Cell and Developmental Biology Commons^™

Targeted Genome-Scale Gene Activation And Gene Editing In Human Cells To Understand Disease Models, Michael De La Cruz

KGI Theses and Dissertations

Since the discovery of sequence directed DNA editing reagents such as CRISPR-Cas9 RNA-guided and TALEN DNA endonucleases, there has been a snowball of advances in the life sciences due to the ability to efficiently edit and control genomes within living cells. CRISPR-Cas9 based genomic tools, which facilitate the high-throughput precise manipulation of genes, allow for unbiased functional genomic screens. We used a human CRISPR-Cas9 Synergistic Activation Mediator pooled library which utilizes an engineered protein complex for transcriptional activation of 23,430 endogenous genes to investigate the development of novel resistance mechanisms to lung cancer targeted therapy, Erlotinib. We set out to …

Novel Low Shear 3d Bioreactor For The Scaled Production Of High Purity Human Mesenchymal Stem Cells, Andrew Burns

KGI Theses and Dissertations

Human mesenchymal stem cells are an ideal candidate for stem cell therapies. They have been researched since the 1960’s and can differentiate into many desired functional cell types without undergoing teratogenesis. However, higher yields are needed for a marketable, successful stem cell therapy. To accomplish this, cells will have to be cultured to expand them to therapeutically relevant dosages for multiple patients. Bioreactor production is an ideal method to solve this problem.

The aim of this thesis is to test and validate a novel bioreactor for the cultivation of human mesenchymal stem cells. In this work, we investigate a novel …

Reversal Of P-Glycoprotein And Breast Cancer Resistance Protein Mediated Multidrug Resistance In Vitro Using In Silico Identified Novel Compounds, Amila Nanayakkara

Biological Sciences Theses and Dissertations

Multidrug resistance (MDR) is a major cause of chemotherapy failure. Overexpression of ATP-binding cassette (ABC) transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two well-studied drug transporters which are associated with MDR. These two transporters also act as a major functional unit of the blood brain barrier to protect the brain from xenobiotics and toxins. Lack of clinically approved P-gp and BCRP inhibitors renders chemotherapy treatments of many MDR cancers ineffective and obstructs drug uptake into the brain.

Using computational methods, we have identified new compounds that inhibit P-gp (Brewer et al., Mol. Pharmacol. 2014). Several of …

Reversal Of P-Glycoprotein And Breast Cancer Resistance Protein Mediated Multidrug Resistance In Vitro Using In Silico Identified Novel Compounds, Amila Nanayakkara

Biological Sciences Theses and Dissertations

Multidrug resistance (MDR) is a major cause of chemotherapy failure. Overexpression of ATP-binding cassette (ABC) transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two well-studied drug transporters which are associated with MDR. These two transporters also act as a major functional unit of the blood brain barrier to protect the brain from xenobiotics and toxins. Lack of clinically approved P-gp and BCRP inhibitors renders chemotherapy treatments of many MDR cancers ineffective and obstructs drug uptake into the brain.

Using computational methods, we have identified new compounds that inhibit P-gp (Brewer et al., Mol. Pharmacol. 2014). Several of …

Network Analyses Of Glomerular Capillaries, Jason Cory Brunson, Justin Sardi, Mark Terasaki

Biology and Medicine Through Mathematics Conference

No abstract provided.

Towards A Better Understanding Of Temporomandibular Disorder, Jessica R. Cox

Graduate Theses/Dissertations

Results from the OPPERA study provided evidence that risk factors such as neck muscle tension, prolonged jaw opening, and female gender increase the likelihood of developing temporomandibular joint disorders (TMJD), which are prevalent, debilitating orofacial pain conditions. Peripheral and central sensitization, which mediate a lowering of the stimulus required for pain signaling, are implicated in the underlying pathology of chronic TMJD. The goal of my study was to investigate cellular changes in the expression of proteins associated with the development of central sensitization. Female Sprague-Dawley rats were injected with complete Freund’s adjuvant in the upper trapezius muscles to promote trigeminal …

Effects Of Herbicides On Zebrafish Embryo Development And Viability, Kayla Ray King

Graduate Theses/Dissertations

Environmental contaminants are chemicals of anthropogenic origin that are found in water, soil, and air, and are harmful to a wide variety of organisms (ORD US EPA, 2018-a). One common group of contaminants are herbicides. Though herbicides are used to control unwanted vegetation in agriculture, aquatic organisms and humans may be exposed to these herbicides through run off into streams and rivers, by drinking contaminated water, by consuming treated crops, by direct exposure, or through bioaccumulation. Thus the effect of these herbicides on animals needs further investigation. In this study, I sought to determine whether six different herbicides, which have …

Kinetics Of Hiv-1 Uncoating In C20 Microglial Cells, Melanie Anne Taylor

Graduate Theses/Dissertations

Uncoating is a poorly understood yet required step of HIV-1 replication that is defined as the disassembly of the viral capsid structure. The goal of this project is to characterize uncoating in C20 microglial cells. These cells are a natural target of HIV-1 that are infected to establish latent viral reservoirs and HIV-associated neurological disorders. A stable C20 cell line that expresses TRIM-CypA was established to study the kinetics of uncoating with the CsA washout assay. The expression of TRIM-CypA was confirmed by western blot and the functionality of the protein was confirmed by a viral infectivity assay. Using this …

Further Characterization Of The Skeletal Phenotype In A Hurler Syndrome Mouse Model And The Ethical Treatment Of Children In Medicine, Anna Marie Mcwoods

Graduate Theses/Dissertations

Mucopolysaccharidosis type I (MPS I) is a rare, autosomal recessive disorder caused by the deficiency of the lysosomal enzyme α-L-iduronidase (IDUA). Absence of IDUA results in the accumulation of dermatan and heparin sulfate and ultimately causes multi-system dysfunction. The most severe form of MPS I is Hurlers syndrome, a rapidly progressive disorder that, if left untreated, is fatal. Current treatment options for diagnosed individuals includes hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT). These treatments are able to ameliorate the majority of symptoms with the exception of the bone phenotype. This investigation aimed to further characterize the bone …

From Spinning Silk To Spreading Saliva: Mouthpart Remodeling In Manduca Sexta (Lepidoptera: Sphingidae), Istvan Miko, Sarthok Rasique Rahman, Anne C. Jones, Mark A. Townley, Brandon Gominho, Sulav Paudel, S David Stupski, Heather M. Hines, Rudolf J. Schilder

Biological Sciences

As a model organism, the tobacco hornworm Manduca sexta (Linnaeus 1763) has contributed much to our knowledge of developmental processes in insects, and major developmental changes between different larval instars are generally well understood. Second and later instars of M. sexta do not produce silk, and their spinneret and accessory labial glands (=Lyonet’s glands), structures thought to be key players in silk production in other lepidopterans, are highly reduced. To our knowledge, mouthparts and labial gland morphology of the silk-producing first instar have never been described. In this study, we compared the mouthpart morphology and transcriptome profile of first and …

Oligodeoxynucleotides Odn 2006 And M362 Exert Potent Adjuvant Effect Through Tlr-9/-6 Synergy To Exaggerate Mammaglobin-A Peptide Specific Cytotoxic Cd8+T Lymphocyte Responses Against Breast Cancer Cells, Duaa Babaer, Suneetha Amara, Brenda S. Mcadory, Owen Johnson, Elbert L. Myles, Roy Zent, Jeffrey C. Rathmell, Venkataswarup Tiriveedhi

Public Health, Health Administration, and Health Sciences Faculty Research

Mammaglobin-A (MamA) is overexpressed in 40–80% of all human breast cancers. Recent phase I clinical trials of the MamA DNA vaccine showed encouraging safety outcomes. However, this vaccine elicited only a modest increase in MamA specific CD8+T lymphocyte (CTL) activation. As vaccine adjuvants play a critical role in enhancing the immunotherapeutic efficiency of vaccines, we tested the potential role of three synthetic CpG oligodeoxynucleotides (ODN2216—class A ODN, ODN2006—class B ODN, and ODN M362—class C ODN) to further enhance MamA specific CTL responses. Towards this, naïve CD8+T cells were obtained from healthy HLA-A2+ human donors. The HLA-A2 specific immunodominant epitope of …

Clinical Implications Of Evolutionary Modeling Of Cancer Progression, Kelsey Heiland

Biology: Student Scholarship & Creative Works

Evolutionary theory of cancer was developed in 1976 by cancer research Peter Nowell and has illuminated the path toward increasing safety and efficacy of clinical treatment strategies. Major foundations that makeup the complexity in Darwinian framework of cancer includes clonal evolution, clonal expansion, and competition. Due to these characteristics, virtually all types of cancer have evolutionary capabilities to reject or adapt and become resistant to pharmaceutical therapies. Despite compelling evidence of these process, evolutionary modeling of cancer continues to be underutilized in clinical settings. Contributions to this include the complex adaptive mechanisms that tools fail to detect or are unable …

Development Of A Sonically Powered Biodegradable Nanogenerator For Bone Regeneration, Avi S. Patel

University Scholar Projects

Background: Reconstruction of bone fractures and defects remains a big challenge in orthopedic surgery. While regenerative engineering has advanced the field greatly using a combination of biomaterial scaffolds and stem cells, one matter of difficulty is inducing osteogenesis in these cells. Recent works have shown electricity’s ability to promote osteogenesis in stem cell lines when seeded in bone scaffolds; however, typical electrical stimulators are either (a) externally housed and require overcomplex percutaneous wires be connected to the implanted scaffold or (b) implanted non-degradable devices which contain toxic batteries and require invasive removal surgeries.

Objective: Here, we establish a biodegradable, piezoelectric …

Development Of A Sonically Powered Biodegradable Nanogenerator For Bone Regeneration, Avi Patel

Honors Scholar Theses

Background: Reconstruction of bone fractures and defects remains a big challenge in orthopedic surgery. While regenerative engineering has advanced the field greatly using a combination of biomaterial scaffolds and stem cells, one matter of difficulty is inducing osteogenesis in these cells. Recent works have shown electricity’s ability to promote osteogenesis in stem cell lines when seeded in bone scaffolds; however, typical electrical stimulators are either (a) externally housed and require overcomplex percutaneous wires be connected to the implanted scaffold or (b) implanted non-degradable devices which contain toxic batteries and require invasive removal surgeries.

Objective: Here, we establish a biodegradable, piezoelectric …

Analyzing The Effect Of Apoptotic Mutations On The State Of The Nascent-Polypeptide Associated Complex In Caenorhabditis Elegans, Monica Gerber

Senior Honors Projects, 2010-2019

Cells experiencing misfolded protein stress can become debilitated and die, contributing to the onset of disease. The nascent polypeptide-associated complex (NAC) is a heterodimeric translational chaperone that protects against misfolded protein stress by mediating proper protein folding and localization during translation. Depletion of this complex results in misfolded protein accumulation in the endoplasmic reticulum (ER). To determine the importance of the NAC to proteostasis, we have previously depleted the complex in C.elegans via RNA interference and observed numerous dose-dependent effects, including apoptosis of neuronal cells and changes in gene expression of hypodermal cells. While we have observed these cell-specific responses …

Molecular Mechanisms Governing Muscle Wasting In Cancer, Aneesha Dasgupta

Theses & Dissertations

Pancreatic cancer is the third-leading cause of cancer-related deaths in the United States. About 80 percent of the pancreatic cancer patients suffer from cachexia and, about one-third die due to complexities related to the syndrome. Cachexia leads to a loss in body weight and cachectic patients are refractory to chemotherapy. Despite recent advances, the mechanisms of pancreatic cancer- cachexia and the potential therapeutic interventions remain poorly evaluated.

Sirtuins represent a class of proteins that are regulated by metabolic fluctuations in tissues. We observed a reduced expression of Sirt1 in spontaneous PDAC mice muscles, human pancreatic cancer muscles, and myotubes treated …

The Cxcr2-Dependent Role Of Cancer-Associated Fibroblasts In Pancreatic Ductal Adenocarcinoma, Mohammad Awaji

Theses & Dissertations

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer, the fourth leading cause of cancer-related deaths in the USA with over 40,000 deaths per year. Unlike other major cancer types, the progress in dealing with PDAC is plodding, attributed mainly to the asymptomatic nature of the disease, the late diagnosis and the ineffectiveness of current therapies. A better understanding of the biology of the disease could permit the discovery of novel diagnostic and therapeutic tools. With that in mind, we present this dissertation that investigates the tumor-stromal interaction underlined by genetic alterations and inflammation. PDAC develop as …

The Role Of Zyxin And Limd1 In Mitosis And Cancer, Jiuli Zhou

Theses & Dissertations

The Hippo signaling pathway, originally discovered in Drosophila, consists of a core kinase cascade and has been subsequently demonstrated to control tissue growth and tumorigenesis. The core of this pathway contains MST1/2 (Mammalian sterile 20-like kinase 1/2), LATS1/2 (large tumor suppressor 1/2) and downstream effector named Yes-associated protein (YAP) and PDZ-binding motif (TAZ). MST1/2 transduce their kinase activity mainly through directly phosphorylating LATS1/2. Once phosphorylated and activated, LATS1/2 subsequently phosphorylate and inhibit YAP/TAZ from translocating to nucleus, thereby suppressing the expression of downstream pro-growth and survival genes. While recent studies provide important insight into the tumor suppressor properties of …

Induction And Metastasis Of Cancer Stem Cells In Pancreatic Cancer, Rama Krishna Nimmakayala

Theses & Dissertations

Pancreatic cancer is one of the most lethal of all types of cancer with an overall 5-year survival rate of less than 8%. Cancer cells in pancreatic tumor are heterogeneous, and it is poorly understood which population is most responsible for the cancer initiation, progression and metastasis. Recent studies provide evidence for the existence of a highly tumorigenic and metastatic cells within a heterogeneous tumor known as the cancer stem cells (CSCs). Studies also provided ample evidence for the existence of distinct types of CSC populations in a heterogeneous tumor with type specific genotypic, phenotypic and functional characteristics. But, it …

Targeting Oncogenic Protein Sythesis In Myc-Driven B Cell Lymphoma, Xuan Zhang

Theses & Dissertations

Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) are aggressive tumors of mature B cells that can be distinguished based on histomorphological, phenotypic, and genetic features. B-cell lymphoma unclassifiable, with features intermediate between BL and DLBCL (BCL-U) is a subset of mature B cell lymphoma contains one or more features that overlap BL and DLBCL. Previous molecular analyses prove there is a biological continuum between BL and DLBCL based on the activity of MYC. Herein, in order to resolve the molecular heterogeneity of BCL-U, we tested whether a targeted expression profiling panel could categorize tumors as BL and DLBCL. …

Human Leukocyte Antigen (Hla) Class I Molecule Components And Amyloid Precursor-Like Protein 2 (Aplp2): Roles In Pancreatic Cancer Cell Migration, Bailee Sliker

Theses & Dissertations

Human leukocyte antigen (HLA) class I molecules are composed of a light chain (beta 2-microglobulin (β₂m)) and HLA heavy chain. The heavy chains of these molecules have three different isotypes (–A, -B, and –C) and are highly polymorphic with thousands of sequence variations termed allotypes. The best-known role for these molecules is within the immune system, however, recent research implicates components of this molecule can function outside of this known immune role by contributing to cell migration. However, no studies have been published thus far investigating this non-immune function in pancreatic cancer. Therefore, I examined the role of …

Identification Of Pathways Required For The Survival Of Inversion(16) Acute Myeloid Leukemia, Yiqian Wang

Theses & Dissertations

Inversion of chromosome 16 [inv(16)] acute myeloid leukemia (AML) generates a fusion gene CBFB-MYH11. Approximately half of inv(16) AML patients eventually relapse mainly due to the existence of leukemia stem cells (LSCs). Previous work using a Cbfb-MYH11 knockin mouse model showed that the LSCs are enriched within CSF2RB^- population. Another gene upregulated by Cbfb-MYH11 encodes the cytokine receptor IL1RL1. Using Cbfb-MYH11 knockin mice, we showed that LSCs exist in multiple sub-populations defined by their immunophenotype, and IL1RL1 is expressed by cell populations with high LSC activity. We also found that treatment of IL-33, the ligand for IL1RL1, promoted …

The Role Of Ros In The Progression And Treatment Of Castration-Resistant Prostate Cancer, Dannah R. Miller

Theses & Dissertations

Prostate cancer is the second leading cause of cancer-related deaths in U.S. men, primarily due to the development of castration-resistant (CR) prostate cancer (PCa), of which there are no effective treatment options. Reactive oxygen species (ROS) plays a critical role in prostate carcinogenesis, including the progression of the CR PCa phenotype. ROS regulates both cell proliferation and apoptosis; a moderate increase in ROS can promote proliferation; however, a substantial rise in ROS levels will result in apoptosis. Oxidase p66Shc is elevated in clinical PCa cells and has been associated with a metastatic phenotype of CR PCa cells, promoting PCa cell …

Developing Targeted Therapy Against Pancreatic Cancer, Garima Kaushik

Theses & Dissertations

Not available.

Anti-Crispr Vs. Crispr: The Evolutionary Arms Race Between Microorganisms, Rachael M. St. Jacques

Masters Theses, 2010-2019

CRISPR arrays are a defense mechanism employed by bacteria against viral invaders. Cas proteins do the work in detecting, capturing, and integrating the viral DNA into the CRISPR array (Barrangou et al., 2007). Anti-CRISPR proteins are produced by phages, viruses that infect bacteria, to stop the bacterial host’s CRISPR-Cas complex from interrupting the phage life cycle (Bondy-Denomy, et al., 2015).

SEA-PHAGES is a course-based bacteriophage research network composed of 120 colleges and known at James Madison University as Viral Discovery. JMU uses the unsequenced Streptomyces griseus ATCC10137 as a host for bacteriophage discovery and propagation, and in this study we …

Yfmk Is A Novel Nε-Lysine Acetyltransferase That Directly Acetylates The Histone-Like Protein Hbsu In Bacillus Subtilis, Valerie J. Carabetta, Todd M. Greco, Ileana M. Cristea, David Dubnau

Rowan-Virtua Research Day

Recently, N^e-lysine acetylation was realized to be a prevalent bacterial post-translational modification (PTM), contrary to the historical notion that this was a rare occurrence. Acetylation can impact protein function in multiple ways, by modifying conformation, interactions, subcellular localization or activity. In bacteria, hundreds of proteins are known to be acetylated, including those involved essential processes such as DNA replication, nucleoid organization, translation, cell shape, central carbon metabolism, and even several virulence factors. Despite the growing recognition that numerous proteins are acetylated, the biological significance of the vast majority of these modifications in any bacteria remains largely unknown. Previously, …

Times Of Action And Evolutionary Conservation Of Heterochronic Genes, Maria Ivanova, Eric G. Moss

Rowan-Virtua Research Day

Specific genes called heterochronic genes control the timing and sequence of developmental events during larval stages of C. elegans. Mutations in heterochronic genes can cause skipping or reiteration of cell fates associated with certain larval stages. lin-14 and lin-28 are two well-studied heterochronic genes. LIN-14 acts during the first larval stage (L1) and controls events of the L1 and L2 stages, LIN-28 acts during the L2 stage and controls its events.

Med12 Mutations Promote Castration-Resistant Prostate Cancer Through Hyperactivated Gli3/Shh Signalling, Soundharrya Muthukumar

Theses & Dissertations

When cells of the prostate gland start growing uncontrollably, it results in cancer due to genetic aberrations. The development and progression of prostate cancer depend on androgenic stimulation. Although prostate cancer is temporarily treated by depriving a tumor of androgen, the patient relapses getting a castration-resistant form of the disease called castration-resistant prostate cancer (CRPC). CRPC can specifically develop through crosstalk of androgen receptor signalling pathways. One such pathway that is of specific interest in this paper is the MED12 regulated SHH signalling pathway. The mediator is a signal processor that helps in the transduction of gene-specific transcription factors to …

Acute Promyelocytic Leukemia: A Case Study Correlating Cytogenetic Abnormality With Prognosis, Alyssa Lamond, Theresa Tellier-Castellone

Senior Honors Projects

Advancements in medical technology today have positively impacted the diagnosis, treatment, and prognosis of cancers. Particularly acute promyelocytic leukemia (APL) has completely improved from having the poorest prognosis to one of the best. Acute promyelocytic leukemia is a malignant disease of hematopoietic tissue classified by WHO as leukemia with >20% blasts from the myeloid lineage, specifically promyelocytes. Determined in 1976, FAB classified AML subtypes M1-M7, with APL being M3. Specific characteristics classify the subtype of AML, with each resulting from a different genetic abnormality. The focus of APL diagnosis, treatment, and prognosis occurs around the known PML-RARα fusion gene. Flow …

A Dedicated Chaperone Mediates The Safe Transfer Of Mitoribosomal Proteins To Their Site Of Assembly, Gabrielle Ashley Hillman

Graduate School of Biomedical Sciences Theses and Dissertations

Mitochondrial ribosomes are functionally specialized for the synthesis of several essential inner membrane proteins of the respiratory chain. While remarkable progress has recently been made towards understanding the structure of mitoribosomes, the unique pathways and factors that facilitate their biogenesis remain largely unknown. This dissertation defines the physiological role of an evolutionarily conserved yeast protein called Mam33 in mitochondrial ribosome assembly. The biomedical relevance of this finding stems from the fact that mutations or changes in its expression of the human ortholog p32 result in mitochondrial dysfunction. In human patients, bi-allelic mutations cause severe multisystemic defects in mitochondrial energy metabolism, …