Molecular Biology Commons^™

Structures Of The Insecticidal Toxin Complex Subunit Xpta2 Highlight Roles For Flexible Domains, Cole L. Martin, David W. Chester, Christopher D. Radka, Lurong Pan, Zhengrong Yang, Rachel C. Hart, Elad M. Binshtein, Zhao Wang, Lisa Nagy, Lawrence J. Delucas, Stephen G. Aller

Markey Cancer Center Faculty Publications

The Toxin Complex (Tc) superfamily consists of toxin translocases that contribute to the targeting, delivery, and cytotoxicity of certain pathogenic Gram-negative bacteria. Membrane receptor targeting is driven by the A-subunit (TcA), which comprises IgG-like receptor binding domains (RBDs) at the surface. To better understand XptA2, an insect specific TcA secreted by the symbiont X. nematophilus from the intestine of entomopathogenic nematodes, we determined structures by X-ray crystallography and cryo-EM. Contrary to a previous report, XptA2 is pentameric. RBD-B exhibits an indentation from crystal packing that indicates loose association with the shell and a hotspot for possible receptor binding or a …

The S1p Receptor 1 Antagonist Ponesimod Reduces Tlr4-Induced Neuroinflammation And Increases Aβ Clearance In 5xfad Mice, Zhihui Zhu, Liping Zhang, Ahmed Elsherbini, Simone M. Crivelli, Priyanka Tripathi, Carmen Harper, Zainuddin Quadri, Stefka D. Spassieva, Erhard Bieberich

Markey Cancer Center Faculty Publications

Background Previously, we showed that the sphingosine-1-phosphate (S1P) transporter spinster 2 (Spns2) mediates activation of microglia in response to amyloid β peptide (Aβ). Here, we investigated if Ponesimod, a functional S1P receptor 1 (S1PR1) antagonist, prevents Aβ-induced activation of glial cells and Alzheimer’s disease (AD) pathology.

Methods We used primary cultures of glial cells and the 5XFAD mouse model to determine the effect of Aβ and Ponesimod on glial activation, Aβ phagocytosis, cytokine levels and pro-inflammatory signaling pathways, AD pathology, and cognitive performance.

Findings Aβ42 increased the levels of TLR4 and S1PR1, leading to their complex formation. Ponesimod prevented the …

Utilizing Crispr Cas9 To Visualize Dopamine Receptors In Caenorhabditis Elegans, Lauren Michelle Velasquez

Electronic Theses, Projects, and Dissertations

Dopamine (DA) is a neurotransmitter with imperative implications in many functions including movement, reward, and cognition. Studying the pathways of dopaminergic neurons at multiple levels allows us to understand the ways in which these systems can go wrong. We study dopamine in a model system such as the worm Caenorhabditis elegans because of its relatively simple and well-characterized nervous system. DA is involved in regulating chemosensory behaviors in worms. The purpose of this research project is to definitively answer the following question: Are the dopamine receptors DOP-1 and DOP-4 expressed in chemosensory neurons? Previous reporter assays show that neither of …

Acute Manipulation Of Erna Level For Dissecting Its Roles In Transcriptional Regulation, Lanxin Bei

Dissertations and Theses (Open Access)

Enhancers are the central genetic elements controlling cell-type and state specific transcription programs to dictate cell fates during development. Mechanistic understanding of enhancer action is important for both biology and disease research. In the human genome, more than 60k human enhancers were found to produce non-coding transcripts named enhancer RNAs (eRNAs). These created a new challenge to understand enhancer functions, which now are not only DNA elements that promote transcription but also RNA-producing transcription units themselves. Importantly, deregulation of eRNAs was associated with various diseases such as cancer, immune disorders, and neurodegeneration. However, the direct role of eRNAs in transcriptional …

Investigating And Characterizing Septin Cellular Regulators During Infection Related Development In M. Oryzae, Rinalda Proko

Graduate Theses and Dissertations

The fungus Magnaporthe oryzae causes a devastating disease of rice and wheat, called blast, which poses a major threat to food security across the planet. To enter plant tissue, M. oryzae produces a remarkable pressure-generating infection cell called an appressorium, which mechanically ruptures the tough cuticle of host leaves through the application of enormous physical force directed onto a narrow penetration structure emerging from its base. Importantly, appressorium form and functionality depends on the regulated assembly of a toroidal ring-like structure, composed of septins, which forms within its base. Septins are a family of membrane-associated GTP-binding proteins that polymerize into …

Unraveling The Biogenesis And Roles Of Iron-Sulfur Proteins During Nitrogen Fixation By Methanosarcina Acetivorans, Jasleen Saini

Graduate Theses and Dissertations

Iron-sulfur (Fe-S) clusters, the ancient cofactors found in proteins across all life forms, play vital roles in numerous cellular processes. Fe-S clusters exhibit structural diversity, ranging from simple to complex forms. Methanogens are anaerobic archaea that produce methane as a metabolic by-product through methanogenesis, a process dependent on numerous Fe-S proteins. Notably, methanogens are capable of fixing nitrogen (diazotrophy) facilitated by the enzyme complex called nitrogenase, which contains both simple and complex Fe-S clusters. While methanogens possess the largest number of Fe-S proteins, the factors involved in Fe-S cluster biogenesis remain largely unknown. Bacteria possess three distinct and well-characterized Fe-S …

The Roles Of Estrogen Receptor Alpha-Interacting Rnas And Exosomes In Breast Cancer, And E-Cadherin In Carcinomas, Brihget Catalina Sicairos-Meza

Graduate Theses and Dissertations

Carcinomas are malignancies of epithelial cells and account for >80% of cancer-related deaths. Approximately 75% of breast cancers express estrogen receptor alpha (ERα). ERα promotes breast cancer progression by enhancing cancer cell proliferation. Tamoxifen is commonly used to treat ERα-positive breast cancers but can result in endocrine therapy resistance. The mechanisms underlying endocrine therapy resistance remain elusive. The Du lab recently found that ERα is an RNA-binding protein. However, the significance of the ERα-RNA interaction is unknown. We fractionated ERα-positive MCF7 cells into cytoplasmic and nuclear fractions, followed by immunoprecipitation of ERα with associated RNAs and RNA-sequencing. We identified many …

Met Alterations In Glioblastoma: Characterization Of Patient-Derived Xenografts And Therapeutic Strategies, Anna Musket

Electronic Theses and Dissertations

Glioblastoma is the most commonly diagnosed central nervous system primary malignancy and it is considered a terminal diagnosis with few treatment options available. Glioblastoma tumors frequently develop treatment resistance due in part to their highly heterogenic nature. The heterogeneity of glioblastoma is partially attributed to the presence of glioma stem-like cells (GSC), which are highly invasive and resistant to chemotherapy and irradiation treatments. Signaling of the receptor tyrosine kinase MET is a known regulator of GSC. Glioblastoma patients have an increasingly poor prognosis that corresponds with increasing MET expression. Both GSC and MET are known to contribute to treatment resistance …

Fatty Acids And Parasitism: Towards A Better Understanding Of Lipid Metabolism In Trypanosoma Brucei, Joshua Saliutama

All Dissertations

Trypanosoma brucei is an extracellular eukaryotic parasite that causes sleeping sickness in humans and cattle. As an extracellular parasite, T. brucei relies on the host’s nutrients to satisfy its growth requirements. The parasite is unusual because it does not uptake most of the host’s lipid species. Instead, T. brucei prefers to perform de novo synthesis of most lipid species. One of the lipid species that T. brucei can both uptake and synthesize is fatty acids. In my thesis work, I investigated the dynamics of fatty acid uptake, metabolism, and utilization of T. brucei. My work starts by determining the …

Biochemical Characterization Of Ssb1, Ssb2, Sycp3, And Meilb2 In Meiotic Recombination, Garrett Buzzard

All Dissertations

The genome is constantly at risk to damage from environmental and normal cellular processes. Double-strand breaks (DSBs) are one of the most deleterious forms of DNA damage as unrepaired DSBs ultimately result in cell death. Homologous recombination (HR) is a conserved DNA repair pathway that utilizes a homologous chromosome as a template for DNA synthesis to repair the DSBs relatively error-free. HR is active in both mitotic and meiotic cells but meiotic HR is initiated by the introduction of programmed DSBs into the genome. The process of HR repairs the DSBs and results in a physical connection between homologous chromosomes …

Reactive Oxygen Species And Weak Magnetic Fields: Unraveling The Mechanisms Of Planarian Regeneration And Tissue Growth, Luke James Kinsey

Dissertations

Reactive oxygen species (ROS) play a critical role in basic cellular behaviors such as apoptosis, proliferation, and differentiation. These molecules once thought to be harmful byproducts of metabolism, are also required for many complex and ubiquitous processes like wound healing, development, and regeneration. This study investigates the influence of ROS on planarian regeneration, with a particular focus on ROS dynamics in stem cell-mediated tissue growth. Data reveal that modulation of ROS levels leads to changes in cellular behaviors and tissue growth outcomes. Inhibition of ROS accumulation blocks stem cell proliferation and tissue growth, preventing planarian regeneration, while partial ROS accumulation …

Role Of Short Interspersed Nuclear Elements (Sines) Of Maternally Imprinted Microrna Clusters In Hemochorial Placental Antiviral Immunity, Ishani Wickramage

USF Tampa Graduate Theses and Dissertations

The evolution of hemochorial placentas in which the fetal trophoblast is bathed in maternal blood has vastly improved fetal development and survival in primates and rodents, due to the enhanced efficiency of nutrient, gas and waste exchange between the mother and the fetus. However, the extensive contact between the fetal tissues and maternal blood in these placentas also poses an increased risk of vertical transmission of pathogens such as viruses from the mother to the fetus. In response to this selection pressure, hemochorial placentas have evolved robust defense mechanisms to protect the immunologically naive fetus. Unlike somatic cells that require …

Study Of Polytopic Membrane Protein Topological Organization As A Function Of Membrane Lipid Composition, Takefumi Morizumi, Kyumhyuk Kim, Hai Li, Elena G Govorunova, Oleg A Sineshchekov, Yumei Wang, Lei Zheng, Éva Bertalan, Ana-Nicoleta Bondar, Azam Askari, Leonid S Brown, John L Spudich, Oliver P Ernst

Faculty, Staff and Student Publications

Kalium channelrhodopsin 1 from Hyphochytrium catenoides (HcKCR1) is a light-gated channel used for optogenetic silencing of mammalian neurons. It selects K+ over Na+ in the absence of the canonical tetrameric K+ selectivity filter found universally in voltage- and ligand-gated channels. The genome of H. catenoides also encodes a highly homologous cation channelrhodopsin (HcCCR), a Na+ channel with >100-fold larger Na+ to K+ permeability ratio. Here, we use cryo-electron microscopy to determine atomic structures of these two channels embedded in peptidiscs to elucidate structural foundations of their dramatically different cation selectivity. Together with structure-guided mutagenesis, we show that K+ versus Na+ …

Proteomic Approaches To Identify Unique And Shared Substrates Among Kinase Family Members, Charles Lincoln Howarth

Dartmouth College Ph.D Dissertations

Protein phosphorylation is a reversible post-translational modification that is a critical component of almost all signaling pathways. Kinases regulate substrate proteins through phosphorylation, and nearly all proteins are phosphorylated to some extent. Crucially, breakdown in phosphorylation signaling is an underlying factor in many diseases, including cancer. Understanding how phosphorylation signaling mediates cellular pathways is crucial for understanding cell biology and human disease.

Targeted protein degradation (TPD) is a strategy to rapidly deplete a protein of interest (POI) and is applicable to any gene that is amenable to CRISPR-Cas9 editing. One TPD approach is the auxin-inducible degron (AID) system, which relies …

Loss Of Pml Nuclear Bodies In Familial Amyotrophic Lateral Sclerosis-Frontotemporal Dementia, Francesco Antoniani, Marco Cimino, Laura Mediani, Jonathan Vinet, Enza M. Verde, Valentina Secco, Alfred Yamoah, Priyanka Tripathi, Eleonora Aronica, Maria Elena Cicardi, Davide Trotti, Jared Sterneckert, Anand Goswami, Serena Carra

Farber Institute for Neuroscience Faculty Papers

Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are two neurodegenerative disorders that share genetic causes and pathogenic mechanisms. The critical genetic players of ALS and FTD are the TARDBP, FUS and C9orf72 genes, whose protein products, TDP-43, FUS and the C9orf72-dipeptide repeat proteins, accumulate in form of cytoplasmic inclusions. The majority of the studies focus on the understanding of how cells control TDP-43 and FUS aggregation in the cytoplasm, overlooking how dysfunctions occurring at the nuclear level may influence the maintenance of protein solubility outside of the nucleus. However, protein quality control (PQC) systems that maintain protein homeostasis comprise …

Structural Diversity And Stress Regulation Of The Plant Immunity-Associated Calmodulin-Binding Protein 60 (Cbp60) Family Of Transcription Factors In Solanum Lycopersicum (Tomato), Vanessa Shivnauth, Sonya Pretheepkumar, Eric J. R. Marchetta, Christina A. M. Rossi, Keaun Amani, Christian Castroverde

Biology Faculty Publications

Cellular signaling generates calcium (Ca2+) ions, which are ubiquitous secondary messengers decoded by calcium-dependent protein kinases, calcineurins, calreticulin, calmodulins (CAMs), and CAM-binding proteins. Previous studies in the model plant Arabidopsis thaliana have shown the critical roles of the CAM-BINDING PROTEIN 60 (CBP60) protein family in plant growth, stress responses, and immunity. Certain CBP60 factors can regulate plant immune responses, like pattern-triggered immunity, effector-triggered immunity, and synthesis of major plant immune-activating metabolites salicylic acid (SA) and N-hydroxypipecolic acid (NHP). Although homologous CBP60 sequences have been identified in the plant kingdom, their function and regulation in most species remain unclear. In …

High-Resolution Cryo-Em Structure Of The Pseudomonas Bacteriophage E217, Fenglin Li, Chun-Feng Hou, Ravi K. Lokareddy, Ruoyu Yang, Francesca Forti, Federica Briani, Gino Cingolani

Department of Biochemistry and Molecular Biology Faculty Papers

E217 is a Pseudomonas phage used in an experimental cocktail to eradicate cystic fibrosis-associated Pseudomonas aeruginosa. Here, we describe the structure of the whole E217 virion before and after DNA ejection at 3.1 Å and 4.5 Å resolution, respectively, determined using cryogenic electron microscopy (cryo-EM). We identify and build de novo structures for 19 unique E217 gene products, resolve the tail genome-ejection machine in both extended and contracted states, and decipher the complete architecture of the baseplate formed by 66 polypeptide chains. We also determine that E217 recognizes the host O-antigen as a receptor, and we resolve the N-terminal portion …

Defining A Factor-Induced Signaling Signature For Endothelial Tubulogenesis, Sprouting, And Pericyte Recruitment, Prisca Kuanting Lin

USF Tampa Graduate Theses and Dissertations

Endothelial cells (ECs) form the inner lining of blood capillary walls. Our laboratory has developed a serum-free, defined Factor system that allows for human ECs to sprout, form lumens and tube networks, recruit pericytes, and induce capillary basement membrane deposition in 3D extracellular matrices. We have shown that the Factor system drives morphogenesis through the activation of multiple signaling pathways; however, we do not know if all factors in the Factor system are needed to induce maximal EC morphogenesis. If so, we have yet to identify why the Factors are critical to the model other than that they induce activation …

Fibrosis-The Tale Of H3k27 Histone Methyltransferases And Demethylases, Morgan D. Basta, Svetlana Petruk, Alexander Mazo, Janice L. Walker

Department of Biochemistry and Molecular Biology Faculty Papers

Fibrosis, or excessive scarring, is characterized by the emergence of alpha-smooth muscle actin (αSMA)-expressing myofibroblasts and the excessive accumulation of fibrotic extracellular matrix (ECM). Currently, there is a lack of effective treatment options for fibrosis, highlighting an unmet need to identify new therapeutic targets. The acquisition of a fibrotic phenotype is associated with changes in chromatin structure, a key determinant of gene transcription activation and repression. The major repressive histone mark, H3K27me3, has been linked to dynamic changes in gene expression in fibrosis through alterations in chromatin structure. H3K27-specific homologous histone methylase (HMT) enzymes, Enhancer of zeste 1 and 2 …

Functional Analysis Of The Gamma-Aminobutyric Acid Type A Receptor Subunit Alpha-1 (Gabra1) Gene During Zebrafish Development., Nayeli Gabriela Reyes-Nava

Open Access Theses & Dissertations

The GABRA1 gene encodes for the alpha-1 (α1) subunit of the Gamma-Aminobutyric acid type A receptor (GABAAR), which are the primary modulators of synaptic inhibition in the central nervous system (CNS). Alpha-1 subunits are essential for maintaining the normal function of native receptors and contribute to over 60% of all GABAARs in the CNS. Remarkably, a broad spectrum of neurodevelopmental and epilepsy-associated disorders have been linked with mutations in the GABRA1 gene. However, the developmental, behavioral, and molecular mechanisms underlying GABRA1-associated epileptic disorders remain to be fully understood. Hence, the overarching goal of this dissertation is to investigate the behavioral …