Molecular Biology Commons^™

Mechanisms Adopted By Dengue-2 Viruses To Induce Autophagy In Mammalian Cells, Sounak Ghosh Roy

Dissertations, Theses, and Capstone Projects

Dengue, the most rapidly spreading flavivirus, threatens to affect almost half of the human global population. We previously showed that dengue-2 protects canine kidney cells (MDCK) from cytotoxic chemicals. We showed, independently, that cell protection, as well as viral replication and maturation, are positively regulated by PI3K-dependent autophagy. However, we had not identified the specific pathway that induces autophagy in infected cells. The current study explores the role of a specific branch of the endoplasmic reticulum (ER) stress-mediated Unfolded Protein Response (UPR), the PERK/eIF2α/ATF4 pathway in the induction of autophagy by …

N-Terminal Domain Of Human Uracil Dna Glycosylase (Hung2) Promotes Targeting To Uracil Sites Adjacent To Ssdna-Dsdna Junctions, Brian P Weiser, Gaddiel Rodriguez, Philip A Cole, James T Stivers

Rowan-Virtua School of Osteopathic Medicine Departmental Research

The N-terminal domain (NTD) of nuclear human uracil DNA glycosylase (hUNG2) assists in targeting hUNG2 to replication forks through specific interactions with replication protein A (RPA). Here, we explored hUNG2 activity in the presence and absence of RPA using substrates with ssDNA-dsDNA junctions that mimic structural features of the replication fork and transcriptional R-loops. We find that when RPA is tightly bound to the ssDNA overhang of junction DNA substrates, base excision by hUNG2 is strongly biased toward uracils located 21 bp or less from the ssDNA-dsDNA junction. In the absence of RPA, hUNG2 still showed an 8-fold excision bias …

Direct Quantification Of Deubiquitinating Enzyme Activity In Single Intact Cells, Nora Safabakhsh

LSU Doctoral Dissertations

Challenges in drug efficacy occur during the treatment of most types of cancer due to the heterogeneity of the tumor microenvironment. This has led to the development of personalized medicine. Due to the clinical success of the proteasome inhibitors Bortezomib and Carfilzomib in treatment of multiple myeloma, interest has shifted towards molecularly-targeted chemotherapeutics for ubiquitin-proteasome system (UPS). Deubiquitinating enzymes (DUBs) are an essential part of this pathway which have been found to promote Bortezomib resistance in multiple myeloma patients. Unfortunately, there is a lack of specific, high throughput biochemical assays to characterize DUB activity in patient samples before and after …

Fars2 Mutations Presenting With Pure Spastic Paraplegia And Lesions Of The Dentate Nuclei, Supreet K. Sahai, Rebecca E. Steiner, Margaret G. Au, John M. Graham, Norikio Salamon, Michael Ibba, Tyler M. Pierson

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Mutations in FARS2, the gene encoding the mitochondrial phenylalanine‐tRNA synthetase (mtPheRS), have been linked to a range of phenotypes including epileptic encephalopathy, developmental delay, and motor dysfunction. We report a 9‐year‐old boy with novel compound heterozygous variants of FARS2, presenting with a pure spastic paraplegia syndrome associated with bilateral signal abnormalities in the dentate nuclei. Exome sequencing identified a paternal nonsense variant (Q216X) lacking the catalytic core and anticodon‐binding regions, and a maternal missense variant (P136H) possessing partial enzymatic activity. This case confirms and expands the phenotype related to FARS mutations with regards to clinical presentation and neuroimaging findings.

Gq Noncanonical Roles In Translational Regulation, Brett Demarco

Electronic Theses and Dissertations

This study investigates protein nucleic acid interactions, focusing on G-quadruplex (GQ) forming DNA/RNA in human disease. GQ structures are formed in DNA/RNA, when four guanine residues form planar tetrads stabilized by Hoogsteen base pairing, that stack forming a GQ structure stabilized by potassium ions. These GQ structures are targeted by the arginine glycine-glycine (RGG) RNA-binding domain. Fragile X mental retardation protein (FMRP), a translation regulator protein implicated in the fragile X syndrome, has an RGG domain and has been previously shown to interact with neuronal GQ forming messenger RNA (mRNA). We have investigated three neuronal FMRP mRNA targets that we …

Microbial Ecology Of South Florida Surface Waters: Examining The Potential For Anthropogenic Influences, Chase P. Donnelly

HCNSO Student Theses and Dissertations

South Florida contains one of the largest subtropical wetlands in the world, and yet not much is known about the microbes that live in these surface waters. These microbes play an important role in chemical cycling and maintaining good water quality for both human and ecosystem health. The hydrology of Florida’s surface waters is tightly regulated with the use of canal and levee systems run by the US Army Corps of Engineers and The South Florida Water Management District. These canals run through the Everglades, agriculture, and urban environments to control water levels in Lake Okeechobee, the Water Conservation Areas, …

Characterization Of Genes Involved In The Biosynthesis Of Phycoerythrin I And Ii In Cyanobacteria, Adam Nguyen

LSU New Orleans Theses and Dissertations

Cyanobacteria are photosynthetic prokaryotes that able to produce oxygen. They have light harvesting complexes called phycobilisomes (PBS). PBS are generally composed of an allophycocyanin core with phycocyanin and phycoerythrin rods connected to the core. PBS are able to efficiently harvest light energy from different wavelengths of visible light due to the evolution of PBP. Phycoerythrin has five chromophores that are attached to six cysteine residues and is essential for efficient green light capture and transfer of energy for use in photosynthesis. The attachment of these chromophores to PBP is facilitated by enzymes known as bilin lyases.

In this study, we …

Structural And Functional Characterization Of Hyper-Phosphorylated Grk5 Protein Expressed From E. Coli, Joseph M. Krampen, John Tesmer, Qiuyan Chen

The Summer Undergraduate Research Fellowship (SURF) Symposium

G protein-coupled receptor (GPCR) kinases (GRKs) are proteins in the cell responsible for regulating GPCRs located on the cell membrane. GRKs regulate active GPCRs by phosphorylating them at certain sites which causes them to stop normal signaling on the membrane. This ultimately affects how the cell responds to its environment. GRK5 is a kinase of particular interest due to its involvement in the pathology of diseases such as cardiac failure, cancers, and diabetes. Understanding the structure and function of GRK5 is essential for discovering ways to manipulate its behavior with these diseases, but not much is known about how GRK5 …

Improving Biomanufacturing Production With Novel Elp-Based Transcriptional Regulators, Juya Jeon, Logan R. Readnour, Kevin V. Solomon

The Summer Undergraduate Research Fellowship (SURF) Symposium

Microbes can be used to produce valuable drugs, chemicals, and biofuels, but their potential has not been fully realized due to low production yields. To improve biomanufacturing processes and yield, we are developing novel, transcriptional regulators using biosynthesis technology in order to improve cellular health and overall production. Our regulator contains elastin-like polypeptides (ELPs), which make ideal sensors since they exhibit a sharp, inverse phase transition to indicators of cell health such as intracellular pH and ionic strength, and external stimuli such as temperature. We hypothesize that ELP can be fused to transcription factors to control expression of target genes. …

Engineering Bioluminescent Sensors Of Cyclic Amp To Study Opioid Signaling, Alexander L. Tesmer, Alexander R. French, Mathew Tantama

The Summer Undergraduate Research Fellowship (SURF) Symposium

Opioids are small signaling molecules which bind to opioid receptors on the surface of cells. The kappa opioid receptor (KOR) is one of three major types of opioid receptors found in human neurons. When an opioid binds to a KOR, a variety of biochemical signaling pathways are activated inside the cell. Each of these pathways are associated with different physiological effects of KOR activation. The production of a small signaling molecule, cyclic adenosine monophosphate (cAMP), is known to be inhibited during KOR activation of the analgesic (pain-killing) signaling pathway. The ability to interrogate the individual responses of KOR signaling pathways …

Cooperative Regulation Of Translational Repression By Fmrp And The Mirna Pathway, Navneeta Kaul

Electronic Theses and Dissertations

Fragile X syndrome (FXS) is the most common inherited monogenic cause of intellectual disability. FXS patients exhibit social and language deficits, hyperactivity, seizures, growth abnormalities, macroorchidism, anxiety, and epilepsy. FXS is caused by the transcriptional silencing of the fragile X mental retardation gene 1 (Fmr1), resulting in the loss of the fragile X mental retardation protein (FMRP). FMRP is a selective mRNA binding protein that plays a role in translation repression. Studies suggest that FMRP utilizes the miRNA pathway to repress translation of its target mRNAs through an unknown mechanism. The aim of my thesis is to investigate …

The Role Of Developmental Timing Regulators In Progenitor Proliferation And Cell Fate Specification During Mammalian Neurogenesis, Jennifer S. Romer-Seibert

Graduate School of Biomedical Sciences Theses and Dissertations

Developmental timing is a key aspect of tissue and organ formation in which distinct cell types are generated through a series of steps from common progenitors. These progenitors undergo specific changes in gene expression that signifies both a distinct progenitor type and developmental time point that thereby specifies a particular cell fate at that stage of development. The nervous system is an important setting for understanding developmental timing because different cell types are produced in a certain order and the switch from stem cells to progenitors requires precise timing and regulation. Notable examples of such regulatory molecules include the RNA-binding …

Inhibition Of Ribosome Biogenesis Through Genetic And Chemical Approaches, Leonid Anikin

Graduate School of Biomedical Sciences Theses and Dissertations

In order to maintain the ability to generate proteins, proliferating cells must continuously generate ribosomes, designating up to 80% of their energy to ribosome biogenesis (RBG). RBG involves transcription of rDNA by RNA polymerases I (Pol I) and III (Pol III), expression of approximately 80 ribosomal proteins, and assembly of these components in a process referred to as ribosome maturation. During maturation, the Pol I transcribed 47S pre-rRNA undergoes a number of processing events, while simultaneously interacting with processing factors and ribosomal proteins that drive pre-ribosome assembly. Inhibition of RBG has become one of the pursued targets for cancer therapy …

Insight Into Translational Activation In Yeast Mitochondria, Julia Lynn Jones

Graduate School of Biomedical Sciences Theses and Dissertations

Mitochondrial function depends on over a thousand proteins, of which the majority are nuclear DNA-encoded and approximately one percent are mitochondrial DNA-encoded. The mitochondrial DNA of Saccharomyces cerevisiae contains eight protein-encoding genes, seven of which are required for proper function of the respiratory complexes and one encodes a ribosomal protein. The bigenomic nature of the oxidative phosphorylation complexes requires coordinated expression and regulation from both the nuclear and the mitochondrial genomes. It is currently unclear how this regulatory network operates. However, it is thought that nuclear genome-encoded messengers localized to the mitochondria aid in this coordination.

A family of proteins …

The N-Terminal Methyltransferase Homologs Nrmt1 And Nrmt2 Exhibit Novel Regulation Of Activity Through Heterotrimer Formation., Jon David Faughn

Electronic Theses and Dissertations

Protein, DNA, and RNA methyltransferases have an ever-expanding list of novel substrates and catalytic activities. Even within families and between homologs, it is becoming clear the intricacies of methyltransferase specificity and regulation are far more diverse than originally thought. In addition to specific substrates and distinct methylation levels, methyltransferase activity can be altered through formation of complexes with close homologs. This work involves the N-terminal methyltransferase homologs NRMT1 and NRMT2. NRMT1 is a ubiquitously expressed distributive trimethylase. NRMT2 is a monomethylase expressed at low levels and in a tissue-specific manner. They are both nuclear methyltransferases with overlapping target consensus sequences …

Bayesian Analytical Approaches For Metabolomics : A Novel Method For Molecular Structure-Informed Metabolite Interaction Modeling, A Novel Diagnostic Model For Differentiating Myocardial Infarction Type, And Approaches For Compound Identification Given Mass Spectrometry Data., Patrick J. Trainor

Electronic Theses and Dissertations

Metabolomics, the study of small molecules in biological systems, has enjoyed great success in enabling researchers to examine disease-associated metabolic dysregulation and has been utilized for the discovery biomarkers of disease and phenotypic states. In spite of recent technological advances in the analytical platforms utilized in metabolomics and the proliferation of tools for the analysis of metabolomics data, significant challenges in metabolomics data analyses remain. In this dissertation, we present three of these challenges and Bayesian methodological solutions for each. In the first part we develop a new methodology to serve a basis for making higher order inferences in metabolomics, …

Deciphering The Role Of Human Arylamine N-Acetyltransferase 1 (Nat1) In Breast Cancer Cell Metabolism Using A Systems Biology Approach., Samantha Marie Carlisle

Electronic Theses and Dissertations

Background: Human arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic metabolizing enzyme found in almost all tissues. NAT1 can additionally hydrolyze acetyl-coenzyme A (acetyl-CoA) in the absence of an arylamine substrate. NAT1 expression varies inter-individually and is elevated in several cancers including estrogen receptor positive (ER+) breast cancers. Additionally, multiple studies have shown the knockdown of NAT1, by both small molecule inhibition and siRNA methods, in breast cancer cells leads to decreased invasive ability and proliferation and decreased anchorage-independent colony formation. However, the exact mechanism by which NAT1 expression affects cancer risk and progression remains unclear. Additionally, consequences …

Uncoupling The Folding And Binding Of An Intrinsically Disordered Protein, Anusha Poosapati, Emily Gregory, Wade M. Borcherds, Lucia B. Chemes, Gary Daughdrill

Molecular Biosciences Faculty Publications

The relationship between helical stability and binding affinity was examined for the intrinsically disordered transactivation domain of the myeloblastosis oncoprotein, c-Myb, and its ordered binding partner, KIX. A series of c-Myb mutants was designed to either increase or decrease helical stability without changing the binding interface with KIX. This included a complimentary series of A, G, P, and V mutants at three non-interacting sites. We were able to use the glycine mutants as a reference state and show a strong correlation between binding affinity and helical stability. The intrinsic helicity of c-Myb is 21%, and helicity values of the …

Chronic Cadmium Exposure Alters Erα Dependency And Drug Sensitivity Of Breast Cancer Cells, Mathew Bloomfield

Dissertations, Masters Theses, Capstones, and Culminating Projects

The global prevalence of breast cancer in women illustrates the importance of identifying factors that contribute to disease onset and progression. Endogenous and environmental agents that interact with estrogen receptor alpha (ERα) have been shown to play a role in breast cancer etiology. Evidence from epidemiological studies and animal models has suggested that cadmium, a heavy metal that can activate ERα, contributes to the development and progression of breast cancer. Additionally, our lab showed that chronic cadmium exposure altered the expression of several ERα-responsive genes and increased the malignancy of MCF7 breast cancer cells. Although these studies support cadmium’s function …

Gut Symbiont Viability In Honey Bees Exposed To Agrochemical Stressors, Bryant Justin Gabriel

Department of Entomology: Dissertations, Theses, and Student Research

The honey bee gut microbiome is essential for protecting this pollinator against abiotic and biotic stressors, including the prevention of harmful gut parasites and pathogens. Previous studies have not only demonstrated a linkage of bee gut dysbiosis to increased immunodeficiencies and pathogen sensitivities, but also report the maladaptation of the gut microbiome in bees exposed to agricultural and apicultural chemistries. There are few techniques available that allow for a simple and reliable analysis of the relative proportions of live and dead gut microbes in bees exposed to these chemistries. Previous techniques for measuring gut symbiont dysbiosis are temporally limited by …