Cystic Fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which cause loss function of the CFTR channel on the apical surface of epithelial cells. ΔF508-CFTR, the major mutation in patients, is misfolded, retained in the endoplasmic reticulum (ER) and degraded. Small molecule corrector compounds partially rescue the trafficking defect of ΔF508-CFTR by allowing escape from the ER and trafficking to the plasma membrane where it exhibits partial function. These compounds may bind directly to the mutant protein and rescue the biosynthetic defect by inducing improved protein conformation. We tested this hypothesis by evaluating the consequence of corrector compound on the conformation of each nucleotide-binding domain (NBD) in the context of the full-length mutant protein in limited proteolytic digest studies. We found that VRT-325 was capable in partially restoring compactness only in NBD1. In comparison, ablation of the arginine framed peptide sequence: R553XR555 (ΔF508-KXK-CFTR) modified the protease resistance of NBD1, NBD2 and the full-length protein. Singly, each intervention led to a partial correction of the processing defect. Together these interventions restored processing of ΔF508-CFTR to near wild-type levels. Importantly however, a defect in NBD1 conformation persisted, as did a defect in channel activation after the combined interventions. This defect in channel activation can be fully corrected by addition of the potentiator: VX-770. The experiments performed partly elucidated ii the molecular mechanism of action for drug therapy and suppressor mutation. It is important to understand these basic concepts in hopes to layout a blue print for future drug design.

Craniofacial nerve injury occasionally causes spread of mechanical hypersensitivity in humans. We modeled this abnormality by transecting the infraorbital nerve (IONX) in male and female mice of the 23 AXB-BXA recombinant inbred lines and their progenitor strains, comparing their responsivity to 7 applications of a 0.2 gram Von Frey filament to the ears, paws and tail. When normalizing their mechano-responsivity on postoperative days 14 and 21 by the preoperative values, subtracting data of sham-operated from IONX mice, highly contrasting line/strain-specific differences were demonstrated. Similar line/strain-specific variability in the spread of mechano-allodynia to the paws post-IONX was demonstrated in our novel 3 minute place-avoidance paradigm, assessing parameters of mobility on a smooth surface versus a pro-allodynic granular surface. These genetically-controlled, widespread changes in mechano-sensitivity caused by IONX were minimally sexually dimorphic and mapped to intervals on chromosomes 5, 9, and 13. Further analysis is needed to identify the causative genes.

The effect of decompression stress on circulating microparticles (MPs) from leukocytes (LMP), platelets (PMP), and endothelial cells (EMP) was investigated in fifteen male naval clearance divers. Venous blood samples were obtained 30 min before and 75 min after exposure to 81msw for 20 min. MPs were isolated by differential centrifugation and immunophenotyped using multiparameter flow cytometry. Venous gas emboli (VGE) were assessed using Doppler ultrasound every 40 min post-dive and subsequently graded using the Kisman Integrated Severity Score (mean KISS=21.92, indicating moderate level of VGE). Following the dive there was increased expression of CD41a, CD106, CD62P and CD31 on MPs, while CD45 and CD141 expression decreased. A positive correlation was found between KISS and CD41a expression post-dive. These results indicate that decompression stress activated platelets, producing PMPs and resulting in potential vascular disruption or injury. The inclusion of MP measures in future DCS-related research may help identify biomarkers of DCS.

In order to elucidate how uncoupling protein 2 (UCP2) influences pancreatic β cells and glucose homeostasis, I have generated and characterized an inducible β cell-specific UCP2 deletion model,MIPCreER×loxUCP2 mice. Male littermates were injected with tamoxifen to induce UCP2 deletion(UCP2 iBKO) or with corn oil (CO). The phenotypes of both short-term (3-4 weeks after the last injection) and long-term (8-9 weeks after the last injection) were determined: Short-term iBKO mice displayed no differences in glucose or insulin tolerance, but enhanced in vivo and in vitro insulin secretion and suppressed islet reactive oxygen species (ROS) levels; while long-term iBKO mice displayed no difference in glucose tolerance, but impaired in vivo and in vitro insulin secretion and enhanced islet ROS levels. In conclusion, short-term UCP2 deletion in β cells promotes insulin secretion, while long-term UCP2 deletion impairs insulin secretion, possibly due to the opposite background of islet ROS.

Remote ischemic preconditioning (rIPC) through transient limb ischemia induces potent cardioprotection against ischemia reperfusion (IR) injury. I examined the delayed phase of protection that appears 24 hours after the initial rIPC stimulus. The primary objective of this study was to establish a mode of sedation and control treatment for delayed rIPC experiments. I used an ex-vivo, Langendorff isolated-mouse heart preparation of IR injury to examine the delayed effects of an intra-peritoneal (IP) injection, sodium-pentobarbital (SP), halothane and nitrous oxide (N2O) anesthesia on post-ischemic cardiac function. Each anesthetic method improved left-ventricular function after IR injury. SP and halothane anesthesia also reduced LV infarct size. Delayed cardioprotection after IP injections was associated with an increase in phosphorylated-Akt levels. The present study shows that IP injections and inhalational anesthesia invoke cardioprotection and, therefore, indicates that these modes of sedation should not be used as control treatments for studies examining the delayed rIPC phenotype.

Canadians live in a society where the sun does not dictate the workday. Our lifestyles must shift to cater to the 24-hour demands of a fast paced global community. As a result our circadian rhythms are altered, leading to dysregulation of key physiological processes responsible for the maintenance of essential functions like energy homeostasis. Energy homeostasis is controlled by neuropeptide-expressing neurons within the hypothalamus. These neurons are affected by circulating hormone and nutrient levels in addition to their endogenous molecular clock machinery that controls cellular processes. Therefore, hypotheses were generated that non-SCN hypothalamic neurons express orexigenic neuropeptides in a rhythmic fashion without external influence from the SCN as a result of internal rhythmicity; and that elevated concentrations of palmitate, a ubiquitous saturated FFA common in a high fat diet, have direct influence on the mRNA levels of circadian clock components Bmal1, Clock, Per2, Rev-erbα and the potent orexigenic neuropeptides NPY, AgRP and ppGhrelin through mechanisms related to HAT, SIRT1 and AMPK. Using the mHypoE-44 neurons, a well characterized cell line that expresses the molecular clock and specific neuropeptides these hypotheses were explored in four studies. Neuropeptide expression within the mHypoE-44 neurons was determined to be rhythmic. NPY and NT demonstrate significant 24-hour rhythms. CRH and ppGhrelin mRNA cycled significantly in an ultradian fashion, oscillating approximately every 18 h. AgRP mRNA did not show a significant rhythm. We identified rhythmic binding of BMAL1 to the NPY promoter, suggesting clock-mediated control of neuropeptide expression. Bmal1 and Clock mRNA levels were elevated with palmitate, whereas Per2 and Rev-erbα mRNA showed significant decreases following palmitate treatment. Palmitate increased the acetylation of both BMAL1 and PER2 proteins. Alteration of AMPK activity altered the mRNA levels of all clock genes assayed and AMPK activation diminished the palmitate-induced changes in Bmal1 mRNA. Palmitate significantly elevated both NPY and ppGhrelin mRNA levels. Chemical modifiers that decrease acetylation altered these systems. AMPK activation reduced the palmitate-induced changes in NPY mRNA levels. These findings demonstrate that non-SCN neurons have rhythmic neuropeptide transcript levels. This thesis elucidates a direct effect of palmitate on the molecular clock and neuropeptide expression at the level of the hypothalamic neuron; and these findings highlight a role for HAT/SIRT1 activation and AMPK in these important processes, which ultimately contribute to the understanding of circadian dysregulation and energy balance.

To learn effectively, an adaptive controller needs to know its sensitivity derivatives — the variables that quantify how system performance depends on the commands from the controller. In the case of biological sensorimotor control, no one has explained how those derivatives themselves might be learned, and some authors suggest they aren’t learned at all but are known innately. Here I show that this knowledge can’t be solely innate, given the adaptive flexibility of neural systems. And I show how it could be learned, using forms of information transport that are available in the brain, by a mechanism I call implicit supervision. I show that implicit supervision explains a wide range of otherwise-puzzling facts about motor learning. It explains how we can cope with conditions that reverse the signs of sensitivity derivatives, e.g. nerve or muscle transpositions, reversing goggles, or tasks like drilling teeth seen in a mirror. It also explains why it is harder to recover from reversals than from other alterations such as magnifying, minifying or displacing goggles. A further prediction of the theory of implicit supervision, in its simplest form, is that each control system — say for gaze stabilization, or saccades, or reaching — has one single, all-purpose estimate of its sensitivity derivatives for all parts of the motion. When that estimate is revised, it should affect all stages of the task. For instance, when you learn to move to mirror-reversed targets then your adapted estimate of e/u should reverse not only your initial aiming but also your online course adjustments: when the target jumps in mid-movement, your path adjustment should be appropriately reversed. Here I put subjects through many trials with jumping targets, and show that, given enough practice, they do learn to reverse their course adjustments, and therefore both initial aiming and later adjustments are governed by revisable estimates of sensitivity derivatives. And I argue that all the available data, from my own experiments and earlier ones, are compatible with a single, adaptable, all-purpose estimate of these derivatives, as in the simplest form of implicit supervision.

Cardiac output (Q) is an important determinant of the cardiovascular system‟s ability to meet the oxygen needs of the body. This dissertation addresses the non-invasive measurement of Q, in healthy children and those with heart and lung disease. 1) The correction factors for collision broadening, downstream difference and end tidal CO2 (PetCO2), used in the CO2 rebreathe (equilibrium) method, were evaluated. In lung disease, one is unable to assume a normal dead space to estimate arterial CO2 (PaCO2), and the use of any of these correction factors alone should be used with caution as they each exert a profound effect on the Q measurement. 2) A new equation to predict PaCO2 from PetCO2 in patients with CF was derived via multiple regression analysis, taking into account disease severity. 3) The validity and reliability of Q measures via the inert gas rebreathing technique (InnocorTM device) were evaluated. The highest intraclass correlation coefficients were attained during exercise (0.7-0.98), indicating excellent reliability of the device. Comparisons of Q measures from the InnocorTM (QInn) to the AMIS mass spectrometer system (QAmis) were made to assess validity. The bias (QInn-QAmis) and limits of agreement (±2SD) were 0.45 ± 1.9 L.min-1 and 0.27 ± 2.1 for children with congenital heart disease and healthy controls respectively, with no systematic differences between the two methods. 4) Assessment of cardiac output in Fontan patients demonstrated that an individualized, atrioventricular (AV) delay optimization was required. Moreover, there was a small but significant improvement in heart function with AV synchronized pacing (DDI) versus ventricular pacing (VVI), suggesting that further study with a larger sample of patients is warranted. The limitations and strengths of the measurement of non-invasive cardiac output in children, primarily via respiratory gas analysis, were delineated and recommendations were made for their use.

The synaptic remodeling of neural circuits is thought to underly memory formation. Both long-term memory formation and remote memory formation are thought to involve a process restructuring of synapses in specific areas of the brain. The transcription factor myocyte enhancer factor 2 (MEF2) has been shown to restrict spine growth in both in vivo and in vitro. It has been suggested that MEF2 is a critical molecule involved in memory formation, however, MEF2‘s role in adult memory formation it is largely unexplored. Thus, we have sought to characterize MEF2’s involvement in the formation of long-term and remote memory formation. We first showed that acute overexpression of MEF2 in the hippocampus blocks long-term spatial memory formation and activity-dependent spine formation. We next found that acute overexpression of MEF2 in the lateral amygdala likewise blocks long-term fear memory formation, suggesting that MEF2 is critical protein involved in synaptic plasticity necessary for long-term memory formation. We next demonstrated the bi-directionality of MEF2 by decreasing MEF2 function in the hippocampus and amygdala and showing a facilitation in memory formation. Together, these results suggest that MEF2 is a critical protein, which regulates spine formation important for the formation of long-term memories. We next investigated whether similar synaptic plasticity mechanisms are involved in the systems consolidation. We acutely overexpressed MEF2 in the anterior cingulate cortex at different time points following contextual fear conditioning. We noted that there was a critical window, where MEF2 blocks spine density increases in the ACC and remote memory formation. Results from this study, suggest that cortical synapses undergo a process of strengthening and remodeling and that MEF2 is a critical regulator involved. Our results demonstrate that MEF2 is involved in the synaptic consolidation of long-term and remote memories.

Postprandial overproduction of apolipoprotein B48 (apoB48)-containing lipoproteins has been observed in states of insulin resistance and is important to the sequelae of cardiovascular disease, but little is understood about factors that regulate their secretion. The glucagon-like peptides (GLPs) are released from ileal enteroendocrine L-cells following lipid ingestion. I hypothesized that the GLPs could acutely affect the production of apoB48-containing triglyceride (TG)-rich lipoproteins (TRL) in the small intestine. Using the Syrian golden hamster, I first characterized the gross effects of the GLPs on TRL secretion in response to an oral fat load and then continued to dissect the mechanisms of these changes using primary intestinal cell cultures and a variety of knockout mouse models. An exogenous GLP-1 receptor (GLP-1R) agonist was found to acutely inhibit chylomicron secretion in both hamsters and mouse models, and extending the bioactivity of endogenously-secreted GLP-1 with a dipeptidyl peptidase-4 inhibitor had suppressive effects in insulin-resistant fructose-fed hamsters. The insulinotropic and delayed gastric emptying functions do not completely account for the hypolipidemic effect of GLP-1R agonism, and the effect of the GLP-1R agonist exendin-4 could be seen directly in the apoB48 secretion of primary enterocytes. In contrast, the sister peptide GLP-2 was a potent acute stimulator of chylomicron secretion in hamsters and mice. The hyperlipidemic effect of GLP-2 could be attributed to an increased rate of luminal FA uptake mediated by the posttranslational modification of the FA transporter CD36, and CD36-deficient mice were found to be refractory to the stimulatory effects of GLP-2. The activity of nitric oxide synthase was also found to be essential to the hyperlipidemic action of GLP-2. I identified a set of intercellular communications that could contribute in mediating the action of GLP-2, in which GLP-2 secreted from the enteroendocrine L-cell stimulates intestinal subepithelial myofibroblasts to release vascular endothelial growth factor, which directly activated the enterocyte to secrete apoB48. In summary, this thesis demonstrates that two co-secreted postprandial hormones have considerable but completely opposite influences on chylomicron production. Changing the balance of the GLPs’ actions in vivo could provide a therapeutic strategy to combat postprandial dyslipidemia.