Molecular Biology Commons^™

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 …

An Expanded Toolkit For Gene Tagging Based On Mimic And Scarless Crispr Tagging In, David Li-Kroeger, Oguz Kanca, Pei-Tseng Lee, Sierra Cowan, Michael T Lee, Manish Jaiswal, Jose Luis Salazar, Yuchun He, Zhongyuan Zuo, Hugo J Bellen

Faculty Publications

We generated two new genetic tools to efficiently tag genes in Drosophila. The first, Double Header (DH) utilizes intronic MiMIC/CRIMIC insertions to generate artificial exons for GFP mediated protein trapping or T2A-GAL4 gene trapping in vivo based on Cre recombinase to avoid embryo injections. DH significantly increases integration efficiency compared to previous strategies and faithfully reports the expression pattern of genes and proteins. The second technique targets genes lacking coding introns using a two-step cassette exchange. First, we replace the endogenous gene with an excisable compact dominant marker using CRISPR making a null allele. Second, the insertion is replaced …

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

University of 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 …

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 Interleukin (Il)-1Β Regulatory Elements And Chromatin Conformation In Macrophage Activation, Woohyun Cho

Electronic Thesis and Dissertation Repository

IL-1β is a potent inflammatory cytokine promptly expressed in activated myeloid cells. PU.1 is a lineage-determining transcription factor that regulates myeloid-specific genes by activating distal enhancers. This study addresses the functional importance of a potential enhancer of IL-1β, and how PU.1 regulates its activity state. A putative enhancer RNA (eRNA) was transcribed from the enhancer, and eRNA knock-down with antisense oligonucleotides inhibited IL-1β production. Furthermore, enhancer-promoter interactions in stimulated macrophages were detected via chromatin conformation capture (3C) analysis. The role of PU.1 was examined in PU.1-overexpressed B16-BL6 cells, which responded to LPS and expressed IL-1β mRNA and eRNAs. Enhancer knock-out …

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 …

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 …

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. …

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 …

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 …

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 …

The Regulation Of Notch Signaling By Src Kinase And Polyphenolic Compounds, Bryce David Lafoya

Boise State University Theses and Dissertations

Cellular signaling pathways provide cells with the means to sense their environment and communicate with other cells. The Notch signaling pathway is comprised of a set of protein machines which work in unison to coordinate cellular processes in response to stimuli coming from neighboring cells and changing microenvironmental conditions. Notch signaling is an important mode of cellular communication which is crucial to many processes involved in development and disease. During Notch activation, information about the extracellular environment is fed into the cell and relayed to the nucleus through a number of biochemical processes. The information-rich messages carried by Notch signaling …

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 …

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 …

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, …

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 …

Canonical Nuclear Factor-Κb Signaling In Satellite Stem Cell Homeostasis And Function., Alex R. Straughn

Electronic Theses and Dissertations

Satellite cells are adult stem cells that are required for the regeneration of skeletal muscle following injury. However, the signaling mechanisms that regulate satellite stem cell homeostasis and function in adult animals remain less understood. Nuclear factor-kappa B (NF-kB) is a major nuclear transcription factor that regulates the gene expression of a plethora of molecules involved in cellular proliferation, differentiation, survival, and the inflammatory immune response. NF-kB can be activated through a canonical or non-canonical pathway. However, the role of canonical NF-kB signaling in the regulation of satellite stem cell function during skeletal muscle regeneration has not been yet investigated …

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 …

The Role Of Rad4 In Dna Repair And Its Interplay With Telomeres In Tetrahymena Thermophila, Emily Nischwitz

MSU Graduate Theses

Telomeres are repetitive parts of the genome that act as a protective end cap to the chromosomes. Telomeres are critical to the integrity and stability of the genome, therefore, ensuring that their sequence is maintained, even after damage, is crucial. Much of the pioneering work responsible for explaining telomeres has been conducted in ciliates, specifically in Tetrahymena thermophila. Telomeres in T. thermophila have a high amount of tandem thymine repeats (GGGGTT) and, thus, are susceptible to ultraviolet light (UV) induced lesions called pyrimidine dimers, which must be repaired by nucleotide excision repair (NER). In humans, Xeroderma Pigmentosum C (XPC) …

Computational Analysis Of Genomic Variants Affecting Predicted Microrna:Target Interactions In Prostate Cancer., Angélica Paola Hernández Pérez

KGI Theses and Dissertations

Prostate cancer (PCa) is the most common cancer of men in the United States and is third only to lung and colon as a cause of cancer death. Clinical behavior of the disease is variable and the combination of prostate-specific antigen (PSA) screening and Gleason score staging are currently the best available molecular and pathology tools to predict outcomes. Cancer biology research establishes microRNAs (miRNAs) as key molecular components in both normal and pathological states. Thus, elucidating miRNAs perturbed by genomic alterations will expand our understanding of the molecular taxonomy of PCa with the aim to complement current practices in …

Pias-Family Proteins Negatively Regulate Glis3 Transactivation Function Through Sumo Modification In Pancreatic Β Cells, Tyler M. Hoard, Xiaoping Yang, Anton M. Jetten, Gary T. Zeruth Dr.

Faculty & Staff Research and Creative Activity

Gli-similar 3 (Glis3) is Krüppel-like transcription factor associated with the transcriptional regulation of insulin. Mutations within the Glis3 locus have been implicated in a number of pathologies including diabetes mellitus and hypothyroidism. Despite its clinical significance, little is known about the proteins and posttranslational modifications that regulate Glis3 transcriptional activity. In this report, we demonstrate that the SUMO-pathway associated proteins, PIASy and Ubc9 are capable of regulating Glis3 transactivation function through a SUMO-dependent mechanism. We present evidence that SUMOylation of Glis3 by PIAS-family proteins occurs at two conserved lysine residues within the Glis3 N-terminus and modification of Glis3 by SUMO …

Production Of A Candidate Recombinant Protein Vaccine For Mannheimia Haemolytica In Lettuce And Tobacco Chloroplasts, Coby K. Martin

Electronic Thesis and Dissertation Repository

The cattle industry worldwide is ravaged by bovine respiratory disease (BRD), a bacterial disease caused by Mannheimia haemolytica. Recent efforts to design vaccines against M. haemolytica focus on a virulence factor, leukotoxin, in addition to surface lipoproteins. Plant-based protein production is a safe and inexpensive alternative to traditional methods. Edible vaccines deliver antigens to pharyngeal tissues, which can provide local immunization against M. haemolytica prior to its progression into the lungs. In this project, a chimeric protein containing M. haemolytica antigens was produced in tobacco chloroplasts as a candidate edible vaccine for BRD. Attempts were made to transform lettuce …

Characterization Of She1 Spindle Role Using Ceullular, Biochemical, And Biophysical Methods, Yili Zhu

Doctoral Dissertations

During development, metaphase spindles undergo large movement and/or rotation to determine the cell division axis. While it has been shown that spindle translocation is achieved by astral microtubules pulling and/or pushing the cortex, how metaphase spindle stability is maintained during translocation remains not fully understood. In budding yeast, our lab has previously proposed a model for spindle orientation wherein the mitotic spindle protein She1 promotes spindle translocation across the bud neck by polarizing cortical dynein pulling activity on the astral microtubules. Intriguingly, She1 exhibits dominant spindle localization throughout the cell cycle. However, whether She1 has any additional role on the …

Extrinsic And Intrinsic Factors In Liver Development, Amrita Palaria

Doctoral Dissertations

Liver is the largest internal organ of the human body. It performs a multitude of functions. Therefore, it is provided with a huge regenerative capacity however, because of the same reason it is also prone to various diseases. Hence, it is essential to understand liver development in order to understand liver regeneration and liver diseases to provide better therapeutic targets and solutions. Liver development is orchestrated by a variety of intrinsic and extrinsic factors. The major focus of this dissertation thesis is to elucidate the role of BMP signals and YY1/VEGFA regulated signals in liver development. Liver organogenesis initiates with …

Examining Shsp-Substrate Capture And Chaperone Network Coordination Through Cross-Linking, Keith Ballard

Doctoral Dissertations

Small heat shock proteins (sHSPs) and related α-crystallins are virtually ubiquitous, ATP-independent molecular chaperones linked to protein misfolding diseases. They comprise a conserved core α-crystallin domain (ACD) flanked by an evolutionarily variable N-terminal domain (NTD) and semi-conserved C-terminal extension/domain (CTD). They are capable of binding up to an equal mass of unfolding protein, forming large, heterogeneous sHSP-substrate complexes that coordinate with ATP-dependent chaperones for refolding. To derive common features of sHSP-substrate recognition, I compared the chaperone activity and specific sHSP-substrate interaction sites for three different sHSPs from Arabidopsis (At17.6B), pea (Ps18.1) and wheat (Ta16.9), for which the atomic solution-state structures …

Effects Of G Protein Signalling Modulator 3 On Cellular Signalling, Aneta A. Surmanski

Electronic Thesis and Dissertation Repository

G protein coupled receptors (GPCRs) promote G protein heterotrimer (Gα·GDP/Gbg) activation.GPCRsignalling is limited via G protein GTPase activity and b-arrestin-receptor interactions. G Protein Signalling Modulators (GPSMs) are proteins that may influence receptor signalling through G protein activity. GPSM3 modulates their activity by binding to Ga_i-GDP, limiting nucleotide exchange and preventing its re-association to Gbg. The impact of GPSM3 on signalling is unknown.We hypothesize that GPSM3 will decrease Ga_i-dependent signalling while promoting Gbg-dependent signalling in G_i-coupled GPCRs.

GPSM3 significantly inhibited b-arrestin recruitment to α_2A-adrenergic and m-opioid receptors via a Gbg-dependent mechanism, …

Water Pharmacophore: Designing Ligands Using Molecular Dynamics Simulations With Water, Sang Won Jung, Minsup Kim, Steven Ramsey, Tom Kurtzman, Art E. Cho

Publications and Research

In this study, we demonstrate a method to construct a water-based pharmacophore model which can be utilized in the absence of known ligands. This method utilizes waters found in the binding pocket, sampled through molecular dynamics. Screening of compound databases against this water-based pharmacophore model reveals that this approach can successfully identify known binders to a target protein. The method was tested by enrichment studies of 7 therapeutically important targets and compared favourably to screening-by-docking with Glide. Our results suggest that even without experimentally known binders, pharmacophore models can be generated using molecular dynamics with waters and used for virtual …