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Pharmacological And Pharmacokinetic Studies Of A KCa2.2 Positive Allosteric Modulator, Mohammad Asikur Rahman

Pharmaceutical Sciences (PhD) Dissertations

Small-conductance Ca²⁺-activated potassium channels (K_Ca2.x) family is widely expressed in neurons, the heart, and endothelial cells. K_Ca2.x channels are named small conductance Ca²⁺-activated potassium channels due to their comparatively low single-channel conductance and are activated solely by rises in intracellular Ca²⁺. The family has three subtypes: K_Ca2.1, K_Ca2.2, and K_Ca2.3, encoded by KCNN1, KCNN2, and KCNN3 genes, respectively. K_Ca2.x channels regulate neuronal excitability and responsiveness to synaptic input patterns. Small-conductance Ca²⁺-activated potassium channels subtype 2 (K_Ca2.2, …

Mechanisms Behind The Chaperone Activity Of Nucleic Acids, Theodore J. Litberg

Electronic Theses and Dissertations

Understanding the interplay between nucleic acids and protein aggregation is integral to the understanding of proteostasis, aging, and neurodegenerative disease progression. Nucleic acids are known to modulate the aggregation of PrP, tau, ⍺-synuclein, and other disease relevant proteins. Although the interactions between misfolded protein and nucleic acids can play a role in disease, this interaction may potentially be beneficial as well. Our group and others have shown nucleic acids can be powerful chaperones. Previous work has shown both RNA and DNA can prevent protein aggregation and RNA can pass off protein clients to the heat shock protein (Hsp) system. Here …

Activation Of The Renin–Angiotensin–Aldosterone System Is Attenuated In Hypertensive Compared With Normotensive Pregnancy, Robin C. Shoemaker, Marko Poglitsch, Hong Huang, Katherine Vignes, Aarthi Srinivasan, Cynthia Cockerham-Morris, Aric Schadler, John Anthony Bauer, John O'Brien

UK CARES Faculty Publications

Hypertension during pregnancy increases the risk of adverse maternal and fetal outcomes, but the mechanisms of pregnancy hypertension are not precisely understood. Elevated plasma renin activity and aldosterone concentrations play an important role in the normal physiologic adaptation to pregnancy. These effectors are reduced in patients with pregnancy hypertension, creating an opportunity to define the features of the renin–angiotensin–aldosterone system (RAAS) that are characteristic of this disorder. In the current study, we used a novel LC-MS/MS-based methodology to develop comprehensive profiles of RAAS peptides and effectors over gestation in a cohort of 74 pregnant women followed prospectively for the development …

Modeling Biphasic, Non-Sigmoidal Dose-Response Relationships: Comparison Of Brain- Cousens And Cedergreen Models For A Biochemical Dataset, Venkat D. Abbaraju, Tamaraty L. Robinson, Brian P. Weiser

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Biphasic, non-sigmoidal dose-response relationships are frequently observed in biochemistry and pharmacology, but they are not always analyzed with appropriate statistical methods. Here, we examine curve fitting methods for “hormetic” dose-response relationships where low and high doses of an effector produce opposite responses. We provide the full dataset used for modeling, and we provide the code for analyzing the dataset in SAS using two established mathematical models of hormesis, the Brain-Cousens model and the Cedergreen model. We show how to obtain and interpret curve parameters such as the ED50 that arise from modeling, and we discuss how curve parameters might change …

Polar Body Membrane Breakdown Occurs Independent Of Macroautophagy In C. Elegans, Shruti Kolli

Electronic Theses and Dissertations

Failure to digest cell corpses can lead to lupus-like autoimmune disorders in mammals. To better understand if LC3-associated phagocytosis (LAP) drives cell corpse breakdown in phagolysosomes, we studied the clearance of the C. elegans polar body during embryonic development. ATG16L1 has separable roles in regulating LC3 recruitment during macroautophagy and LAP. We demonstrated that the ATG16L1 C. elegans orthologs, ATG-16.1 and ATG-16.2, function redundantly to promote polar body membrane breakdown. We also discovered that truncating the LAP-specific WD40 domain of ATG-16.2 unexpectedly disrupts autophagy. Furthermore, we confirmed that polar body membrane breakdown occurs independent of macroautophagy. We also showed that …

Investigating The Effects Of E219 Mutations In M. Tuberculosis Indole-3-Glycerol Phosphate Synthase, Cinthya Moran

Theses, Dissertations and Culminating Projects

Tuberculosis (TB) is a serious infectious disease caused by Mycobacterium tuberculosis. Tuberculosis drug resistance is increasingly a problem, but recent research has provided results that suggest Indole-3-Glycerol Phosphate Synthase (IGPS) could be a potential drug target. IGPS is an enzyme that catalyzes the fourth step in the tryptophan biosynthesis pathway, a crucial process for optimal Mycobacterium tuberculosis growth. In this research, we studied IGPS from Mycobacterium tuberculosis (MtIGPS) to shed light on a particular conserved residue that has not been studied in depth in any IGPS homolog. My investigation focuses on understanding the role of the active site residue E219. …

Phenotypic And Transcriptomic Characterization Of Rice Snrk1 Mutants Developed By Crispr/Cas9 Mutagenesis, Maria Clara Faria Chaves

Graduate Theses and Dissertations

SnRK1 is a heterotrimeric protein kinase that is composed of a catalytic subunit (α) and two regulatory subunits (β and βγ), and it has a main role in regulating energy homeostasis in the plant by modulating anabolic and catabolic process. SnRK1 phosphorylates and alters the activities of enzymes involved in metabolism and regulates gene expression by altering the activity of chromatin-remodeling enzymes or the transcription factors. Rice contains three functional paralogs of SnRK1α: SnRK1αa (LOC_Os03g17980), SnRK1αb (LOC_Os08g37800), and SnRK1αc (LOC_Os05g45420). This study focused on the function of these SnRK1 paralogs by evaluating the phenotypic and transcriptomic characteristics and the disease …

Functional Analysis Of Two Campoletis Sonorensis Ichnovirus Vinnexins In Drosophila Melanogaster, Peng Zhang

All Dissertations

Campoletis sonorensis Ichnovirus (CsIV) is produced by the ichneumonid parasitoid Campoletis sonorensis (the primary host). Female parasitoid wasps restrictedly replicate viruses in their ovaries and inject them into a larval lepidopteran (secondary host) during oviposition. This symbiotic virus of C. sonorensis is essential for successful parasitization. CsIV is characterized by having a large, segmented double-stranded DNA genome with a large number of genes, many of which are associated with one of five multigene families. The CsIV vinnexin gene family is one of them and has four members, including CsIV-vnxD and CsIV-vnxG. Vinnexins are homologues of insect Innexins, which form gap …

Epitranscriptomic Regulation In Breast Cancer And Pcb-Induced Liver Disease., Belinda Petri

Electronic Theses and Dissertations

Post-transcriptional RNA modifications including N6-methyladenosine (m6A) regulate mRNA stability, splicing, and translation. My research examined m6A in two disease models: breast cancer (BCa) and non-alcoholic fatty liver disease (NAFLD). Acquired resistance to endocrine therapies (ET) develops in approximately 20% of BCa patients with estrogen receptor α positive (ER+) tumors following treatment. The mechanisms by which tumor cells evade ET are not completely understood. Using a cell line model, we investigated the role of an m6A reader protein, HNRNPA2B1 (A2B1) that is upregulated in ET-resistant ER+ BCa cells. Stable overexpression of A2B1 in ET-sensitive MCF-7 cells (MCF-7-A2B1), results in ET resistance, …

Determining The Existence And Regulation Of Microlipophagy In Primary Brown And White Adipocytes, Sara C. Arenas De Leon

Biomedical Sciences ETDs

Lipids are one of life’s four main macromolecules and provide essential functions to cells.

The degradation of lipid droplets and mobilization of lipids occurs through lipolysis. Emerging evidence demonstrates evidence of a selective form of autophagy in lipolysis. The process of microlipophagy has only recently been described, and many features of its regulation are still poorly understood. Our recent study showed that inhibiting mammalian target of rapamycin complex 1 (mTORC1) pathway by depletion of Raptor in adipocytes led to an influx of lysosomes and accumulation of lipid droplets within lysosome. Here, we expand on these previous findings. We were able …

Molecular Characterization Of Compartment Boundary Formation During Epithelial Remodeling In Drosophila Melanogaster, Samia Parveen

Graduate Theses and Dissertations

The ability of epithelial cells to self-organize is crucial for the development of proper tissue structures and organs in multi-cellular organisms. Epithelia consist of inter-connected cells that form flat sheets, and specific groups of cells within these sheets are partitioned into distinct compartments via compartment boundaries (CBs). These CBs are specialized interfaces between cells characterized by stable, high-tension actomyosin cables, which act as fences to prevent movements between adjacent compartments during tissue reorganization. In the early Drosophila embryo, CBs are established in response to the asymmetrical localization of two cell-surface proteins the Leucine-Rich Repeat (LRR) protein Tartan and the Teneurin …

X-Ray Crystal Structure Of E399q,E708q Ecm16 Double Mutant, Gileydis Guillama

Open Access Theses & Dissertations

The use of antibiotics has undoubtedly been a boon for humanity in combating infections and microbial threats. However, their widespread utilization has contributed to the emergence and spread of antibiotic resistance, which now poses a significant public health challenge. Streptomyces bacterium, produce diverse secondary metabolites with antibacterial, antifungal, antiviral, antitumoral, and immunosuppressant activities. Among these compounds is echinomycin, a nonribosomal peptide antibiotic synthesized by Streptomyces lasalocidi, which inhibits DNA replication and transcription by intercalating the DNA duplex at CpG steps. A gene called ecm16 was identified in the echinomycin biosynthetic gene cluster, which provides echinomycin self-resistance. Ecm16 recognizes DNA duplexes …

Structural Study Of Lsd13 And Lsd14 Type I Modular Polyketide Synthases From The Lasalocid A Biosynthesis Pathway, Dayan Viera

Open Access Theses & Dissertations

Bioactive natural products often possess a complex structure and have a large size which make their chemical synthesis highly challenging. To overcome this challenge, we are developing a biosynthetic method for production of natural products and their analogs. This technology is expected to decrease the synthesis time from years to weeks and reduce the cost of drug production by multiple orders of magnitude. We are investigating the biosynthesis pathway of lasalocid A, an anticancer antibiotic, to develop this technology. Specifically, we aim to characterize the enzymes structurally and biochemically in this pathway, thus paving the way for their rational engineering. …

Co-Targeting Nucleus Accumbens Associate 1 And Nf-Κb Signaling Synergistically Inhibits Melanoma Growth, Lixiang Gu, Xingcong Ren, Chrispus Ngule, Xiaofang Xiong, Jianxun Song, Zhiguo Li, Jin-Ming Yang

Markey Cancer Center Faculty Publications

Nucleus-accumbens-associated protein-1 (NAC1) is a cancer-related transcriptional factor encoded by the NACC1 gene, which is amplified and overexpressed in various human cancers and has been appreciated as one of the top potential cancer driver genes. NAC1 has therefore been explored as a potential therapeutic target for managing malignant tumors. Here, we show that NAC1 is a negative regulator of NF-κB signaling, and NAC1 depletion enhances the level of the nuclear NF-κB in human melanoma. Furthermore, the inhibition of NF-κB signaling significantly potentiates the antineoplastic activity of the NAC1 inhibition in both the cultured melanoma cells and xenograft tumors. This study …

Metabolic Disruption Induced By Mtor Signaling Pathway Inhibition In Regulatory T-Cell Expansion For Clinical Application, Roberto Gedaly, Gabriel Orozco, Alexandre P. Ancheta, Mackenzie Donoho, Siddharth Desai, Fanny Chapelin, Aman Khurana, Lillie J. Lewis, Cuiping Zhang, Francesc Marti

Markey Cancer Center Faculty Publications

Background: Regulatory T cell (Treg) therapy is considered an alternative approach to induce tolerance in transplantation. If successful, this therapy may have implications on immunosuppression minimization/withdrawal to reduce drug-induced toxicity in patients. The aim of this study was to assess the efficacy of the mTORC1/C2 inhibitor, AZD8055, in the manufacturing of clinically competent Treg cells and compare the effects with those induced by rapamycin (RAPA), another mTOR inhibitor commonly used in Treg expansion protocols.

Methods: Primary human Treg cells were isolated from leukapheresis product. Cell viability, expansion rates, suppressive function, autophagy, mitochondrial unfolded protein response (mitoUPR), and cell metabolic profile …

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 …

Fbg Αc 389 – 402 Modulates Factor Xiii Crosslinking In The Fibrinogen Αc Region., Francis Dean Orlina Ablan

Electronic Theses and Dissertations

Fibrinogen (Fbg) is a coagulation protein critical for clot formation. Coagulation Factor XIII (FXIII) is a calcium-dependent transglutaminase that crosslinks reactive glutamines (Q) and lysines (K) between fibrin and other anti-fibrinolytic proteins. In the presence of Ca²⁺, FXIII could be activated non-proteolytically (FXIII-A°), or proteolytically by thrombin (FXIII-A*). Significant increases in clot stability and red blood cell retention are linked to FXIII activity in the fibrinogen αC region (Fbg Aα 221 – 610). This region contains several FXIII-reactive glutamines and lysines, as well as a binding site for FXIII-A* (Fbg αC 389 – 402) that includes a key …

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 …

Elucidating Acetate Metabolism: Identification Of Transporters And Enzymes Required For Acetate Utilization In The Fungal Pathogen Cryptococcus Neoformans, Perry L. Kezh

All Dissertations

Cryptococcus neoformans is the leading cause of fungal meningitis world-wide. While exposure to this environmental sporophyte is common during childhood, those who are immune compromised are at risk of infection. Following inhalation, this basidiomycetous fungus subsequently colonizes other organs though hematogenous dissemination, eventually crossing the blood brain barrier and colonizing the brain where it causes as cryptococcal meningitis. Changes in the availability of carbon sources stemming from the movement from soil to the lungs induce changes in fungal metabolism. Specifically, alveolar macrophages, which present a first line of defense against infection, provide a glucose-/amino acid-poor environment. As such, the use …

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 …

Disparities In Antifibrotic Medication Utilization Among Veterans With Idiopathic Pulmonary Fibrosis, Bhavika Kaul, Joyce S Lee, Laura A Petersen, Charles Mcculloch, Ivan O Rosas, Venkata D Bandi, Ning Zhang, Alison M Dedent, Harold R Collard, Mary A Whooley

Faculty, Staff and Students Publications

BACKGROUND: Two antifibrotic medications, pirfenidone and nintedanib, are approved for the treatment of idiopathic pulmonary fibrosis (IPF). Little is known about their real-world adoption.

RESEARCH QUESTION: What are the real-world antifibrotic utilization rates and factors associated with uptake among a national cohort of veterans with IPF?

STUDY DESIGN AND METHODS: This study identified veterans with IPF who received care either provided by the Veterans Affairs (VA) Healthcare System or non-VA care paid for by the VA. Patients who had filled at least one antifibrotic prescription through the VA pharmacy or Medicare Part D between October 15, 2014, and December 31, …

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 …

Noncanonical Binding Of Transcription Factors: Time To Revisit Specificity?, Md Abul Hassan Samee

Faculty, Staff and Students Publications

Transcription factors (TFs) are one of the most studied classes of DNA-binding proteins that have a direct functional impact on gene transcription and thus, on human physiology and disease. The mechanisms that TFs use for recognizing target DNA binding sites have been studied for nearly five decades, yet they remain poorly understood. It is classically assumed that a TF recognizes a specific sequence pattern, or motif, as its binding sites. However, recent studies are consistently finding examples of noncanonical binding, that is, TFs binding at sites that do not resemble their sequence motifs. Here we review the current literature on …