Ph.D. - Biomedical Sciences (Physiology)
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ItemChronic 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)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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ItemDetection of cortical arousals in sleep EEG([Honolulu] : [University of Hawaii at Manoa], [December 2010], 2010-12)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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ItemDifferential gene expression in mice with misexpression of Six2 associated with frontonasal dysplasia([Honolulu] : [University of Hawaii at Manoa], [August 2012], 2012-08)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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ItemADH response to peripheral and central cortisol administration( 1987)Cortisol affects water balance, but whether this effect is mediated through antidiuretic hormone (ADH) is unclear. This study examines the response of plasma ADH (pADH) in two groups of conscious dogs; one received cortisol centrally (ivt) in the third ventricle at 300 ng/min, the other peripherally (iv) at 4.16 µg/kg/min, in 4 states of water balance, i.e., dehydration, normal hydration, 5% NaCl iv infusion (0.05 ml/kg/min), and after a water load (40 ml/kg given iv over 30 min), as compared to control experiments without cortisol. Cortisol, either ivt or iv, had no affect on pADH or plasma osmolality (pOsm) during dehydration or normal hydration. Ivt cortisol infusion caused a progressive decline in plasma cortisol (pCort) while iv cortisol infusion increased pCort (control 2.0 µg%, ivt pCort 0.5 µg%, iv pCort 17 µg%, P<0.01). During the 5% NaCl iv infusion, pADH and pOsm increased similarly in both the control and ivt cortisol experiments from 1.0 to 1.9 µU/ml and 295 to 305 mOsm/kg H2O, respectively (P<0.01). The increase in pADH seen with 5% NaCl infusion was delayed in the iv cortisol experiment as compared to the iv control (75 min versus 45 min, P<0.01). This delay was also seen in pOsm; 45 min in iv cortisol versus 15 min in iv control (P<0.01), indicating that the elevated pCort apparently delays the development of increased pOsm and the subsequent increase in pADH. During a water load, the cumulative urine excreted was 99% of that ingested with iv cortisol (P<0.05), 82% in the control, and 70% with ivt cortisol; in all three cases similar decreases in pADH and pOsm occurred. The free water clearance (FWC) was augmented in the iv cortisol infusion and attenuated in the opposite situation of pCort insufficiency which was established during the ivt cortisol infusion. Thus, the present study demonstrates that cortisol has a peripheral effect in that elevated plasma cortisol 1) delays the rise in pOsm during hypertonic saline infusion 2) increases FWC during a water diuresis but 3) does not alter the pADH versus pOsm relationship, therefore 4) affects the ability to excrete a water load independent of ADH. These data are compatible with a mechanism in which excess cortisol enhances the Na+ "leak" pathway of the cells by increasing the membrane permeability to Na+, thereby increasing the osmolar content of the cells.
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ItemThe role of complement and neutrophils in air bubble-induced lung injury( 1995)Pulmonary air embolism causes vascular obstruction and induces biochemical reactions leading to lung injury. In the present study, by using isolated and perfused rat lungs, we investigated the involvement of the complement system and polymorphonuclear leukocytes (P:MN) in the alterations of segmental vascular resistances, lung weight gain, and filtration coefficient (K), a measure of vascular permeability. After establishing ventilation with air and 5% CO2, the lung was removed en bloc and suspended in a humidified chamber at 37°C. Lung weight, arterial and venous pressures were monitored continuously. Lungs were perfused with physiological salt solution (PSS) containing 4% Ficoll. We used 6 series of perfusates containing: 1) PSS, 2) PMN, 3) plasma, 4) decomplemented plasma, 5) PMN and plasma, and 6) PMN and decomplemented plasma. Air embolism, induced by a 0.76-ml air infusion to arterial catheter in 20 min, increased arterial pressure without altering capillary and venous pressure, suggesting that the increased arterial resistance alone was responsible for the pulmonary hypertension. In lungs perfused with both PMN and normal plasma, air embolism increased Kf by 145 ± 190% which was significantly greater than those in lungs perfused with either PMN (91 ± 8%), plasma (90 ± 8%), or PMN and decomplemented plasma (80 ± 9%). Air embolism increased Kf by 45 ± 12% in the lungs perfused with decomplemented plasma, which was the least among groups. These results suggest that air embolism damages the lung by hypertension, activation of the complement, and activation of PMN, singly or in combinations. The modulation role of PMN in air bubble-induced lung injury was investigated by pretreating the lungs with blocking agents of the cytotoxic substances released from PMN. The lungs perfused with both P:MN and normal plasma served as the control group. In lungs pretreated with scavengers, air embolism increased Kr by 108 ± 25% which was not different from the control group. Air embolism induced little change of Kf in the lungs pretreated with indomethacin (17 ± 8%) or isoproterenol (0 ± 9%), suggesting that air embolism increases pulmonary vascular permeability involving the release of arachidonic acid metabolites.
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ItemBioenergietics of the bottlenose dolphin (Tursiops truncatus)( 1995)There are numerous species of marine mammals found throughout the earth's oceans and waterways. The study of marine mammal energetics is an attempt to define the flow of energy in homeotherms adapted to an aqueous environment. Their lifestyle is different than terrestrial mammals and must compensate for the challenging thermoregulatory requirements of water survival. Field studies of free-ranging animals are difficult, since continual direct observation and measurements are impossible. It is important to conduct controlled laboratory studies, from which to build a foundation of physiological principles on these mammals. The objectives of this research project were to investigate the bioenergetic scheme of the bottlenose dolphin under controlled laboratory-like conditions. Morphometric data in a large bottlenose dolphin population established an equation to predict age from length measurements in juvenile animals. The decrease in caloric intake seen with aging is in agreement with terrestrial mammals, and the importance of nutritional planning based on calories per kilogram body weight versus total calories fed was explained. Increased activity levels required increased caloric consumption, the amount being related to the degree of activity. A recently developed method to assess blubber volume using ultrasound are compared to standard measurements of body condition, weight-length ratio and condition index, and found to be a valid technique in this species. Blubber insulation was found to be more important than surface area in controlling environmental heat loss. Using stable isotopes of water, body composition, lean body mass and fat mass, were determined. An increase in fat mass, with a concurrent decrease in lean body mass, was observed in adult bottlenose dolphins, similar to terrestrial mammals. Also, consumption of a high fat diet contributed to an increase in body fat. Metabolic rates declined with age in bottlenose dolphins, and a significant increase was observed with low fat diets. Finally, the use of doubly labeled water to determine metabolic rate in free-ranging bottlenose dolphins was compared to the intake balance method and found to be an accurate technique in this species. The drawbacks of controlled laboratory trials is that, although all attempts are made to mimic diets and conditions that exist in the wild, the methods are only an approximation of wild conditions. The advantages are the ability to isolate and evaluate individual parameters and ensure adequate and timely data collection. It is essential to combine laboratory and field studies in order to elucidate the bioenergetic mechanisms and physiological adaptations of marine mammals. Although marine mammals have evolved specialized compensatory adaptations to ensure success in an aquatic environment, they follow the same general principles observed in terrestrial mammals. This research supports the concept of evolution and the relationship of all homeotherms, despite differences in ecological niches.