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Articles 1 - 30 of 120
Full-Text Articles in Biochemistry, Biophysics, and Structural Biology
Progress Toward The Total Synthesis Of Lyconadin A, Yu Zhang
Progress Toward The Total Synthesis Of Lyconadin A, Yu Zhang
Theses and Dissertations
Lyconadin A is a pentacyclic Lycopodium alkaloid isolated from the club moss Lycopodium complanatum with antitumor properties. We have developed a novel 7-exo/6-exo acyl radical cascade cyclization as a method of making the bicyclo[5.4.0]undecane ring system of lyconadin A. The model products are trans-fused ring systems, while a cis-fused ring system is needed in lyconadin A. We have discovered a method to convert the trans-fused model cascade cyclization product into the desired cis isomer. Based on Donohoe's pyridone synthesis, we developed a method for the construction of 5-alkyl and 3,5-dialkyl-6-carbomethoxy-2-pyridones, the former of which …
Overexpression And Partial Purification Of Serine Hydroxymethyltransferase, Kimberly Rose Stevens
Overexpression And Partial Purification Of Serine Hydroxymethyltransferase, Kimberly Rose Stevens
Chemistry and Biochemistry
No abstract provided.
Light-Related Photosynthetic Gene Expression And Enzyme Activity In The Heterokont Alga Vaucheria Litorea And Its Symbiotic Partner The Sacoglossan Mollusc Elysia Chlorotica, Kara M. Soule
Electronic Theses and Dissertations
Photosynthesis is comprised of tightly coupled reactions and therefore requires strict matrices of regulation, particularly involving alterations in gene expression and enzyme activity within the nucleus and plastid. Extensive research has been carried out on these light-regulated mechanisms in plants and green algae, however, much less is known in the red algal lineage, including heterokonts. The goal of this study was to investigate the influence of light on photosynthetic gene expression and select enzyme activity in the heterokont alga Vaucheria litorea and its symbiotic partner Elysia chlorotica, a photosynthetic sacoglossan mollusc (sea slug). Elysia chlorotica harbors V. litorea plastids …
Applications Of Variable Number Tandem Repeat Genotyping In The Validation Of An Animal Medical Model And Gene Flow Studies In Threatened Populations Of Reptiles, Candace D. Smith
Applications Of Variable Number Tandem Repeat Genotyping In The Validation Of An Animal Medical Model And Gene Flow Studies In Threatened Populations Of Reptiles, Candace D. Smith
Graduate Theses and Dissertations
We used variable number tandem repeats (VNTR) to validate the chicken as a human medical model for Pulmonary Arterial Hypertension. We identified seven regions on four chromosomes and interrogated for VNTR markers that significantly associate with Pulmonary Hypertension Syndrome/ascites. In those regions, we identified 7 candidate genes; AGTR1, ACE, p38MAPK, SST, 5HT2B, NET1, and CALM3 for further analysis as significantly contributing QTL for ascites/PHS. We also used variable number tandem repeats to measure gene flow and gather evidence for multiple paternity in a population of Timber rattlesnakes, Crotalus horridus. We were able to verify 1 VNTR that can be used …
Characterization And Functional Regulation Of Bioactive Peptides In Avian Macrophages And Heterophils, Lakshmi Kannan
Characterization And Functional Regulation Of Bioactive Peptides In Avian Macrophages And Heterophils, Lakshmi Kannan
Graduate Theses and Dissertations
Oligopeptides and low molecular weight polypeptides play central roles as effectors and signal transducers acting as hormones, neurotransmitters, growth factors, toxins, and antimicrobial factors that are important for the survival of the organism. Owing to the ubiquitous involvement of peptides in many key regulatory processes, we have been interested to identify native peptides in different cells and tissues and understand their functions. To conduct our studies, we used avian macrophages and heterophils as models of specialized cells which constitute central components of innate immunity. These studies involved (a) qualitative identification and characterization of the peptides associated with high intensity mass …
Cloning And Expression Of Porcine Dicer And Argonaute-2, Heather Stowe
Cloning And Expression Of Porcine Dicer And Argonaute-2, Heather Stowe
All Theses
In vitro-produced embryos exhibit aberrations in development, but the reasons for these developmental problems are unknown. Recently, a class of small non-coding RNA called microRNA (miRNA) has been described and reported to have roles in normal mammalian embryonic development. These miRNAs are encoded in the genome, transcribed by RNA pol II and processed into fragments approximately 22 nt in length by ribonuclease enzymes, the final one being a protein called Dicer. miRNA work through the RNA-induced silencing complex (RISC), of which the argonaute gene family are key proteins. Argonaute-2 (Ago2) has been identified as the only member possessing endonuclease activity, …
The Physiological And Biochemical Understanding Of 5’-Amp Induced Deep Hypometabolism, William G. O'Brien
The Physiological And Biochemical Understanding Of 5’-Amp Induced Deep Hypometabolism, William G. O'Brien
Dissertations & Theses (Open Access)
Our laboratory’s ability to induce deep hypometabolism (DH) via an injection of adenosine monophosphate (5’-AMP) followed by placement in an ambient temperature (Ta) around 15oC currently can last about 3-9 hours. While we have insight into how 5’-AMP induced hypometabolism is initiated, it remains unclear how arousal from hypometabolism is controlled. Other laboratory members have been unable to prolong this process safely and effectively with previous attempts of re-injecting a dose of 5’-AMP upon arousal or by decreasing the Ta. While these methods worked in suppressing arousal, the mortality rate is also increased. To gather a better understanding of the …
The Role Of Multiple Ccaat-Binding Factors In Candida Albicans Gene Expression, Lashall Lynn Bates
The Role Of Multiple Ccaat-Binding Factors In Candida Albicans Gene Expression, Lashall Lynn Bates
Graduate Theses and Dissertations
The CCAAT-binding factor is a heterooligomeric transcription factor that is evolutionarily conserved in eukaryotes. In yeast, the DNA-binding component that interacts with the CCAAT consensus sequence in promoters consists of the subunits termed Hap2p, Hap3p and Hap5p. In yeast and fungi, a fourth subunit, Hap4p, is required for regulating gene expression. The goal of this research is to understand the function of the Candida albicans CCAAT-binding factor and how it relates to virulence and pathogenicity. C. albicans is a human opportunistic pathogen responsible for a variety of mucosal and systemic infections that result in significant morbidity and mortality, particularly in …
Low-Dose Of Sodium Arsenite Causes Delayed Differentiation In C2c12 Mouse Myoblast Cells Through The Repression Of The Transcription Factor Myogenin, Amanda Steffens
Low-Dose Of Sodium Arsenite Causes Delayed Differentiation In C2c12 Mouse Myoblast Cells Through The Repression Of The Transcription Factor Myogenin, Amanda Steffens
All Theses
A number of epidemiological studies have correlated arsenic exposurwith cancer, skin diseases, cardiovascular diseases, and adverse developmental outcomes such as stillbirths, spontaneous abortions, neonatal mortality, low birth weight, and delays in the use of musculature. The current study used C2C12 mouse myoblast cells to examine whether low concentrations of arsenic could alter their differentiation into myotubes, which would indicate that arsenic has the ability to act as a developmental toxicant. Myoblast cells were exposed to 20nM sodium arsenite and allowed to differentiate into myotubes and expression of the muscle-specific transcription factor myogenin, along with the expression of myosin light chain …
Applications Of Bret: The Detection And Visualization Of Protein Interactions And Intramolecular Conformational Changes, Lindsay Anne Staron
Applications Of Bret: The Detection And Visualization Of Protein Interactions And Intramolecular Conformational Changes, Lindsay Anne Staron
Masters Theses
Bioluminescence is a phenomenon in which chemical energy is converted into light energy. Here, the oxidation of a luciferin substrate, catalyzed by a luciferase enzyme, results in the emission of a photon. This biological process is exploited in a technology referred to as Bioluminescence Resonance Energy Transfer (BRET). As its name implies, BRET depends on a nonradiative energy transfer event that occurs between a donor luciferase and an acceptor fluorophore. Fusion of the donor and acceptor molecules to a protein(s) of-interest allows one to identify and monitor molecular events, such as protein interactions or hormone binding events, based solely on …
Synthesis And Evaluation Of N6,5'-Bis-Ureido-5'-Amino-5'-Deoxyadenosine Derivatives: Novel Nucleosides With Antiproliferative And Protein Kinase Binding Activities, Marcelio Oliveira
Synthesis And Evaluation Of N6,5'-Bis-Ureido-5'-Amino-5'-Deoxyadenosine Derivatives: Novel Nucleosides With Antiproliferative And Protein Kinase Binding Activities, Marcelio Oliveira
Theses and Dissertations
A new series of N6,5'-bis-ureido-5'-amino-5'-deoxyadenosine derivatives was prepared and evaluated for anticancer activities using the NCI 60 panel of human cancers. Certain of the derivatives showed promising activities (low micromolar GI50's) against several of the representative cancers. These included cell lines from the following general cell types in the NCI 60: Leukemia, Breast, Central Nervous System, Non-Small Cell Lung, Ovarian, Prostate, Renal, and Colon cancers. Select compounds were also screened for their affinities for protein kinases. The synthesis of the compounds was straightforward and involved N6 acylation with arylisocyanates, preceded by activation and nucleophilic substitution of the 5'-position to give …
The Regulation Of Alternative Splicing By Oncogenic Signaling Pathways., Jacqueline Shultz
The Regulation Of Alternative Splicing By Oncogenic Signaling Pathways., Jacqueline Shultz
Theses and Dissertations
In the presented study, we demonstrate that the alternative splicing of caspase 9 was dysregulated in a large percentage of NSCLC tumors and cell lines. These findings led to the hypothesis that survival pathways activated by oncogenic mutation regulated this mechanism. Indeed, the oncogenic PI3-Kinase/Akt pathway was demonstrated to regulate the alternative splicing of caspase 9. Further mechanistic studies demonstrate that multiple Akt isoforms can regulate the alternative splicing of caspase 9 in NSCLC. Akt was additionally shown to mediate the exclusion of the exon 3,4,5,6 cassette of caspase 9 via the phospho-state of the RNA trans-factor, SRp30a. Mutagenesis studies …
Pore Size Characterization Of Monolithic Capillary Columns Using Capillary Flow Porometry, Yan Fang
Pore Size Characterization Of Monolithic Capillary Columns Using Capillary Flow Porometry, Yan Fang
Theses and Dissertations
A simple capillary flow porometer (CFP) was assembled for pore structure characterization of monolithic capillary liquid chromatography columns based on ASTM standard F316-86. Determination of differential pressures and flow rates through dry and wet samples provided the necessary information to determine the through-pore throat diameter, bubble point pore diameter, mean flow pore diameter, and pore distribution. Unlike measurements in bulk using traditional techniques to provide indirect information about the pore properties of monolithic columns, monoliths can be characterized in their original chromatographic forms with this system. The performance of the new CFP was first evaluated by characterizing the pore size …
Gas Phase Chiral Recognition, Characterization Of Porous Polymer Monolith Nanospray Ionization, And The Negative Mode Crafti Method Using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry, Nannan Fang
Theses and Dissertations
Our group has been studying chiral recognition in gas phase using mass spectrometry for more than 10 years. We are interested in gas phase studies of fundamental interactions because the gas phase avoids complications and masking effects that may arise upon solvation. Therefore, the results of gas phase experiments can be directly compared with those of high-level computational studies. In chapter 2, I studied the roles of hydrogen bonding and pi stacking in gas phase chiral recognition between aromatic crown molecules and aromatic amines. High affinity between host and guest doesn't necessarily result in better recognition. If the affinity is …
Total Synthesis Of (-)-Acutumine, Fang Li
Total Synthesis Of (-)-Acutumine, Fang Li
Theses and Dissertations
Acutumine is a tetracyclic alkaloid isolated from the Asian vine Menispermum dauricum with selective T-cell cytotoxicity and antiamnestic properties. We have developed a total synthetic route to this congested alkaloid, during which we also found a novel, stereoselective radical-crossover reaction that combines an intramolecular radical conjugate addition with a subsequent enolate hydroxylation. Key features of this synthesis also include a reagent-controlled diastereoselective ketone allylation, an anionic oxy-Cope rearrangement to form a congested quaternary sterocenter, a pyridine-mediated selective ozonolysis, and a Lewis acid promoted Michael-type cyclization.
Development Of Biocompatible Polymer Monoliths For The Analysis Of Proteins And Peptides, Yun Li
Development Of Biocompatible Polymer Monoliths For The Analysis Of Proteins And Peptides, Yun Li
Theses and Dissertations
Biocompatibility is an important issue for the development of chromatographic stationary phases for the analysis of biomolecules (including proteins and peptides). A biocompatible stationary phase material is a material that resists nonspecific adsorption of biomolecules and does not interact with them in a way that would alter or destroy their structures or biochemical functions. The monolithic column format is a good alternative to typical spherical particle packed columns for capillary liquid chromatography of biomacromolecules. Several novel anion-exchange polymer monoliths for the analysis of proteins were synthesized for improved biocompatibility. Two novel polymeric monoliths were prepared in a single step by …
Real-Time Virus Analysis Via Image Charge Detection Surface Induced Dissociation Tandem Mass Spectrometry, Seth T. Call
Real-Time Virus Analysis Via Image Charge Detection Surface Induced Dissociation Tandem Mass Spectrometry, Seth T. Call
Theses and Dissertations
This thesis reports on the development of a novel mass spectrometer combining image charge detection with surface induced dissociation for real-time analysis of intact viruses. Protonated viruses produced using electrospray are accelerated and subsequently impact on a solid surface. Capsid peptides released during the impact are analyzed using time-of-flight mass spectrometry. Image charge detection is used to measure the mass and charge states of structurally intact, electrosprayed viruses prior to impact. Since virus capsids are composed of loosely-bound proteins, collision of viruses with surfaces at moderate impact energies could release intact proteins. The masses and numbers of different protein types …
Design And Characterization Of A Human Exposure Chamber And Inversion Episodes In Salt Lake City, Utah In January/February Of 2009, Roman Yuri Kuprov
Design And Characterization Of A Human Exposure Chamber And Inversion Episodes In Salt Lake City, Utah In January/February Of 2009, Roman Yuri Kuprov
Theses and Dissertations
Research on health effects of particulate matter (PM) has been a very active area in the last two decades. One plausible mechanism by which exposure to PM affects human health includes modification of autonomic endothelium function. Decreased endothelium activity causes heightened risks of cardiovascular disease. A human exposure chamber designed to conduct experiments to quantify diminished function of endothelium from short term exposure to PM is described. The chamber consists of two stages for containment and pre-treatment of PM and exposure of human subjects. Concentrations of CO, CO2, NO, NO2, O3, and PM2.5, are monitored and controlled in the exposure …
Structure-Function Studies Of The Large Subunit Of Ribonucleotide Reductase From Homo Sapiens And Saccharomyces Cerevisiae, James Wesley Fairman
Structure-Function Studies Of The Large Subunit Of Ribonucleotide Reductase From Homo Sapiens And Saccharomyces Cerevisiae, James Wesley Fairman
Doctoral Dissertations
Sufficient pools of deoxyribonucleotide triphophates (dNTPs) are essential for the high fidelity replication and repair of DNA, the hereditary material for a majority of living organisms. Ribonucleotide reductase (Rnr) catalyzes the rate-limiting step of de-novo DNA synthesis, the reduction of ribonucleosides to deoxyribonucleosides. Since the cell relies primarily upon ribonucleotide reductase for its dNTPs, both the cellular levels and activity of Rnr are heavily regulated, especially when DNA damage occurs or during replication blocks in the cell cycle. If dNTP pools become too high, too low, or imbalanced, genomic instability results, leading to either the formation of cancerous cells or …
Community College Student Participation In Undergraduate Research: An Explanatory Case Study For Faculty And Research Mentors, Dana L. Peterson
Community College Student Participation In Undergraduate Research: An Explanatory Case Study For Faculty And Research Mentors, Dana L. Peterson
Electronic Theses and Dissertations
This study adapted the current model of science undergraduate research experiences (URE's) and applied this novel modification to include community college students. Numerous researchers have examined the efficacy of URE's in improving undergraduate retention and graduation rates, as well as matriculation rates for graduate programs. However, none have detailed the experience for community college students, and few have employed qualitative methodologies to gather relevant descriptive data from URE participants. This study included perspectives elicited from both non-traditional student participants and the established laboratory community. The purpose of this study was to determine the effectiveness of the traditional model for a …
Nodulin 26-Like Intrinsic Protein Nip2;1 And Nip7;1: Characterization Of Transport Functions And Roles In Developmental And Stress Responses In Arabidopsis, Won-Gyu Choi
Doctoral Dissertations
Nodulin-intrinsic proteins (NIPs) are plant-specific, water and solute transporters with homology to soybean nodulin 26. In this study, it is shown that Arabidopsis NIP2;1 (AtNIP2;1) expression is acutely stimulated upon waterlogging (70-fold in whole seedlings within 1 hr) and hypoxia (> 1000-fold in roots within 2 hr). Subcellular localization of a AtNIP2;1::YFP fusion protein shows localization to the plasma membrane. Analysis of AtNIP2;1 protein in Xenopus oocytes shows that it is a transporter of lactic acid, a fermentation end product.
Experiments with T-DNA insertional mutants in the AtNIP2;1 promoter showed that reduced AtNIP2;1 expression induced higher lactic acid …
Genomic, Proteomic And Metabolomic Approaches To Study Drought Responses In Aquilegia, David Henry
Genomic, Proteomic And Metabolomic Approaches To Study Drought Responses In Aquilegia, David Henry
All Dissertations
Global population is expected to increase 30% by 2040, which will result in an increased need for crop production to feed the growing population. Combined with projected increased drought conditions worldwide, plant genetic research is necessary to gain a deeper knowledge of the molecular factors involved in plant drought response in order to engineer crop species with improved drought tolerance. Aquilegia has been recently developed as a model species for gene exploration based on its ability to thrive in a wide variety of environments including arid locations. An attractive asset of Aquilegia is its evolutionary position, equidistant between rice and …
Nudibranchs Of The Ross Sea, Antarctica: Phylogeny, Diversity, And Divergence, Christopher Shields
Nudibranchs Of The Ross Sea, Antarctica: Phylogeny, Diversity, And Divergence, Christopher Shields
All Theses
The Southern Ocean (SO) surrounding Antarctica is extremely cold and geographically isolated. The phylogenetic affinities of only a few SO taxa have been examined in detail; in these, a high degree of endemism and radiation within the SO has been established using molecular phylogenetic methods. In order to address these Antarctic paradigms, we used Bayesian inference to construct phylogenetic trees of nudibranch molluscs based on mitochondrial cytochrome-c oxidase I (COI) and 18S ribosomal DNA. We gathered sequences from temperate (COI n=37; 18S n=31) and polar (COI n=21; 18S n=22) species and then combined them with sequences retrieved from GenBank (COI …
Characterization Of The Toc Complex By Blue Native Page:Oligomeric And Dynamic Changes Of The Toc Complex, William I. Crenshaw
Characterization Of The Toc Complex By Blue Native Page:Oligomeric And Dynamic Changes Of The Toc Complex, William I. Crenshaw
Masters Theses
The majority of chloroplast proteins are nuclear encoded and transcribed on cytosolic ribosomes, and therefore must be post-translationally imported into the chloroplast. Preproteins are directed to the chloroplast via a cleavable Nterminal extension known as a transit peptide. This transport is mediated by the Toc and Tic complexes (Translocon at the Outer/Inner Chloroplast envelope membrane), functioning in tandem to transport preproteins into chloroplasts relying on the hydrolysis of ATP and GTP. The Toc complex is composed of the β-barrel channel protein Toc75 and the homologous GTPase receptors Toc34 and Toc159. GTP hydrolysis is necessary for the formation of the early …
Lysophosphatidic Acid Is A Mediator Of Interleukin-6 Production In Ovarian Cancer Cells, David Dang
Lysophosphatidic Acid Is A Mediator Of Interleukin-6 Production In Ovarian Cancer Cells, David Dang
Theses and Dissertations
Lysophosphatidic acid (LPA) is a naturally occurring bioactive lysophospholipid that mediates a broad range of cellular processes such as cell proliferation, survival, migration and invasion. LPA also plays a potential role in human oncogenesis as suggested by elevated expression of its receptors and its producing enzymes in malignant tissues. In the current study, we demonstrated that LPA is a potent mediator of interleukin-6 (IL-6) production in ovarian cancer. IL-6 is a pleiotropic cytokine which is thought to be an important mediator of ovarian cancer development and progression. Here, we demonstrated that IL-6 levels are indeed increased in the plasma of …
A Top-Down Proteomic Approach For The Discovery Of Novel Serum Biomarkers Of Pregnancy-Related Disease, Karen Merrell
A Top-Down Proteomic Approach For The Discovery Of Novel Serum Biomarkers Of Pregnancy-Related Disease, Karen Merrell
Theses and Dissertations
The serum fraction of blood is an ideal material in which to search for novel biomarkers for disease. It is easily obtained through relatively non-invasive means, routinely collected, and a rich treasure-trove of information about the health of an individual. Cells react to signal molecules, take up nutrients, and release waste products, fragments that are the result of proteolysis, and other molecules out into the bloodstream. If these components are unique to the cells in question, that part of the complex mixture that is the blood stream can potentially characterize the health of the tissue or organ those cells are …
The Regulation Of Platelet Activating Factor Acetylhydrolase By Oxidized Phospholipids, Rachael Griffiths
The Regulation Of Platelet Activating Factor Acetylhydrolase By Oxidized Phospholipids, Rachael Griffiths
Theses and Dissertations
Platelet-activating factor acetylhydrolase (PAFAH) is elevated in atherosclerosis and may play a role in pathogenesis of this disease. Molecular mechanisms regulating the expression of this lipoprotein-associated PLA2 are indistinct. Mildy oxidized low density lipoprotein (oxLDL) and monocytes (the primary source of PAFAH) are co-localized in early atheromas. Monocytes are activated by oxidized phospholipids (oxPL) in the oxLDL particle. We hypothesized that oxPL-activated monocytes are the source of increased levels of PAFAH in atherosclerosis. We found that PAFAH expression is significantly induced by OxPAPC and in particular long-chain fractions of oxPAPC in monocytes and cytokine-differentiated DC, but not cytokine-differentiated MO. Furthermore, …
Analysis Of Secreted Phosphoprotein-24 And Its Effects During Osteoblast Differentiation In A Mesenchymal Stem Cell Model, Vasquez Jochen Granja
Analysis Of Secreted Phosphoprotein-24 And Its Effects During Osteoblast Differentiation In A Mesenchymal Stem Cell Model, Vasquez Jochen Granja
Theses and Dissertations
Musculoskeletal diseases, in particular osteoporosis, are increasingly becoming more prevalent in the U.S. due to the ageing population (Figure1). It is estimated that one-sixth of 300 million people in U.S. suffer from bone disorders or loss. About 10 million of those people above age 50 suffer from osteoporosis. Patients that suffer from osteoporosis have high morbidity and mortality rates. For instance, patients have decreased bone mineral density (BMD), a measurement of bone density that reflects the strength of bone as represented by calcium content. A decrease in BMD typically leads to an increased risk of bone fractures. In particular, hip …
Synergistic Growth Inhibition And Enhancement Of Cell Death By Combination Of Melanoma Differentiation Associated Gene-7 (Mda-7/Il-24) And Cisplatin In Ovarian Cancer Cell Lines, Renyan Liu
Theses and Dissertations
Ovarian cancer is the most lethal gynecological malignancy among women. The current first-line treatments for ovarian cancer are cisplatin, carboplatin and paclitaxel. However, resistance to these platinum-based drugs occurs in the large majority of initially responsive tumors, resulting in fully chemoresistant, fatal disease. Therefore, the resistance to cisplatin therapy has been a critical hurdle in the management of recurrent ovarian cancer. The mechanisms responsible for cisplatin resistance are not completely understood. In the search for new therapies to overcome/bypass cisplatin resistance, melanoma differentiation gene-7 (MDA-7) IL-24, which is a new cytokine, has anti-cancer efficacy by suppressing cell growth and inducing …
Intracellular Targets Of Sphingosine-1-Phosphate, Graham Michael Strub
Intracellular Targets Of Sphingosine-1-Phosphate, Graham Michael Strub
Theses and Dissertations
The bioactive lipid mediator sphingosine-1-phosphate (S1P) has emerged as a key regulator of a variety of important physiological functions, including cell growth, cell survival, cell motility, angiogenesis, lymphocyte trafficking, and mast cell function. S1P is formed by two different sphingosine kinases (SphKs) and binds to a family of 5 differentially expressed G-protein coupled receptors (S1PRs). The majority of research to date has focused on the activation of these receptors, but there is compelling evidence to suggest that S1P exerts intracellular functions independent of S1PRs. However no bona fide intracellular targets of S1P have been identified. In my dissertation, I have …