Accurate chromosome segregation is critical as unequal distribution of the genomic DNA results in impaired cell function or cell death. Kinetochores, the multi-protein structures assembled on centromeric DNA, drive chromosome segregation. Chromosome segregation is under supervision of mitotic spindle checkpoint. The mitotic spindle checkpoint is a surveillance mechanism ensuring that cells enter anaphase with all kinetochores properly attached to spindle microtubules and thereby preventing missegregation. Some checkpoint proteins are localised at kinetochore where they generate and enhance the checkpoint signal. Mps1 (Mph1 in S. pombe) and Aurora B (Ark1 in S. pombe) kinases are required for precise chromosome segregation and mitotic spindle checkpoint in fission yeast. In this study we investigate the roles of Mph1 and Ark1 in regulating the S. pombe kinetochore protein Spc7, which is the homologue of human Blinkin/KNL1. We demonstrated that both kinases target the N-terminus of Spc7. Loss of phosphorylation on the candidate phosphosites results in sensitivity to microtubule depolymerizing drugs indicating mitotic defects. As Blinkin has been proposed to be a docking platform for checkpoint proteins, we tested the possibility that Mph1 kinase is involved in kinetochore targeting of checkpoint proteins, Bub1 and Bub3. Our results demonstrate that Mph1-dependent phosphorylation of Spc7 at conserved MELT motifs is required for Bub1 and Bub3 kinetochore localisation. We were able to reconstitute the interaction between Spc7 and the Bub proteins in vitro demonstrating that the Spc7 phosphorylation is sufficient for Bub1 and Bub3 association with Spc7, most likely with Bub3 making the Spc7 contact. Mimicking phosphorylation at the MELT motifs leads to constitutive Bub1 localisation at kinetochores. We also showed that the N-terminus of Spc7 has microtubule binding activity regulated by Ark1 kinase. Mimicking phosphorylation at Ark1 sites results in reduced amount of recombinant Spc7 co-precipitating with microtubules in microtubule binding assays. Moreover, two stretches of basic residues, that contribute to Spc7 microtubule binding activity, have been mapped in the extreme Nterminus of Spc7. Spc7 also interacts with PP1 phosphatase, Dis2 in S. pombe, which is required for checkpoint silencing, but the mechanism of this interactions remains to be determined. These findings allow us to speculate on Spc7 role(s) in coupling microtubule binding with spindle checkpoint activation and silencing.

Mitochondrial Ca2+ buffering is an important physiological modulator of neuronal signaling and bioenergetics, but this propensity toward Ca2+ regulation proves pathological during excitotoxic insult. Specifically, excessive mitochondrial Ca2+ uptake is a key component of glutamate toxicity within the penumbra surrounding the ischemic core following stroke. This mitochondrial toxicity and Ca2+ dyshomeostasis may be visualized in real time as delayed calcium deregulation (DCD). DCD is a predictor of neuronal, excitoxic death, and is composed of three phases: 1) an initial response; 2) a latent period of elevated, but stable cytosolic Ca2+; and 3) failure of mitochondrial Ca2+ retention, termed deregulation. The duration of the latent period is an index of neuronal resistance. Mitochondria are dynamic organelles that rapidly and reversibly undergo fission and fusion (MFF). MFF is tightly regulated by the phosphoregulation of fission inducing Drp1 at serine 656. Drp1-S656 phosphorelation is mediated by PKA/AKAP1, and it is dephosphorylated by PP2A/Bβ2. Phosphorylation of Drp1-S656 inactivates this contractile GTPase resulting in inhibition of mitochondrial fission and a shift toward elongated mitochondria. This PKA/AKAP1 dependent Drp1-S656 phosphorylation has proven to be neuroprotective. Likewise, attenuation of PP2A/Bβ2 signaling enhances neuronal survival during ischemia and excitotoxic insult. Based on the mitochondrial buffering role in excitotoxicity and MFF modulation of neuronal survival, we began investigating the role of Ca2+ buffering as a function of MFF during glutamate toxicity. Noted above, resistance to excitoticity is visualized by the duration of the DCD latent period. Overexpression of AKAP1 in cultured hippocampal neurons greatly prolonged DCD latency in a PKA dependent manner, while Bβ2 ablation prolonged DCD latency by hours. Pharmacological modulation of PKA required PDE4 inhibition to reproduce the AKAP1 observations. Preliminary experiments studying the effect of Bβ2 overexpression on matrix Ca2+ load suggests possible mechanism of MFF regulated of matrix Ca2+ accumulation. Using mtPericam DRG neurons as a model system for individual mitochondrial Ca2+ recording, we discovered impaired extrusion kinetics in mitochondria fragmented by both Drp1 and Bβ2 overexpression. Ca2+ uptake was comparable to that of control. Extreme elongation of mitochondria via dominant negative Drp1-K38A enhanced recovery. Understanding these observations, however, requires knowledge of the mitochondrial Ca2+ buffering mechanism. Mitochondrial uptake candidates include MCU and ccdc109b. Our neuronal characterization of MCU confirms a role in mitochondrial Ca2+ buffering, but not a requirement; other components must be involved. Ccdc109b remains an inconclusive candidate, but may be an important regulator of MCU. Mitochondrial efflux transporters include Letm1 and NCLX. Though Letm1 observations are hindered by control artifact, preliminary evidence supports a role in extrusion. The role of NCLX is complicated by possible tissue specificity. Functional expression experiments utilizing Na+ free Li+ external solution suggests absence of NCLX in hippocampal neurons; DRG neurons were capable of Li+ exchange. The above observations confirm the significance of mitochondrial Ca2+ extrusion in neuronal survival. Understanding the mechanisms and regulation of mitochondrial Ca2+ transport has the potential to provide novel therapeutic targets in pathologies of excitotoxic etiology.

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Thesis (M.S.)--University of Nevada, Reno, 2005. / "August, 2005." Includes bibliographical references. Library also has microfilm. Ann Arbor, Mich. : ProQuest Information and Learning Company, [2005]. 1 microfilm reel ; 35 mm. Online version available on the World Wide Web.

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In a lead-cooled fast reactor (LFR), small bubbles (in the order of one mm or less) may enter the coolant from a leaking steam generator. If such a leakage is undetected the small bubbles may eventually coalesce into a larger bubble in local stagnation zones under the active core. If such a bubble or void releases and passes through the core, it could drive the reactor into prompt criticality. It is therefore desirable to be able to detect the initial stages of such void formation. In this thesis, a methodology to detect such leaks is presented together with a study on void-induced reactivity effects in various LFR's. The methodology developed is based on information from two fission chambers positioned radially outside the core. The fissile content of the fission chambers consist either of 235U or 242Pu making them sensitive to different parts of the neutron spectrum. It is shown that the information from the fission chambers can be used to obtain an early indication of the presence of a small leak within typically a month. Furthermore, it is shown that for all but the smallest LFR’s, prompt criticality due to voids passing the core cannot be excluded. One conclusion is that the methodology may form an attractive complement to the general monitoring system of future LFR’s but, as is noted, it has potential for further developments.

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