Molecular Biology Commons^™

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 …

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, …

Paraoxonase 2 Is Critical For Non-Small Cell Lung Carcinoma Proliferation., Aaron Whitt

Electronic Theses and Dissertations

Non-small cell lung carcinoma (NSCLC) comprises 85% of lung cancer diagnoses and is plagued by drug resistance. Thus, elucidating the underlying mechanisms of NSCLC is paramount to expand future treatment options. Paraoxonase 2 (PON2), an intracellular enzyme with arylesterase and lactonase functions, has well-established anti-atherosclerotic activity. Recent studies show PON2 is overexpressed in a variety of tumors and confers drug resistance, although these interactions have not been thoroughly examined in NSCLC. Thus, we sought to investigate the role of PON2 in cellular proliferation using PON2-knockout mice, primary mouse cells, and NSCLC cell lines. Using these approaches, we demonstrate that PON2 …

Spatial Reorganization Of Histone-Like Nucleoid Structuring Proteins Caused By Silver Nanoparticles, Meaad Alqahtany

Graduate Theses and Dissertations

Silver nanoparticles (AgNPs) and ions (Ag+) can be the new generation of antibiotics due to their antimicrobial effects against bacteria and other microbes. Many studies have shown that AgNPs and suppress the growth of bacteria and damage the cell walls of the microbes; therefore, treating bacterial cells with AgNPs may be a promising method to terminate multi-resistant bacteria. In this work, the effect of AgNPs with two different surface coatings on the spatial reorganization of histone-like nucleoid structuring (H-NS) proteins in Escherichia coli bacteria was investigated using quantitative super-resolution fluorescence microscopy to understand the toxicity and antimicrobial mechanism of AgNPs. …

The Role And Regulation Of Alternative Polyadenylation In The Dna Damage Response, Michael R. Murphy

Dissertations, Theses, and Capstone Projects

Cellular homeostasis is achieved by the dynamic flux in gene expression. Post-transcriptional regulation of coding and non-coding RNA offers a fast method of adapting to a changing cellular environment, including deadenylation, microRNA (miRNA) pathway, and alternative polyadenylation (APA). In this dissertation, I explored some of the mechanisms involved in the post-transcriptional regulation of gene expression. The main hypothesis in these studies is that a single APA event after DNA damage is governed by specific conditions and factors outside of current known regulators of APA, and that the resultant transcript has a role in the DNA damage response (DDR). My aims …

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

Dissertations and 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 …

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

Dissertations and 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. …

Cellular Localization Of Rad51d Mutant Proteins And The Application Of Art To Increase Scientific Literacy In America, Claire L. Chabot

Senior Theses

Ovarian cancers are the leading cause of death from cancer of the female reproductive system. Approximately 50% of ovarian cancers have defects in the homologous recombination (HR) DNA repair pathway that is required for the repair of DNA double-stranded breaks. The status of HR genes, such as BRCA1, BRCA2, and the RAD51 family, contributes to ovarian cancer development as well as treatment decisions regarding chemotherapy, radiation, and immunotherapy. The overarching goal of this project is to identify new insights into HR that can integrate with Precision Medicine Initiatives and align with the goals of the Cancer Moonshot 2020 Program. I …

Delineation Of Events In Centripetal Migration During Drosophila Oogenesis, Travis Tait Parsons

UNLV Theses, Dissertations, Professional Papers, and Capstones

All multicellular organisms initially start out as a single cell. This cell must use the genetic information encoded in its DNA to multiply in number and build itself into a complex multicellular organism. How this process occurs is the focus of developmental biology, a field that seeks to understand how a combination of genetic information and environmental conditions shape a cell from its beginnings as a zygote all the way to maturity. A fundamental part of this process is the ability of cells to work together in order to build complex tissues and organs. Cells achieve this coordination by using …

Hormone Signaling, Gene Expression, And Mitochondrial Hormone Receptor Expression In Avian Muscle (Cells), Kentu Rushadd Lassiter

Graduate Theses and Dissertations

Mitochondria are vital to the proper growth and function of muscle cells since they’re responsible for the majority of ATP production used for cellular energy. Previous studies have investigated how differences in mitochondrial function affects feed efficiency (FE) in broilers phenotyped for High and Low FE. Low FE broilers have been shown to have increased levels of reactive oxygen species (ROS), thus contributing to higher levels of oxidative stress and damage seen in these birds. Global gene and protein expression studies conducted on breast muscle of the High FE and Low FE phenotypes have suggested that differences in mitochondrial function …

Gamete Nuclear Migration In Animals And Plants, Umma Fatema, Mohammad F. Ali, Zheng Hu, Anthony J. Clark, Tomokazu Kawashima

Plant and Soil Sciences Faculty Publications

The migration of male and female gamete nuclei to each other in the fertilized egg is a prerequisite for the blending of genetic materials and the initiation of the next generation. Interestingly, many differences have been found in the mechanism of gamete nuclear movement among animals and plants. Female to male gamete nuclear movement in animals and brown algae relies on microtubules. By contrast, in flowering plants, the male gamete nucleus is carried to the female gamete nucleus by the filamentous actin cytoskeleton. As techniques have developed from light, electron, fluorescence, immunofluorescence, and confocal microscopy to live-cell time-lapse imaging using …

Mechanisms Of Oriented Cell Division And Their Roles In Tissue Development, Evan Blake Dewey

Biology ETDs

Properly executed cell division is crucial to development, maintenance, and longevity of multicellular organisms. Defects in both symmetric and asymmetric divisions can lead to improper developmental patterning, as well as genomic instability, disruption of tissue homeostasis, and cancer. Our research focuses on how regulators orchestrate proper cell divisions. Mushroom Body Defect (Mud) is one such regulator, and here we describe how Mud is regulated via the Hippo signaling pathway kinase Warts (Wts), showing Wts phosphorylates Mud to enhance interaction with the polarity protein Partner of Inscuteable, promoting spindle orientation activity. We next focus on another regulator, Shortstop (Shot), describing a …

Novel Insights Into The Multifaceted Roles Of Blm In The Maintenance Of Genome Stability, Vivek M. Shastri

USF Tampa Graduate Theses and Dissertations

Genomic instability is a hallmark of disorders in which DNA replication and repair genes are dysfunctional. The tumor suppressor RECQ helicase gene BLM encodes the 3’-5’ DNA Bloom syndrome helicase BLM, which plays important roles during DNA replication, recombination and repair to maintain genome stability. Mutations within BLM cause Bloom syndrome, an autosomal recessive disorder characterized by growth defects, immunodeficiency, >10-fold higher sister chromatid exchange compared to normal cells, and an increased predisposition to a wide range of cancers from an early age. Single nucleotide polymorphisms or SNPs in BLM have been reported to be associated with susceptibility to a …

Ultrafine Carbon Nanoparticles Activate Inflammasome Signaling And Cell Death In Murine Macrophages, Alexander Soloniuk, Hadley Lamascus, Jay Brewster, John Mann

Seaver College Research And Scholarly Achievement Symposium

Carbon black (CB) is the primary nanoparticulate component of air pollution from fossil fuel combustion. This work examines the cellular impact of ultrafine carbon (carbon black, CB) nanoparticles, that range in size down to 30 nm, upon murine macrophages. The size analysis of the carbon black nanoparticles was performed using atomic force microscopy (AFM) and transmission electron microscopy (TEM) techniques. RAW246.7 macrophage cells were exposed to CB doses ranging from 50 – 200 ug/ml in complete media. Analysis of cell survival over time revealed elevated rates of significant nuclear degradation and cell lifting after 48 hours of exposure, and in …

Investigating The Role Of The Chromosome 19 Microrna Cluster In Human Trophoblast Differentiation And Infantile Hemangioma, Ezinne Francess Mong

USF Tampa Graduate Theses and Dissertations

Trophoblast differentiation and invasion is essential for normal implantation and establishment of the maternal-fetal interface, which allows for proper nutrient exchange and support of the fetus. For this to occur, cytotrophoblasts must undergo an epithelial to mesenchymal transition and differentiate into migratory and invasive extravillous trophoblasts (EVTs) that invade the maternal decidua and myometrium. Trophoblast differentiation, migration and invasion is highly regulated by a complex network of signaling pathways, adhesion molecules and transcription factors and is important for the remodeling of maternal spiral arteries from low flow, high resistance to high flow, low resistance vessels to allow optimal perfusion of …

Conserved Glycine Residues Control Transient Helicity And Disorder In The Cold Regulated Protein, Cor15a, Oluwakemi Sowemimo

USF Tampa Graduate Theses and Dissertations

COR15A is a cold regulated disordered protein from Arabidopsis thaliana that contributes to freezing tolerance in plants by protecting membranes. It belongs to the (LEA) Late Embryogenesis Abundant group of proteins that accumulate during the later stage of seed development and are expressed in various parts of the plant. During freezing-induced cellular dehydration, COR15A transitions from a disordered structure to a mostly α-helical structure that binds and stabilizes chloroplast membranes when cells dehydrate due to freezing. We hypothesize that increasing the transient α-helicity of COR15A under normal conditions will increase its ability to bind and protect chloroplast membranes when cells …

Biochemical And Proteomic Approaches To Determine The Impact Level Of Each Step Of The Supply Chain On Tomato Fruit Quality, Robert T. Madden

USF Tampa Graduate Theses and Dissertations

Fresh fruits and vegetables (FFVs) are the most frequently wasted foods because of their perishability and handling requirements. However, there is a lack of information on how much each step of the supply chain impacts FFVs quality, particularly on tomatoes, and what measures need to be taken for an immediate and effective impact on waste reduction. There is also no information on how the supply chain affects the proteome of the tomato and what proteins are differentially regulated by the most impactful steps of the supply chain. The objectives of the work presented on this thesis were to evaluate the …

The Drosophila Neuroblasts: A Model System For Human Ribosomopathies, Sonu Shrestha Baral

LSU Doctoral Dissertations

This dissertation describes the use of Drosophila neuroblasts (NBs) to model human ribosomopathies; the overall goal is to understand why specific stem cell and progenitor cell populations are the primary targets in nucleolar stress as seen in the ribosomopathies. Chapter 1 provides an overview of relevant literature. Chapter 2 describes nucleolar stress in Drosophila neuroblasts as a model for human ribosomopathies. For this, we induce nucleolar stress by using the UAS-GAL4 system to express RNAi that depletes Nopp140 transcripts, and we also employ homozygous, CRISPR-Cas9-generated Nopp140 gene disruptions with a systemic null phenotype (Nopp140-/-). Embryonic lethality was observed …

Integrated Molecular Characterization Of Lung Adenocarcinoma With Implications For Immunotherapy, Nicholas T. Gimbrone

USF Tampa Graduate Theses and Dissertations

This dissertation covers a variety of the genetic and molecular abnormalities of lung adenocarcinoma with an emphasis on STK11 loss and its implications on immunotherapy response. Given that lung cancer is the leading cancer killer, novel therapies are in great demand. In particular, immunotherapy has shown some of the most promise in the last decade but remains limited due to nearly 80% of patients not significantly responding. This dissertation aims to molecularly characterize lung adenocarcinoma while attempting to explain the reason why patients with STK11 loss do not respond to immunotherapy.

In the first chapter we discuss the relationship between …

Deletion Of Mgr2p Affects The Gating Behavior Of The Tim23 Complex, Oygul Mirzalieva, Shinhye Jeon, Kevin Damri, Ruth Hartke, Layla Drwesh, Keren Demishtein-Zohary, Abdussalam Azem, Cory D. Dunn, Pablo M. Peixoto

Publications and Research

The TIM23 complex is a hub for translocation of preproteins into or across the mitochondrial inner membrane. This dual sorting mechanism is currently being investigated, and in yeast appears to be regulated by a recently discovered subunit, the Mgr2 protein. Deletion of Mgr2p has been found to delay protein translocation into the matrix and accumulation in the inner membrane. This result and other findings suggested that Mgr2p controls the lateral release of inner membrane proteins harboring a stop-transfer signal that follows an N-terminal amino acid signal. However, the mechanism of lateral release is unknown. Here, we used patch clamp electrophysiology …

Insights Into The Ribosomal, Extra-Ribosomal And Developmental Role Of Rp L13a In Mammalian Model, Ravinder Kour

ETD Archive

Ribosomal protein L13a plays an extra-ribosomal function in translational silencing of GAIT (IFN-gamma-activated inhibitor of translation) element bearing mRNAs encoding inflammatory proteins but the underlying molecular mechanism of translational silencing and ribosomal incorporation of L13a remains poorly understood. Also, our laboratory showed that L13a acts as a physiological defense against uncontrolled inflammation in macrophage-specific knockout (KO) mice. However, the consequence of a total knockout of L13a in mammals remains unexplored. Therefore, our current study is focused on (i) identifying the amino acid residue(s) of L13a essential for incorporation and translational silencing of target mRNAs and (ii) studying the consequences of …

The Role Of The Cell-Surface Protease Tmprss13 In Colorectal Cancer, Fausto Alexander Varela

Wayne State University Dissertations

Colorectal cancer (CRC) is one of the most common and deadly cancers in both men and women in the United States. Extracellular proteolysis is often dysregulated in cancer including (CRC), resulting in degradation of extracellular matrix, as well as cleavage, processing, or shedding of cell adhesion molecules, growth factors, and cytokines. Several members of the type II transmembrane serine protease (TTSP) family have been shown to play critical roles in cancer progression; however, many family members have not yet been characterized in malignancy. We identified TMPRSS13 transcript to be upregulated in CRC compared to normal colon. This increase was confirmed …

Cornus Officinalis Significantly Improves Oxidative Capacity And Promotes The Calcium-Dependent Transcription Factor, Nfatc2, In Human 1.1b4 Pancreatic Cell Line, Arielle Sharp, Alexis Coiner, Brant Burkhardt

Molecular Biosciences Faculty Publications

Type 1 diabetes (T1D) is an autoimmune disease resulting in the destruction of pancreatic β cells (β-cells) and subsequent loss of insulin production. The only treatment for T1D is using exogenous insulin coupled with continual glucose monitoring following significant autoimmune destruction of β-cells. Novel interventional therapies are needed that can preserve and protect existing pancreatic β cells in individuals with early identified T1D autoimmunity. Our initial in-vitro evidence indicates Cornus officinalis (CO) may be able to serve in this function. What sets ethnopharmacology apart from conventional medicine is the simultaneous targeting of multiple mechanisms using a single herb due to …

Structural Basis Of Phosphatidylcholine Recognition By The C2–Domain Of Cytosolic Phospholipase A,2Α, Yoshinori Hirano, Yong-Guang Gao, Daniel J. Stephenson, Ngoc T. Vu, Lucy Malinina, Dhirendra K. Simanshu, Charles E. Chalfant, Dinshaw J. Patel, Rhoderick E. Brown

Molecular Biosciences Faculty Publications

Ca²⁺-stimulated translocation of cytosolic phospholipase A2α (cPLA2α) to the Golgi induces arachidonic acid production, the rate-limiting step in pro-inflammatory eicosanoid synthesis. Structural insights into the cPLA2α preference for phosphatidylcholine (PC)-enriched membranes have remained elusive. Here, we report the structure of the cPLA2α C2-domain (at 2.2 Å resolution), which contains bound 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) and Ca2+ ions. Two Ca2+ are complexed at previously reported locations in the lipid-free C2-domain. One of these Ca2+ions, along with a third Ca2+, bridges the C2-domain to the DHPC phosphate group, which also interacts with Asn65. Tyr96 plays a key role in lipid …

Conserved Glycines Control Disorder And Function In The Cold-Regulated Protein, Cor15a, Oluwakemi T. Sowemimo, Patrick Knox-Brown, Wade M. Borcherds, Tobias Rindfleisch, Anja Thalhammer, Gary W. Daughdrill

Molecular Biosciences Faculty Publications

Cold-regulated (COR) 15A is an intrinsically disordered protein (IDP) from Arabidopsis thaliana important for freezing tolerance. During freezing-induced cellular dehydration, COR15A transitions from a disordered to mostly α-helical structure. We tested whether mutations that increase the helicity of COR15A also increase its protective function. Conserved glycine residues were identified and mutated to alanine. Nuclear magnetic resonance (NMR) spectroscopy was used to identify residue-specific changes in helicity for wildtype (WT) COR15A and the mutants. Circular dichroism (CD) spectroscopy was used to monitor the coil–helix transition in response to increasing concentrations of trifluoroethanol (TFE) and ethylene glycol. The impact of the COR15A …

Functional Inhibition Of Acid Sphingomyelinase Disrupts Infection By Intracellular Bacterial Pathogens, Chelsea L. Cockburn, Ryan S. Green, Sheela R. Damle, Rebecca K. Martin, Naomi N. Ghahrai, Punsiri M. Colonne, Marissa S. Fullerton, Daniel H. Conrad, Charles E. Chalfant, Daniel E. Voth, Elizabeth A. Rucks, Stacey D. Gilk, Jason A. Carlyon

Molecular Biosciences Faculty Publications

Intracellular bacteria that live in host cell–derived vacuoles are significant causes of human disease. Parasitism of low-density lipoprotein (LDL) cholesterol is essential for many vacuole-adapted bacteria. Acid sphingomyelinase (ASM) influences LDL cholesterol egress from the lysosome. Using functional inhibitors of ASM (FIASMAs), we show that ASM activity is key for infection cycles of vacuole-adapted bacteria that target cholesterol trafficking—Anaplasma phagocytophilum, Coxiella burnetii, Chlamydia trachomatis, and Chlamydia pneumoniae. Vacuole maturation, replication, and infectious progeny generation by A. phagocytophilum, which exclusively hijacks LDL cholesterol, are halted and C. burnetii, for which lysosomal cholesterol accumulation is bactericidal, …

Functional Analysis Of The Replication Fork Proteome Identifies Bet Proteins As Pcna Regulators, Sarah R. Wessel, Kareem N. Mohni, Jessica W. Luzwick, Huzefa Dungrawala, David Cortez

Molecular Biosciences Faculty Publications

Identifying proteins that function at replication forks is essential to understanding DNA replication, chromatin assembly, and replication-coupled DNA repair mechanisms. Combining quantitative mass spectrometry in multiple cell types with stringent statistical cutoffs, we generated a high-confidence catalog of 593 proteins that are enriched at replication forks and nascent chromatin. Loss-of-function genetic analyses indicate that 85% yield phenotypes that are consistent with activities in DNA and chromatin replication or already have described functions in these processes. We illustrate the value of this resource by identifying activities of the BET family proteins BRD2, BRD3, and BRD4 in controlling DNA replication. These proteins …

P53 Phosphomimetics Preserve Transient Secondary Structure But Reduce Binding To Mdm2 And Mdmx, Robin Levy, Emily Gregory, Wade Borcherds, Gary W. Daughdrill

Molecular Biosciences Faculty Publications

The disordered p53 transactivation domain (p53TAD) contains specific levels of transient helical secondary structure that are necessary for its binding to the negative regulators, mouse double minute 2 (Mdm2) and MdmX. The interactions of p53 with Mdm2 and MdmX are also modulated by posttranslational modifications (PTMs) of p53TAD including phosphorylation at S15, T18 and S20 that inhibits p53-Mdm2 binding. It is unclear whether the levels of transient secondary structure in p53TAD are changed by phosphorylation or other PTMs. We used phosphomimetic mutants to determine if adding a negative charge at positions 15 and 18 has any effect on the transient …

Identification Of Proteins Interacting With Zebrafish Miro Orthologs In The Yeast Two-Hybrid System, Yiran Tao

Honors Program Theses

The motility of mitochondria is required for providing energy and maintaining basic cellular functions in neurons. Mitochondrial Rho GTPase (Miro) is an essential component of the mitochondrial trafficking machinery, a protein complex that anchors mitochondria to the microtubule motor protein for long-distance transport. As a protein localizing to the mitochondrial outer membrane, Miro regulates the morphology and the motility of mitochondria. Upon mitochondrial depolarization, the PINK1/Parkin pathway is activated to arrest mitochondrial movement by targeting Miro for phosphorylation and ubiquitination in a calcium-sensitive manner. However, it remains unclear what effects the PINK1-dependent phosphorylation has on the structure and binding properties …