Ph.D. - Biomedical Sciences (Physiology)

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    Chronic exposure to an insulin-containing lipogenic stimulus results in ectopic cytoplasmic lipid accumulation and altered pro-inflammatory function in mast cells
    ([Honolulu] : [University of Hawaii at Manoa], [May 2013], 2013-05) Greineisen, William Ernest
    This thesis presents evidence that mast cells (MC) chronically exposed to insulin respond by developing steatotic levels of cytosolic lipid bodies, suggesting that immunocytes, like hepatocytes and myocytes, are sites of lipid sequestration in response to dysregulated insulin levels. This ectopic lipid accumulation influences mast cell functionality, biasing mast cell phenotype towards the production of bioactive lipid mediators (LTC4) and away from the release of histamine and other secretory granule components. In the current study we present an analysis of the whole cell and lipid body lipidome in control, and insulin-exposed mast cells. Our data show a significant upregulation in lipid-associated pro-inflammatory precursor molecules in response to chronic insulin exposure. We also show that the lipid body population in these cells are heterogeneous to a previously unsuspected degree. Moreover, due to the intimate relationship between the endoplasmic reticulum (ER) and lipid body production, we tested the hypothesis that the ER may be altered in response to chronic insulin exposure. Indeed, our data show that (in a manner analogous to observations in hepatocytes from obese models) the ER is reprogrammed towards a lipogenic phenotype, is morphologically distended, is compromised as a calcium store and exhibits certain indicators of a unfolded protein response (UPR)/ER stress response in response to chronic insulin. Taken together, these data show that chronic insulin exposure in a model mast cell system drives lipidomic remodeling in a manner that alters lipid body formation and mast cell proinflammatory function.
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    Detection of cortical arousals in sleep EEG
    ([Honolulu] : [University of Hawaii at Manoa], [December 2010], 2010-12) Collin, Herve
    Introduction: Cortical arousals (CA) are a transient part of the sleep-wake system that may play an important role in characterizing sleep fragmentation and disorders, such as obstructive sleep apnea. The American Association of Sleep Medicine (AASM) (1992) rules describe electroencephaolgram (EEG) frequency ranges, as well as electromyogram (EMG) signal morphology to identify CA; however, because of a lack of reliability in arousal detection, even among well-trained human scorers employing the AASM rules, CA has had limited efficacy in describing healthy sleep and/or in diagnosing sleep pathology. The purpose of this study is to increase the reliability of CA detection utilizing Power Spectrum Density (PSD). The exact frequency bands needed for CA detection for each sleep stage will be identified. It will be tested whether or not, the submental activity is necessary in slow wave sleep (SWS) to increase the reliability of CA detection. Methods: Previously recorded 30-second EEG sleep epochs from healthy adult subjects (N = 99) were examined in this study. The average and standard deviation of the relative powers of all frequency bands (Delta, Theta, Alpha, Sigma, Beta1, Beta2, and Gamma) were computed for all epochs and sorted by sleep stages. Using EEG activity, the relative power of specific frequency bands was compared to the average plus a multiple of the standard deviation for the purpose of detecting CA in each sleep epoch. EMG activity was also included for all sleep stages. The average and standard deviation of the relative amplitudes were then computed for all epochs and sorted by sleep stages. For each sleep stage, CA detection was achieved by comparing the standard deviation of the amplitudes to a multiple of the averages of the standard deviations. This experimental scoring technique was then compared with EEG epoch data scored by the Sleep Heart Center Study (SHHS) sleep scientists. An estimate of reliability was obtained using the Cohen kappa, sensitivity, and specificity measures. Results: Optimum CA detection entailed using a combination of different explicit frequency ranges for different respective stages. Based on the reliability calculated from the Cohen kappa, the optimum frequency bands for stage1 were: Beta1 (16-24 Hz), stage2: Beta1 (16-24 Hz), stage3: Beta1 and Gamma (24-48 Hz), stage4: Beta1 and Gamma (24-48 Hz), and stage REM: Delta, Alpha, Beta1, and Gamma (0-4, 8-12, 16-24 and 32-48 Hz). It was also found that the use of EMG for NREM sleep stages increased the sensibility. The corresponding statistical measures for all sleep stages were: Sleep Stage Cohen kappa Sensitivity Specificity Stage1 0.16±0.025 63±3% 56±2% Stage2 0.35±0.025 61±3% 81±1% Stage3 0.48±0.050 60±5% 96±1% Stage4 0.73±0.170 73±15% 98±2% Stage REM 0.48±0.030 58±3% 93±1% Conclusion: Careful consideration of the frequency band is necessary in order to increase the reliability of the detection of CA in EEG. Submental activity was also found to increase the reliability of CA detection in sleep stages SWS. Moreover, incorporating submental activity similarly increases most statistical measures. In conclusion, the AASM rules for detecting CA in EEG would be improved if the criteria included specific frequency ranges and submental activity in SWS sleep stages.
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    Differential gene expression in mice with misexpression of Six2 associated with frontonasal dysplasia
    ([Honolulu] : [University of Hawaii at Manoa], [August 2012], 2012-08) Hynd, Thomas Eugene
    We have previously described the Br mutant mouse displaying heritable frontonasal dysplasia. Linkage analysis mapped the mutation near the homeobox transcription factor Six2, normally expressed in the facial and metanephric mesenchyme during development. The purpose of this study is to determine expression patterns of Six2, as well as possible downstream targets of Six2, in the developing midface. The three sets of facial prominences (medial, lateral, and maxillary) from embryos at gestational day 11.5 (E11.5) were dissected and RNA extracted for qRT-PCR assays and microarray analysis. Medial nasal prominences (MNP) and E13.5 kidneys were also taken for cell culture. Results from qRT-PCR indicated Six2 expression is highest in the MNP at E11.5 and demonstrated haploinsufficient down-regulation in each of the three facial prominence sets in the Br mouse at this age. Microarray results suggested the misregulation of several genes in the Br midface, including Six3, another member of the Six family of transcription factors. MNP and kidney qRT-PCR and immunohistochemistry for Six3 substantiated its upregulation in the microarray. Additionally, Shh and Flrt2 were confirmed misexpressed in the developing midface, both of which have been previously shown to play critical roles in craniofacial development. RNA interference on Six2 in E11.5 MNP and E13.5 embryonic kidney cultures did not demonstrate misexpression of Six3, suggesting Six2 is not a direct regulator of Six3 and that the Br mutation may be located in a transcriptional activation domain of Six2 that also inhibits Six3 transcription. Further sequencing analysis will be needed to confirm the type and location of the Br mutation. This work was supported, in part, by NIH R01DK064752 & NCRR 5P20RR024206.
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