Thesis (Ph. D.)--Case Western Reserve University, 2008. / [School of Medicine] Department of Pharmacology. Includes bibliographical references.

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Glucose is a primary and essential energy source for humans. It is broken down from complex carbohydrates in the diet and absorbed across the gut epithelium into the blood stream. Glucose homeostasis is important as hyperglycermia causes damage of pancreatic and peripheral cells. In response to a meal glucose is principally taken up by fat and muscle tissues and this response is activated by insulin release from pancreatic beta cells. Insulin stimulates the translocation of GLUT4 from the intracellular storage vesicles to the plasma membrane in fat and muscle cells. Although many proteins have been implicated in this process, the key insulin-regulated substrate has not been determined yet. In the present study, the phosphoserine/threonine binding protein 14-3-3 was used as a tool to affinity-purify insulin-stimulated phosphoproteins from 3T3-L1 adipocytes. By using mass spectrometry 38 proteins were identified, reflecting the important role of 14-3-3 in mediating many insulin-regulated processes. Among the potential phosphoproteins was Myosin 1C (Myo1c), an actin-associated molecular motor, which has previously been implicated in insulin-stimulated GLUT4 trafficking in adipocytes. I showed that insulin stimulates the activation of CaMKII which phosphorylates Myo1c at S701 in a Ca2+/PI3K-dependent manner. Myo1c phosphorylation induced its interaction with 14-3-3-proteins, reduced calmodulin-binding and stimulated its in vitro ATPase activity. Insulin-dependent stimulation of Myo1c phosphorylation and its ATPase activity were both required for GLUT4 translocation. By using yeast two-hybrid techniques, I identified a candidate ligand of the Myo1c tail, Armcx5, and demonstrated the in vivo interaction in 3T3-L1 adipocytes. The siRNA-mediated knockdown of Armcx5 inhibited insulin-stimulated glucose uptake and GLUT4 translocation. These results suggest that the regulation of Myo1c and its ligand Armcx5 are essential in insulin-regulated GLUT4 trafficking, possibly playing a key role in vesicle fusion.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2010. / Title from PDF title page (viewed on June 28, 2010). Includes bibliographical references (p.179-204).

Large numbers of people suffer from nervous system injuries and neurodegenerative diseases each year, with little success in regaining lost neural functions. Attempts to successfully regenerate nervous tissue in the mammalian Central Nervous System have meet with limited success. Simpler models have thus been useful in determining conserved mechanisms in the enhancement of neural regeneration. One such mechanism is intracellular calcium signaling. We used <italic>Caenorhabditis elegans</italic> as a model system to study the effects of optogenetic stimulation on regeneration. Using a femtosecond laser we cut individual <italic>C. elegans</italic> axons <italic>in vivo</italic> and then periodically stimulated the neurons by activating the genetically encoded light activated channel, Channelrodopsin-2. We found that periodic photo-activation could increase regeneration over 24h by at least 31%. We repeated these experiments with dantrolene treatment and in <italic>unc-68(e540)</italic> mutants to assess the effects from a lack of internal calcium ion signaling in these worms. In both cases, we found a complete suppression of stimulated regeneration when calcium signaling was blocked. This indicates that intracellular calcium ion signaling is crucial in the initiation of neural regeneration in the first 24 hours and mediates the enhanced outgrowth we observe with periodic photo-activation. The importance of intracellular calcium ion signaling can lead to further studies to enhance the stimulation of neural regeneration, and improve therapies for patients with neural damage and loss of neural functions.

The extensive relationship between modulation of intracellular Ca2+ content and the control of cell proliferation (Boynton et al. 1974; Whitfield et al. 1979; Berridge and Irvine 1984), differentiation (Bridges et al. 1981; Holliday et al. 1991) and death (Orrenius et al. 2003) has led to much examination into the relationship between Ca2+ signaling pathways and the onset of various pathological conditions, including cancer, cardiac hypertrophy, immunodeficiency, neurodegeneration. Control of Ca2+ signals is achieved via an extensive combination of pumps, channels and exchangers which regulate the concentration of Ca2+ within not only the cytosol but also all intracellular compartments. Accordingly, a great deal of research has focused on the mechanisms which regulate these channels and pumps, and recently the primary mechanism for Ca2+ influx in non-excitable cells has been identified. This process, termed Store-operated calcium entry (SOCe), is a key evolutionarily conserved mechanism whereby decreases in endoplasmic reticulum Ca2+ content (sensed by the ER Ca2+ sensor, STIM1) leads to the influx of Ca2+ across the plasma membrane through the Orai family of Ca2+ channels. However, many questions remain about how this Ca2+ signaling pathway is regulated. In this thesis, I provide evidence regarding the transcriptional and molecular mechanisms regulating SOCe. Initial studies in my thesis work aimed to identify some of the key events leading to dysregulation of Ca2+ homeostasis in the kidney specific pediatric malignancy, Wilms' Tumor. I found that STIM1 expression levels and SOCe signals are significantly reduced in Primary Wilms' Tumor samples. Subsequent analysis of these phenomena led me to the finding that STIM1 expression is under the control of the transcription factors Wilms' Tumor Suppressor 1 (WT1) and Early Growth Response 1 (EGR1). Subsequent investigations were carried out with the purpose to assess how activation of the EGR1 transcription factor alters long term Ca2+ signals. Indeed, I found that receptor-mediated activation of EGR1 leads to induction of STIM1 expression and increases in SOCe. However, unexpectedly through these analyses, I propose a novel role for STIM1 that STIM1 interacts with the Plasma Membrane Ca2+ ATPase (PMCA) through its C-terminal proline-rich domain and reduces PMCA-mediated Ca2+ clearance, effectively creating local, augmented Ca2+ gradients. This coordinated control of Ca2+ entry and exit from the cell has wide-ranging implications for Ca2+ signaling in multiple cell types. / Biochemistry

All organisms require rapid and flexible signaling mechanisms in order to effectively respond to biotic and abiotic stress. Calcium ions (Ca2+) have proven to be important components of signaling networks. Observations of stimulus-specific oscillations of cytosolic Ca2+ during signal transduction suggest that Ca2+ signals directly encode information. These stimulus-specific oscillations, known as Ca2+ signatures, can be interpreted by an array of Ca2+-binding sensors and effectors, which subsequently regulate appropriate cellular responses. While progress has been made regarding the classic Ca2+-sensor calmodulin (CaM), less research has been directed towards the CaM-like family of Ca2+-sensors (CMLs). This family – unique to plants – is suspected to regulate a multitude of stress and developmental pathways; however, to date very few members of this family have had their functions elucidated by the identification of downstream targets and upstream regulators. In the present study, I investigate the regulation of CML39, which has previously been shown to strongly respond to the stress hormone jasmonic acid (JA) in Arabidopsis. Bioinformatic tools predict a large number of putative JA-responsive cis-elements within the CML39 promoter. Deletion analysis of CML39 promoter fragments in planta reveals that some cis-elements respond in a tissue-specific manner. Analysis of transgenic MYC2 loss-of-function (myc2) mutants demonstrates that MYC2 – the preeminent JA-responsive transcription factor – is not necessary for CML39 promoter activity. Collectively, these data reveal a complex tissue-specific pattern of CML39 regulation and provide a foundation for the future identification of relevant transcription factors. / Thesis (Master, Biology) -- Queen's University, 2013-09-24 21:06:30.592

The hypothalamus is an integral part of the brain's regulation of mammalian physiology and behavior. Among many functions, this regulatory center activates the sympathetic nervous system, maintains appropriate body temperature, controls food intake, and controls release of hormones from the pituitary gland. Deep within the hypothalamus lie a paired cluster of cells, the suprachiasmatic nuclei (SCN), which function as the chief circadian pacemaker. The goal of the present thesis research was to study rhythmically controlled ATP gliotransmission. I used an immortalized SCN2.2 hypothalamic cell line to determine the mechanism by which ATP signaling is regulated in this context. Additionally, this research aimed to elucidate if clock-controlled ATP gliotransmission is fundamentally distinct from stimulus-evoked calcium-dependent mechanisms that regulate intercellular ATP signaling among astrocytes. In this thesis, I show that there are multiple ATP signaling mechanisms present among SCN2.2 cells. cAMP-dependent signaling mediates clock-controlled ATP accumulation but not stimulus-evoked ATP signaling. In addition, pharmacological studies suggest that disparate purinergic receptor-mediated mechanisms are involved in the regulation of clock-controlled versus stimulus-evoked ATP signaling. Rhythmic accumulation of ATP in SCN2.2 cultures is modulated by calcium-dependent processes. Peaks in ATP accumulation coincide with elevated mitochondrial calcium levels, while troughs in ATP accumulation coincide with periods of high cytosolic calcium levels, suggesting a possible mechanistic link between circadian shifts in intracellular calcium handling and ATP handling in SCN2.2 cells. Clock-controlled ATP accumulation in SCN2.2 cells is not a by-product of rhythmic cell cycle or rhythmic cell death. Overall, my research suggests that the ATP accumulation rhythm in SCN2.2 cells is likely an output of the biological clock, mediated by astrocytic calcium signaling processes, and not an output of cell division or cell death. Estimation of ATP accumulation in SCN2.2 cultures at peak time points suggests that clock-controlled ATP release is critical to the function of astrocytes in the mammalian brain, perhaps in the regulation of brain metabolism, the regulation of sleep/wake physiology, or the integration of both.

Thesis (Ph. D.)--Case Western Reserve University, 2004. / [School of Medicine] Department of Physiology and Biophysics. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 77-99).