraged upon pharmacological inhibition of the RB-EZH2 complex. First, I identified potential phosphorylation sites in the …

Illuminating Transfer Rna Variants As Genetic Modifiers In Models Of Human Disease, Jeremy T. Lant

Electronic Thesis and Dissertation Repository

Transfer RNAs (tRNAs) physically link the genetic code to an amino acid sequence, by recruiting amino acids to three-nucleotide codons in messenger RNAs. To ensure that the genetic code is translated as intended, tRNAs must be accurately aminoacylated and faithfully recognize codons in the ribosome during protein synthesis. Given the critical function of tRNAs, it has often been assumed that mutations in human tRNA genes would be either lethal to cells or not significantly impair tRNA function. My goal was to rigorously test this assumption in mammalian cell models, prompted by the recent discovery of unprecedented variation in human tRNA …

The Regulation Of Pannexin1 And Pannexin2 In The Skin In Health And Disease, Rafael E. Sanchez Pupo

Electronic Thesis and Dissertation Repository

Pannexins (PANX1, 2, 3) are a family of channel-forming glycoproteins that mediate intracellular and paracrine signaling. In contrast to PANX2, PANX1 has been extensively investigated in the skin, modulating cell differentiation, wound healing, and melanoma development. PANX1 and PANX2 can co-exist in the same cell and form mixed channels where their glycosylation seems to regulate their intermixing. N-glycosylation and caspase cleavage have been proposed as modulators of the function of PANX1, but their effects on PANX2 are unknown. We explored the PANX2 expression in mouse skin and showed that a Panx2 splice variant (PANX2-202) is continuously expressed throughout aging skin. …

Characterization And Modulatory Influence Of Pyruvate Kinase Muscle Isoforms 1 And 2 Within The Murine Pluripotent Continuum, Joshua G. Dierolf

Electronic Thesis and Dissertation Repository

Mouse embryonic stem cells (mESCs) and mouse epiblast stem cells (mEpiSCs) represent opposite ends of a pluripotency continuum, respectively referred to as naïve and primed pluripotent states. A third, recently discovered intermediate state has been described as the ‘formative state’. Metabolism has been traditionally regarded as a by-product of cell fate; however, recent evidence now supports metabolism as promoting stem cell fate. Pyruvate kinase muscle isoforms 1 and 2 (PKM1 and PKM2) catalyze the final, rate limiting step of glycolysis generating adenosine triphosphate (ATP) and pyruvate; however, the precise role(s) of these isozymes in naïve, formative, and primed pluripotency is …

Dnajc7, A Molecular Chaperone Protein That Modulates Protein Misfolding In Amyotrophic Lateral Sclerosis (Als), Meaghan Kathleen Stoltz

Electronic Thesis and Dissertation Repository

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease associated with protein misfolding and dysregulated cellular protein quality control mechanisms. Molecular chaperones, and heat shock proteins (Hsp), are key players in maintaining cellular protein quality control. DNAJC7 is an understudied cytosolic Hsp40 that works together with Hsp70 and Hsp90 to regulate proper protein folding or degradation. Of note, mutations in the gene encoding DNAJC7 were discovered to cause familial ALS. We asked whether ALS-associated mutations in DNAJC7 compromise its function as a chaperone, which may cause the toxic accumulation of misfolded proteins. This study attempts to uncover the functions of DNAJC7 …

Characterization Of Mechanotransduction In Annulus Fibrosus Cells, Min Kyu M. Kim

Electronic Thesis and Dissertation Repository

IVD degeneration is a multifactorial pathological process associated with back pain. While biomechanical factors are important regulators of IVD homeostasis, mechanical loading also contribute to the onset of IVD degeneration. Importantly, the mechanotransduction pathways that mediate cell type-specific responses to mechanical loading are not well understood. Transient receptor potential vanilloid 4 (TRPV4) is a multimodally activated cell surface cation channel implicated as a mechanoreceptor regulating the mechano-response in other musculoskeletal cell types. Using both in vitro and in vivo models, the current study aimed to characterize the role of TRPV4 in annulus fibrosus (AF) cell mechanotransduction. Using a mechanically dynamic …

The Amino Terminal Domains Of Sheep Cx46 Or Cx50 Determine Their Gap Junction Channel Open Stability And Unitary Channel Conductance, Benny Yue

Electronic Thesis and Dissertation Repository

Connexins form intercellular communication channels known as gap junctions (GJs), which are found throughout the vertebrate species. GJs formed by different connexins harbor unique channel properties that have not been fully defined. High-resolution structures of native Cx46 and Cx50 GJs from sheep (sCx46 and sCx50) were recently resolved. Molecular dynamics studies identified the NT domains, especially the 9^th position, as key determinants in the differences of energetic barrier to K⁺ permeation in sCx46 and sCx50 GJs. We studied functional properties of GJs formed by sCx46, sCx50, NT domain swapped chimeras (sCx46-50NT and sCx50-46NT), and point variants at the …

Thyroxine-Dependent And -Independent Effects On Premature Aging And Myelination In Atrx Mutant Mice, Megan E. Rowland

Electronic Thesis and Dissertation Repository

ATRX is an ATP-dependent chromatin remodeler required to safeguard genomic integrity. Conditional deletion of Atrx in the mouse embryonic forebrain and anterior pituitary in Atrx^Foxg1Cre mice phenocopies mouse models of progeria which display increased DNA damage, coupled with reduced lifespan, growth and subcutaneous fat. These mice also have severely low circulating levels of insulin like growth factor 1 (IGF-1) and (T4) which have been reported in models of premature aging. Based on evidence that Igf1 is activated by the ligand-bound thyroid hormone receptor, I tested whether T4 supplementation could restore IGF-1 levels and ameliorate premature aging phenotypes in Atrx …

Characterizing The Role Of Thymine Dna Glycosylase In Transcriptional Regulation And Cancer In Vivo, Mohammad Haider Hassan

Electronic Thesis and Dissertation Repository

Cytosine methylation (5mC) is essential for transcriptional control and genomic stability and is often used as a prognostic marker in cancer. Although 5mC has long been considered a relatively stable epigenetic mark, recent studies have demonstrated that it can be reversed enzymatically by TET proteins which oxidize 5mC into 5-hydroxymethylcytosine (5-hmC), and then to 5-formylcytosine (5-fC) and 5-carboxylcytosine (5caC). This mechanism is known as active DNA demethylation and the base excision repair enzyme Thymine DNA Glycosylase (TDG) plays an essential role in this process by removing 5-fC and 5-caC which are subsequently replaced by the unmethylated cytosine. Importantly, homozygous loss …

Building Muscle: The Role Of Insulin-Like Growth Factor Binding Protein-6 And Insulin-Like Growth Factors In The Differentiation Of Placental Mesenchymal Stem Cells Into Skeletal Muscle, Doaa Aboalola

Electronic Thesis and Dissertation Repository

Human placenta is a readily available source for isolation of adult mesenchymal stem cell (MSC), for potential use in regenerative therapies. MSC fate is influenced by the microenvironment in vivo, and insulin-like growth factors (IGFs) are critical components of the stem cell niche, as they regulate proliferation and differentiation into different lineages including bone, fat, and skeletal muscle. Insulin-like growth factor binding protein-6 (IGFBP-6), relative to other IGFBPs, has high affinity for IGF2 and is believed to be the main modulator of IGF-2 function. However, the role of IGFBP-6 in muscle development has not been clearly defined. In this …

The Effects Of Acetylenic Tricyclic Bis-(Cyano Enone) On Cell Migration, Eddie Chan

Electronic Thesis and Dissertation Repository

Although cancer survival rates have significantly improved over the past few decades, the improvements are primarily due to early diagnosis and inhibiting cancer growth. Limited progress has been made in the treatment of cancer metastasis, which contributes to 90% of cancer related deaths, and therapeutic agents targeting the various aspects of metastasis are lacking. One potential approach is to utilize small pharmacological compounds to inhibit tumour cell motility, as a strategy against tumour cell migration, invasion, and metastasis. The acetylenic tricyclic bis-(cyano enone), TBE-31, has been shown to be a promising chemopreventative compound. However, its effects on cell migration are …

The Association Between Oxidative Stress, Cellular Differentiation And Galectins In Human Promyelocytic Leukemia Cells (Hl-60), James R. Vinnai

Electronic Thesis and Dissertation Repository

Galectins are a group of β-galactoside-binding proteins involved in different cellular processes including stress responses and differentiation. The role and expression of galectins under oxidative stress and during neutrophilic differentiation was examined in HL-60 cells. Galectin gene (LGALS), and galectin protein expression were determined using RT-qPCR and immunoblotting, respectively. Neutrophilic differentiation was measured via a spectrofluorometric assay. DNA methylation and JNK signaling were investigated as galectin regulatory mechanisms. Menadione-induced oxidative stress, DMSO-induced differentiation, DNA hypomethylation and JNK signaling all promoted similar galectin expression profiles. Antioxidant N-acetylcysteine attenuated the menadione-induced galectin expression but only partially attenuated DMSO-induced galectin expression. …

Characterization Of The Catalytic Ck2 Subunits With Substitutions At Residues Involved In Inhibitor Binding, Paul Desormeaux

Electronic Thesis and Dissertation Repository

CK2 is a constitutively active, ubiquitously expressed and pleiotropic serine/threonine protein kinase that is implicated in many cellular functions including tumorigenesis. CK2 has two catalytic subunits, CK2a and CK2a’, that carry out its function in the cell. Previous studies have indicated that inhibitor-refractory mutants have been effective in recovering residual CK2 activity, in the presence of inhibitors, when compared to wild type CK2. Based on these observations, inhibitor-refractory mutants were created for both CK2a and CK2a’ and tested with various concentrations with two CK2-specific inhibitors, CX-4945 and inhibitor VIII. The CK2a triple mutant (V66A/I174A/H160D) was tested in inducible U2OS Flp-In …

Cal And Magi Pdz Protein Regulation Of Crfr1 And 5-Ht2ar Trafficking And Signaling, Maha Mahmoud Hammad

Electronic Thesis and Dissertation Repository

PDZ (PSD95/Disc Large/Zona Occludens) domain-containing proteins are scaffolding proteins that play important roles in regulating the activity of G protein-coupled receptors. Corticotropin Releasing Factor Receptor 1 (CRFR1) and Serotonin 2A Receptor (5-HT_2AR) are two GPCRs that are commonly associated with mental disorders. Both receptors also contain a class I PDZ-binding motif at the carboxyl terminal tail. In the first chapter, we investigate the effects of CAL (CFTR-associated ligand) on regulating the trafficking and signaling of CRFR1. We demonstrate a role for CAL in inhibiting CRFR1 endocytosis, cell surface expression, and CRF-mediated ERK1/2 signaling via the CRFR1 PDZ-binding motif. …

Characterization Of Numb As A Regulator Of Anaplastic Lymphoma Kinase, Ran Wei

Electronic Thesis and Dissertation Repository

Cellular events rely on protein-protein interactions that are often mediated by modular domains which recognize particular sequence motifs in binding partners. The NUMB protein is the first described cell fate determinant and multifaceted adaptor that is involved in a wide variety of cellular events. NUMB mainly mediates protein interactions via its modular PTB domain. Here we present a systematic investigation of the NUMB-PTB interactome by employing an integrative strategy combining both protein and peptide arrays. We profiled NUMB-PTB binding specificity and interacting proteins genome-wide. The receptor tyrosine kinases (RTKs) are found highly enriched in the interactome, raising the possibility that …

Hd2d Is A Regulator Of Abscisic Acid Responses In Arabidopsis, Joshua A. Farhi

Electronic Thesis and Dissertation Repository

Histone deacetylases have important roles in development and stress response in plants. To further investigate their function, the HD2D gene, of the plant specific HD2 family, was studied. An hd2d-1 mutant and two HD2D overexpression lines were used in this study. Germination was delayed in hd2d-1 and HD2D overexpression seeds only in the presence of ABA. HD2D was found to positively regulate the expression of members of the ABA-response pathway (ABI1, ABI5, and RD29A) leading to increased resistance to drought and salinity treatments. Furthermore, HD2D expression delayed flowering by positively regulating FLC expression. Using bimolecular fluorescence complementation, the HD2D protein …

Characterizing The C-Terminal Region Of Human Adenovirus E1a: An Undiscovered Country, Michael J. Cohen

Electronic Thesis and Dissertation Repository

Human Adenovirus (HAdV) E1A is the first protein expressed during viral infection. The primary function of E1A is to reprogram the cell for viral replication, but it is additionally capable of transforming primary rodent cells in co-operation with other oncogenes such as HAdV E1B. Despite extensive study, little is known about the function and cellular targets of the C-terminal region of E1A. Importantly, this region is required for the transforming ability of E1A with E1B, but can also suppress transformation with Ras. Previous studies showed that interaction with the C-terminal Binding Protein (CtBP) plays a role in both functions described …

Analyzing A-Series Gangliosides In Neurons Following Exposure To Glutamate, Dae Hee Park

Electronic Thesis and Dissertation Repository

Neurons within different brain regions have varying levels of vulnerability to external stress and therefore respond differently to injury. A potential reason to explain this may lie within a key lipid class of the cell’s plasma membrane called gangliosides. These glycosphingolipid species have been shown to play various roles in the maintenance of neuronal viability. The purpose of this study is to use electrospray ionization mass spectrometry (ESI-MS) technique and immunohistochemistry to evaluate the temporal changes in the expression profiles of various ganglioside species during the course of neurodegeneration in rat primary cortical neurons exposed to glutamate toxicity. Primary embryonic …

Translesion Synthesis And Mutations: On The Mutagenic Properties Of The Two Dna Lesions, 8-Oxo-G And Pt-Gg, And The Functions Of Y-Family Dna Polymerases And Rev3l On The Bypass Of Each Of The Dna Lesions In Mammalian Cells, Lizhen Guo

Electronic Thesis and Dissertation Repository

I studied the capabilities of the two DNA lesions 8-oxo-guanine and cisplatin intrastrand crosslinked 1,2-d(GpG) or Pt-GG to cause mutations in mammalian cells. Using isogenic cell lines generated from mice with selective gene knockouts of distinct DNA polymerases as models, I deduced the biological functions of the translesion DNA polymerases Pol eta, Pol kappa, Pol iota, Rev1 and Rev3L on bypassing each of the lesions 8-oxo-G and Pt-GG. My study takes advantage of the Next Generation Sequencing (NGS) technology to determine mutagenic effects of the DNA lesions in vivo and effects of translesion DNA polymerases on bypassing the lesions. Through …

Elucidating The Signalling Pathway Of Mer Tyrosine Kinase Receptor In Efferocytosis, Ekenedelichukwu Azu

Electronic Thesis and Dissertation Repository

Efferocytosis is the clearance of apoptotic cells and is necessary for homeostasis. Mer Tyrosine Kinase (MerTK) is a crucial efferocytic receptor whose loss is associated with chronic inflammatory diseases and autoimmunity. While previous studies have shown that MerTK mediates efferocytosis through a unique mechanism that requires integrins, MerTK signalling pathway remains unknown. Given this unusual internalization mechanism, I hypothesized that MerTK signals and engages integrins through a novel signalling pathway different from that used by other phagocytic receptors. Therefore, this study aimed to identify the signalling pathways activated by MerTK, utilizing conventional cell biology and pharmacological approaches.

I found that …

Sirna Targeting Of Thymidylate Synthase, Thymidine Kinase 1 And Thymidine Kinase 2 As An Anticancer Therapy: A Combinatorial Rnai Approach, Christine Di Cresce

Electronic Thesis and Dissertation Repository

Thymidylate synthase (TS) is the only de novo source of thymidylate (dTMP) for DNA synthesis and repair. Drugs targeting TS protein are a mainstay in cancer treatment but off-target effects and toxicity limit their use. Cytosolic thymidine kinase (TK1) and mitochondrial thymidine kinase (TK2) contribute to an alternative dTMP-producing pathway, by salvaging thymidine from the tumour milieu, and may modulate resistance to TS-targeting drugs. We have previously shown that TS antisense molecules (oligodeoxynucleotides, ODNs, and small interfering siRNA, siRNA) sensitize tumour cells, both in vitro and in vivo, to TS targeting drugs. As both TS and TKs contribute to cellular …

Molecular Mechanisms Underlying The Early Life Programming Of The Liver, Gurjeev Sohi

Electronic Thesis and Dissertation Repository

Clinical studies have demonstrated that intrauterine growth restriction (IUGR) offspring, faced with a nutritional mismatch postpartum, have an increased risk of developing the metabolic syndrome. The maternal protein restriction (MPR) rat model has been extensively studied to investigate the adverse effects of a nutritional mismatch in postnatal life of IUGR offspring. Previous studies have demonstrated that MPR leads to impaired function of the liver, an important metabolic organ. However the underlying mechanisms which predispose these offspring to the metabolic syndrome remain elusive. In the following studies, low protein diet during pregnancy and lactation led to IUGR offspring with decreased liver …

Characterization Of A Tumour Suppressor Function Of Ranbpm, Elnaz Atabakhsh

Electronic Thesis and Dissertation Repository

Ran-binding protein M (RanBPM) is an evolutionarily conserved nucleocytosolic protein that has been proposed to regulate various cellular processes, including protein stability, gene expression, receptor-mediated signalling pathways, cell adhesion, development, and apoptosis. Despite the multitude of functions attributed to RanBPM however, little is known regarding the precise mechanisms by which RanBPM executes these cellular roles. In this work, we seek to address this matter by describing functions for RanBPM in the regulation of apoptotic and pro-survival signalling pathways, and in cellular transformation.

We first identify RanBPM as a pro-apoptotic protein that regulates the activation of the intrinsic apoptotic signalling pathway …

Identification And Characterization Of The Arabidopsis Homolog Of The Yeast Trex-2 Complex, Gang Tian

Electronic Thesis and Dissertation Repository

Nuclear pore complexes (NPCs) are vital to nuclear-cytoplasmic communication in eukaryotes. The yeast Thp1-Sac3-Cdc31-Sus1 complex, also known as the TREX-2 complex, is anchored to the NPC via the nucleoporin Nup1, and is essential for mRNA export. In this study, the Arabidopsis homolog of the yeast TREX-2 complex was discovered. Physical and functional evidence support the identification of the Arabidopsis orthologs of the yeast Thp1 and Nup1. Of three Sac3 Arabidopsis homologs, two are putative TREX-2 components. Surprisingly, none are required for mRNA export as is the yeast Sac3. Physical association with TREX-2 was observed for the two Cdc31 homologs, but …

Mass Spectrometry-Based Proteomics Analysis Of The Matrix Microenvironment In Pluripotent Stem Cell Culture, Christopher Hughes

Electronic Thesis and Dissertation Repository

The stem cell microenvironment contains soluble factors, support cells, and components of the extracellular matrix (ECM) that combine to effect cellular behavior. Mass spectrometry based proteomics offers the opportunity to directly assay components of extracellular microenvironments, thereby providing a sensitive means for obtaining insight into the stem cell niche. In this study we present the generation and analysis of human embryonic stem cell (hESC) and human induced pluripotent stem cell (hiPSC) matrix microenvironments using an MS-based proteomics approach.

One of the primary limitations in the proteomics analysis of hESCs and hiPSCs is the reproducible generation of sufficient cell numbers amenable …