UMD Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/3
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Item ARABIDOPSIS THALIANA GLUTAMATE RECEPTOR-LIKE 3.7 UNDERLIES ROOT MORPHOLOGY AND SIGNALING VIA MEMBRANE POTENTIAL HOMEOSTASIS(2021) Barbosa-Caro, Juan Camilo; Feijó, José A; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Plants perceive highly variable environments and biotic interactions through membrane receptors like the GLutamate Receptor-like (GLR) family, related to the ionotropic Glutamate Receptors that underlie information transmission in neurons. GLRs underpin information transduction and morphological adaptations in plants. However, mechanistic understanding is scarce. In Arabidopsis thaliana roots, we investigated how GLRs underlie amino acid-induced electric and Ca2+ excitability. We also assessed the contribution of GLR3.7 in root hair elongation. We present GLRs as mediators of a local, glutamate-induced electric and Ca2+ response in roots, with the same initiation kinetics of wound-induced Slow Wave Potentials (SWP). We identify GLR3.7 as mediator of root hair elongation through maintenance of membrane depolarization at the growing cell apex. These results propose a parallel between glutamate-triggered signals and SWP initial phase as local and chemically induced, and posit GLR3.7 as a possible contributor to Ca2+ homeostasis in root hair apical growth.Item INTRACELLULAR REGULATION OF ATRIAL EXCITATION CONTRACTION COUPLING IN NORMAL AND ARRHYTHMOGENIC HEARTS(2017) Garber, Libet; Lederer, Jonathan W; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Atrial fibrillation (AF) is the most common arrhythmia with a prevalence of 1-2% of the US population and it is the most important single risk factor for an ischemic stroke. Despite decades of research, successful termination of the arrhythmia remains difficult. The challenge is in part due to our incomplete understanding of atrial myocyte Ca2+ signaling and underlying disease mechanisms. In the atria, like all cardiac tissue, the conducted action potential (AP) underlies triggering of the [Ca2+]i transient, which is responsible for activating contraction. The process that links electrical activity to Ca2+ signaling and contraction is known as excitation-contraction coupling (ECC). The objective of this dissertation is to understand the mechanism of excitation contraction coupling in atrial myocytes. To achieve this goal, we (1) developed tools to specifically study atrial cell biology, (2) we studied the role of altered Ca2+ buffering on ionic membrane currents and Ca2+ signaling, (3) we investigated the role that reactive oxygen species (ROS) plays in altered Ca2+ signaling and the morphology of the AP and (4) we measured intracellular sodium concentration ([Na+]i ) and studied Na+ and Ca2+ signaling in a transgenic murine model of AF. This work includes mathematical modeling of atrial cell electrical and Ca2+ signaling to define our quantitative understanding of the processes involved. Our results indicate that increased Ca2+ buffering plays a major role in speeding the inactivation of the L type Ca2+ current (ICa,L ). This work also shows that low concentrations of H2O2 for a brief period increases atrial Ca2+ spark rate, changes spark characteristics and decreases the duration of the AP. We quantified for the first time the [Na+]i in murine atrial cells both at rest and during field stimulation in control and transgenic mice. Our results indicate that [Na+]i is significantly lower in atrial myocytes in comparison to their ventricular counterparts, which reveal important differences in how [Na+]i is regulated in atrial cells. Moreover, our work demonstrates that [Na+]i and [Ca2+]i homeostasis are profoundly affected during AF. The results further our understanding of mechanisms that modulate excitation-contraction coupling in atrial myocytes in normal and pathophysiological conditions.Item THE ROLE OF EXCITATION-CONTRACTION COUPLING FAILURE IN MUSCLE FATIGUE AND WEAKNESS OF DYSTROPHIC SKELETAL MUSCLE(2011) Mazala, Davi Augusto Garcia; Chin, Eva R; Kinesiology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Alterations in intracellular calcium (Ca2+) are thought to play an important role in skeletal muscle weakness associated with muscular dystrophy due to the activation of Ca2+-regulated proteases (calpains). It was hypothesized that impairments in Ca2+ regulation are exacerbated in dystrophic muscle and that calpain inhibition could attenuate the muscle weakness induced by fatiguing contractions. Single muscle fibres from control and dystrophic mice lacking dystrophin (mdx) and utrophin plus dystrophin (Utr-/-/mdx) were used. Fibres from Utr-/-/mdx mice had similar peak tetanic Ca2+ compared to control and mdx mice, however Utr-/-/mdx mice took longer to clear the released Ca2+. All fibres showed similar time to fatigue but fewer mdx and Utr-/-/mdx fibres remained excitable 1hr after fatiguing contractions. Exposure to a calpain inhibitor improved Ca2+ levels in dystrophic fibres (mdx; trend only in Utr-/-/mdx) after fatigue. Together, these data indicate that calpains play a role in prolonged muscle weakness after fatiguing contractions.Item Mitochondrial outer membrane permeability to metabolites influences the onset of apoptosis(2007-05-08) Tan, Wenzhi; Colombini, Marco; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Apoptosis is a process in multicellular organisms to signal and induce death of specific cells, while avoiding inflammatory reactions. It is an important way to recycle the materials of unwanted cells and maintain cell balance. The execution phase of apoptosis can be initiated by proteins released from mitochondria (such as cytochrome c). Results reported here are consistent with this release being influenced by changes in the mitochondrial outer membrane permeability to metabolites. Phosphorothioate oligonucleotides induce cell death and block VDAC, a protein in the mitochondrial outer membrane that facilitates metabolite flow. These properties seem to be linked in that both require the phosphorothioate modification, both are enhanced by an increase in oligonucleotide length, and both are insensitive to nucleotide sequence. VDAC reconstituted into planar phospholipid membranes is blocked by phosphorothioate oligonucleotides with a 1:1 stoichiometry. They block the pore of the channel through interacting with the inner wall of the pore. The rate of binding occurs at a 100 μs scale but the binding is usually unstable. However, some conformational change stabilizes the complex resulting in long-term complete blockage of VDAC. In mitochondria, this blockage interferes with metabolite flow and inhibits the respiration of mitochondria. It is very specific for VDAC at sub-micromolar concentrations of phosphorothioate oligonucleotide and under these conditions there is minimal effect on enzymatic processes in the mitochondrial inner membrane. The ability of PorB from Neisseria meningitidis to inhibit apoptosis by moving to the mitochondrial outer membrane, was investigated in light of VDAC's role in apoptosis. PorB is unable to alter VDAC's gating properties but does allow ATP to cross membranes. Thus it may restore metabolite flux when VDAC channels close early in apoptosis. Attempts to test this in yeast were not successful. VDAC gating influences transmembrane Ca2+ flux. The closed states favor calcium permeation and the open state limits calcium flux. In mitochondria this gating could influence the rate of Ca2+-dependent mitochondrial swelling and subsequent cytochrome c release. Thus, the mitochondrial outer membrane permeability regulated by VDAC gating may play an important role in mitochondrial function and control of apoptosis.