Browsing by Subject "Homeostasis"
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Item Activity-Dependent Regulation of Inhibition from Different Inhibitory Subtypes(2007-08-08) Bartley, Aundrea Frances; Gibson, Jay R.Neuronal activity, in the form of action potential firing, is critical in the maturation and maintenance of neocortical circuitry. A negative feedback mechanism by which neuronal circuits adapt to changing levels of average activity on a time scale of hours to days is known as homeostatic plasticity. At the simplest level, homeostatic adaptations occur to maintain firing rate of neurons at a particular set-point. To better understand homeostatic plasticity at the network level, one must understand the activity-dependent adaptations that occur in the different neocortical cells types. To this end, I examined the regulation of inhibitory neurons and their synapses. I used chronic pharmacological block of activity in a neocortical slice cultures to examine the role activity plays in regulating feedback inhibition defined by two biochemical inhibitory neuron subtypes - parvalbumin-positive (Parv+) and somatostatin-positive (Som+). The cellular and synaptic components of local feedback inhibition were examined. I found that chronic activity blockade caused the following: 1) an increase in the intrinsic excitability of Som+ neurons through the downregulation of 2 substhreshold currents. While not thoroughly examined in Parv+ neurons, a similar, but weaker, increase in excitability may occur in these neurons as well. These< changes are consistent with a homeostatic maintenance of firing rate in these neurons. 2) a differential regulation of monosynaptic inhibition based on subtype that was frequency dependent. At low frequency action potential firing, Parv+ mediated inhibitory drive was downregulated while Som+ was unchanged. Both subtypes were likely downregulated at high frequency firing. 3) an upregulation of excitatory drive onto both Parv+ and Som+ neurons. This was most dramatic at low frequency firing where both subtypes displayed an almost 3-fold increase. This is also consistent with homeostatic maintenance of firing rate in inhibitory neurons. 4) based on the above, a clear change in recurrent inhibition occurred at low frequency firing. First, net recurrent inhibition was increased for both subtypes, but the relative influence of the two changed, such that Som+ recurrent inhibition contributed more relative to that of Parv+ circuitry. At high frequency firing, a slight, but less resolvable, increase in net recurrent inhibition may have occurred in both subtypes without any change in relative contribution. 5) all of the synaptic changes were likely due to increases in presynaptic release probability and/or decreases in synapse number.Item Cellular Responses to Inorganic Phosphate in Physiological and Pathological Processes(2013-04-11) Schwartz-Moretti, Joel 1984-; Moe, Orson W.; Kuro-O, Makoto; Carroll, Thomas J.; Huang, Chou-LongPhosphate is an essential chemical component of all known living organisms and is present in a variety of biological molecules such as nucleic acids and phospholipids. In vertebrates, phosphate is one of the primary ionic constituents of bone mineral. Phosphate homeostasis is maintained by a balance of absorption in the intestine, storage in pools such as bone and soft tissue, and excretion in the kidney. The flow of phosphate through these sites is controlled by several interacting hormone systems, including parathyroid hormone, vitamin D and the recently discovered endocrine hormone fibroblast growth factor 23. Loss of appropriate control of phosphate homeostasis can result in structural defects in bone if phosphate supply is inadequate, or can lead to mineralization of soft tissues if phosphate is present in excess. In human diseases or animal models characterized by impaired function of fibroblast growth factor 23 or its co-receptor Klotho, phosphate is not sufficiently eliminated by the kidney and excess phosphate accumulates. This results in ectopic mineralization in soft tissues and a variety of other pathological consequences, ultimately increasing risk of death. Previous research in the areas of osteoblast development and pathological mineralization in vascular tissue has indicated that excess extracellular phosphate causes changes in cellular behavior. We performed a series of experiments to examine in detail what effects excessive extracellular phosphate might have both on individual cells and on the organism as a whole. We found that treatment with elevated extracellular phosphate caused acute activation of cellular signaling pathways and induced expression of the mineral binding protein osteopontin in osteoblasts and fibroblasts. We found evidence that these responses are likely not the result of interaction of cells with phosphate per se but with calcium phosphate precipitates that form in the experimental conditions used. We found that diets containing higher than normal phosphate had adverse effects on mice, such as weight loss and kidney fibrosis. There is accumulating clinical evidence that such insoluble calcium phosphate particles arise in conditions of phosphate excess such as chronic kidney disease. Greater understanding of the formation and clearance of such particles may aid in the management of pathologies caused by phosphate excess.Item Disturbances in renal autoregulation and the susceptibility to hypertension-induced chronic kidney disease(2004-08-05) Palmer, Biff F.Item FGF23 at the intersection of phosphate and iron homeostasis(2023-08-25) Wolf, MylesItem Mechanisms of Genome Buffering and Cell Fate Coordination in Adult Tissue Homeostasis(2016-07-26) Tuladhar, Rubina; Amatruda, James F.; Scherer, Philipp; DeBerardinis, Ralph J.; Lum, LawrenceSelf-renewal competency of adult stem cells is essential for tissue homeostasis. The corruption of genes essential for genome preservation or for niche-stem cell interactions frequently results in loss of stem cell viability and disease. The two components of my thesis focus on understanding adult stem cell preservation - the integration of metabolism and intercellular communication mediated by the Wnt family of secreted signaling molecules, and epigenetic mechanisms that buffer the proteome against insertion/deletion (INDEL)-type genetic mutations. Wnt-mediated signaling is essential for embryogenesis and the maintenance of adult tissues. Lipidation of Wnt proteins by the acyltransferase Porcupine (Porcn) is crucial for secretory pathway exiting. Using chemically based approaches, I have demonstrated that Porcn active site features conserved across animals enforce ω-7 cis fatty acylation of Wnt proteins. Deviant acylation of a Wnt protein using an exogenously supplied trans fatty acid cripples its ability to traverse the secretory pathway due to a previously unappreciated stereoselectivity of the Wnt chaperone Wntless (WLS) for fatty acids. My findings provide a mechanistic account of chemical specificity observed in Porcn inhibitors, and delineate a universal mechanism for integrating communal cell fate decision-making with metabolic fitness. As part of my efforts to generate isogenic cells for the expression of LKB1, a tumor suppressor that regulates Wnt protein production, I encountered the emergence of foreign LKB1 proteins subsequent to the introduction of INDELs by the DNA editing enzyme CRISPR-Cas9. I demonstrate that these novel proteins are the products of: a) the installation of internal ribosomal entry sites (IRES), b) the induction of exon skipping due to compromised exon splicing enhancers (ESEs), and c) the conversion of pseudo-mRNAs to protein-coding mRNAs due to the unwanted elimination of premature termination codons. I propose that these molecular events serve as compensatory mechanisms employed by cells to restore proteome integrity in the face of INDEL-type challenges to the genome posed by pathogens and environmental mutagens. Taken together, these two projects will: a) delineate intervention strategies premised upon the attack of an universally conserved point of intersection between metabolism and cell-to-cell communication, b) facilitate the personalization of medicine, and c) accelerate tissue engineering initiatives.Item Regulation of the cGAS-STING Pathway in Health and Disease(2018-11-27) Pokatayev, Vladislav Andreyevich; van Oers, Nicolai S. C.; Conrad, Nicholas; Chen, Zhijian J.; Yan, NanThe innate immune system senses non-self or altered-self molecular structures through pattern recognition receptors in order to eliminate pathogens or damaged cells, and restore an organism to its basal physiology. Nearly all nucleated cell types can sense intracellular viral nucleic acids. These sensors detect either viral RNA through RIG-I like receptors or DNA through the cGAS-STING signaling pathway. Antiviral immune pathways are vital for resolution of viral infections; however, their dysregulation may give rise to various immune-mediated diseases. The neuro-inflammatory autoimmune disease Aicardi-Goutières Syndrome (AGS) develops from mutations in genes encoding several nucleic acid processing proteins, including RNase H2. Defective RNase H2 may induce accumulation of self-nucleic acid species which trigger chronic inflammation leading to AGS pathology. We created a knock-in mouse model with an RNase H2 AGS mutation in a highly conserved residue of the catalytic subunit, Rnaseh2aG37S/G37S (G37S), the most severe Rnaseh2a mutation categorized as it abolishes nuclease activity to less than 10% of WT RNase H2, to understand disease pathology. Importantly, I found that the G37S mutation induces a cellular anti-viral state, and an increased expression of interferon-stimulated genes dependent on the cGAS-STING signaling pathway. G37S homozygotes are perinatal lethal, and ablation of STING in G37S mice results in partial rescue of the perinatal lethality and complete rescue of the immune phenotype. This study motivates inhibitors of the cGAS-STING pathway in the goal of resolving Rnaseh2a-mediated AGS. As my previous work implicates STING in the development of AGS, I performed a genetic screen to identify novel regulators of this protein. I discovered that TOLLIP, a protein previously identified as a regulator of extracellular Toll-like receptor pathways, can function as a positive regulator of the cGAS-STING pathway. TOLLIP antagonizes STING protein degradation through a regulatory pathway controlled by the protein IRE1α. In Tollip-/- cells, IRE1α is activated and induces lysomal-mediated degradation of STING. Chronic activation of this degradative pathway blunts the cellular response to cGAS or STING agonists. These findings have implications in vivo, as deleting Tollip in a mouse model for AGS, the Trex1-/- mouse, can rescue symptoms of the disease. These findings have clinical importance, as novel therapeutics against TOLLIP can be developed to treat auto-inflammation caused by dysregulation of the cGAS-STING signaling pathway.Item The Role of Foxo Transcription Factors in B Cell Development and Activation(2010-01-12) Hinman, Rochelle Marie; Satterthwaite, Anne B.A functional immune system depends on a diverse, self tolerant B cell repertoire. Mature B cells distributed throughout secondary lymphoid organs respond to antigenic stimuli by dividing and differentiating into plasma cells and other effector cell types. Signaling from the B cell receptor (BCR) plays a critical role at several points during this developmental process. Cell survival, proliferation, differentiation, death, anergy, and receptor editing may occur in response to BCR stimulation. A variety of factors, including signal strength and duration, cytokine presence, and co-stimulation determine the ultimate B cell fate. In this thesis, the roles Foxo transcription factors play in maintaining B cell homeostasis will be explored. Foxo1, Foxo3, and Foxo4 have both anti-mitogenic and pro-apoptotic properties. The transcription factors are posttranslationally controlled via Akt. When a mature B lymphocyte is stimulated through the BCR, Akt-mediated phosphorylation of Foxos results in their exclusion from the nucleus. In the absence of Foxo nuclear activity, the B cell progresses into the cell cycle. We have discovered a second PI3K-dependent means of control for Foxos, at the level of mRNA expression. Downstream of the BCR, this means of control is unique and functionally relevant. Mature B cells proliferating in response to anti-IgM downregulate Foxo mRNA expression. This is via activation of the PI3K/Btk/BLNK/PLC-gamma2 pathway. Conversely, Foxo mRNA expression is upregulated in immature B cells, both when the tonic/basal signal through the BCR is disrupted and when the BCR is engaged with anti-IgM. Overexpression of Foxo3 mRNA in an immature B cell line promotes anti- IgM induced apoptosis. Primary immature B cells from Foxo3-/- mice have decreased apoptotic response to BCR crosslinking. Thus, at the immature stage of development our work has revealed a potential role for Foxo3 in promoting clonal deletion. Foxo3-/- mice also have reduced frequencies of pre-B and mature recirculating B cells in the blood and bone marrow. The mice demonstrate increased basal levels of IgG2a, IgG3, and IgA. Thus, Foxo3 deficiency affects numerous aspects of B cell development.Item SAM Homeostasis Is Regulated by CFIm-Mediated Splicing of MAT2A(August 2021) Scarborough, Anna Maurine; Tu, Benjamin; Mendell, Joshua T.; Green, Carla B.; Conrad, NicholasS-adenosylmethionine (SAM) is the methyl donor for nearly all cellular methylation events. Cells regulate intracellular SAM levels through intron detention of MAT2A, the only SAM synthetase expressed in most cells. The N6-adenosine methyltransferase METTL16 promotes splicing of the MAT2A detained intron by an unknown mechanism. Using an unbiased CRISPR knock-out screen, we identified CFIm25 (NUDT21) as a regulator of MAT2A intron detention and intracellular SAM levels. CFIm25 is a component of the cleavage factor Im (CFIm) complex that regulates poly(A) site selection, but we show it promotes MAT2A splicing independent of poly(A) site selection. CFIm25-mediated MAT2A splicing induction requires the RS domains of its binding partners, CFIm68 and CFIm59 as well as binding sites in the detained intron and 3´ UTR. These studies uncover mechanisms that regulate MAT2A intron detention and reveal a previously undescribed role for CFIm in splicing and SAM metabolism.Item Structural Studies of Integral Membrane Proteins Involved in GPCR Signaling and Sterol Homeostasis(2018-11-27) Clark, Lindsay D.; Rice, Luke M.; Rosenbaum, Daniel M.; Gardner, Kevin H.; Jiang, YouxingMembrane proteins are crucial molecules for cellular survival, and can take on multiple and diverse roles within the native membrane. In this dissertation, I will detail my efforts to understand and study two different types of membrane proteins. First, I will discuss my research developing and applying a strategy to use NMR spectroscopy to study specific receptors within the large family of G protein-coupled receptors. This strategy enabled the first methyl-TROSY experiments on a wild-type human GPCR, and have significant value for future drug discovery efforts on this important class of membrane proteins. Second, I will discuss my endeavors to understand the important role of the protein Scap, which can both sense and respond to differences in cholesterol levels within the ER membrane. Scap is a central player in the SREBP pathway, which is targeted by multiple classes of pharmaceuticals, including statins. Through efforts described in the second half of this dissertation, I have been able to demonstrate the first biochemical characterization of the full-length mammalian Scap/Insig complex, which has led to the first structural characterization of this important machinery. The long-term goal of both of these projects is aimed at having a more complete understanding of how these important membrane proteins respond to ligands and other environmental changes within their native cell membrane. This information will further our ability to diagnose and treat diseases ranging from insomnia and chronic pain to atherosclerosis and hypercholesterolemia.Item Studies of Bile Acid-Like Signaling Pathways in Mammals and Nematodes(2010-01-12) Zhi, Xiaoyong; Mangelsdorf, David J.Bile acids are not only detergents for lipid solubilization and absorption, but also important signaling molecules. They regulate biological events in mammals by acting on nuclear receptors and membrane-bound receptors. Bile acid homeostasis is maintained in part through a FXR-SHP signaling circuit, in which SHP functions as a transcriptional corepressor. The mechanism whereby SHP represses was one focus of my thesis research. I used a number of biochemical strategies including tandem affinity purification to identify SHP interacting proteins. I also successfully solubilized SHP recombinant protein, which was used to generate crystals that diffracted to 3.2 Angstroms. Bile acid-like molecules function in nematodes to control a variety of life history traits such as dauer and infective L3 formation through the nuclear receptor DAF-12. Although DAF-12 homologues from different nematode species are functionally and structurally conserved, they show differential pharmacological responses to ligands. To that end, I solved the X-ray crystal structure of the hookworm Ancylostoma ceylanicum DAF-12 ligand binding domain and revealed the molecular basis underlying species specific-ligand binding for DAF-12. Furthermore, DAF-12 was shown to be structurally similar to the bile acid sensor FXR, suggesting bile acid-like signaling pathways have been conserved across evolution. In conclusion, my studies provide new insights into how bile acids are sensed and regulated in mammals and nematodes.Item Vibrio Effector Protein, VopQ Targets the Host Lysosome to Manipulate Autophagy(2014-07-23) Sreelatha, Anju; Tu, Benjamin; Orth, Kim; Goodman, Joel M.; Shiloh, MichaelVibrio parahaemolyticus is a gram-negative marine bacterium that is the major cause of gastroenteritis due to the consumption of contaminated raw or undercooked seafood. Vibrio parahaemolyticus harbors two Type III secretion systems. The first, T3SS1, orchestrates a temporally regulated cell death mediated by autophagy, membrane blebbing, followed by cell rounding and eventual lysis of the host cell. One T3SS1 effector protein, VopQ is both necessary and sufficient to induce rapid autophagy during the first hour of infection. Herein, I characterize the biochemical activity of the virulence factor VopQ, a novel Vibrio parahaemolyticus protein with no homology to any proteins outside of the Vibrio species. VopQ binds to the conserved Vo domain of the V-ATPase that is enriched on the lysosomal membrane and causes deacidification of the lysosomes within minutes of entry into the host cell. VopQ forms an ~18 angstrom gated channel that facilitates outward-rectified flux of ions across lipid bilayers. These studies show how a bacterial pathogen uses a novel, targeted pore forming effector to alter autophagic flux by manipulating the partitioning of small molecules and ions. Additionally, we demonstrate that VopQ is also a potent inhibitor of vesicular membrane fusion using in vitro membrane fusion. The inhibition of membrane fusion appears to be independent of VopQ’s pore-forming activity. VopQ inhibits the final step of membrane fusion by inhibiting trans-SNARE complex formation. In order to delineate the two inhibitory functions of VopQ, deacidification and membrane fusion, I use mutational, biochemical and crystallographic studies. Elucidating the molecular mechanism of VopQ not only provides a better understanding of Vibrio parahaemolyticus pathology but also offers new insight into the host cell mechanisms of autophagy and vesicle fusion.