Oxygen is a necessary part of our everyday lives and is important for normal eye function. Blood flow through the retinal vasculature supplies oxygen to the inner retina. The resistance of the retinal vessels can change, increasing and decreasing blood flow by dilation and constriction of the vessel. The response of retinal hemodynamics to vasoactive stimuli is termed vascular reactivity. To investigate vascular reactivity characteristics, a system that prospectively targets a certain level of oxygen is employed. We characterize how the retinal vessels respond over time to hypoxia as well as define vascular reactivity to different oxygen concentrations in healthy participants. We demonstrate that the vessels increase diameter fully after 6 minutes and flow after 10 minutes. The relationship between retinal hemodynamics and arterial partial pressure of oxygen (PaO2) is demonstrated in healthy humans. Future studies should investigate these changes in diseased models to better understand when the retinal vasculature response may be insufficient.

Atrial arrhythmias are very common clinically relevant conditions that are strongly associated with aging and parasympathetic tone. Additionally, ATP-sensitive K+ (KATP) channel activation has been reported to facilitate the development of re-entrant atrial arrhythmias. Since KATP channels are direct effectors of Gαi/o and RGS4 is an inhibitor of Gαi/o-signaling, we here investigate whether KATP channel activity is increased under decreased RGS4 activity in a manner that enhances susceptibility to AF. We show that loss of RGS4 facilitates the induction of atrial arrhythmias under parasympathetic challenge both in whole animals and isolated atrial tissues. Furthermore, using both genetic disruption (Kir6.2 ablation) and pharmacologic blockade (tolbutamide), we show that loss of functional KATP channels decreases the incidence of pacing-induced re-entry and prolongs repolarization in RGS4-deficient atria. Our findings are consistent with the conclusion that enhanced KATP channel activity may contribute to pacing-induced re-entrant rotors in the RGS4-deficient mouse model.

Microglia, immune cells of the central nervous system, activate in response to pathophysiological stimuli. One of their reactive phenotypes is to migrate to site of injury where they could have either beneficial or detrimental effects. However, little is known regarding the mechanisms underlying microglial migration and how they traverse the unique extracellular environment in brain tissue to reach their destination. Our laboratory first discovered that microglia express structures called podosomes, which can adhere to as well as degrade extracellular matrix. In this study, I further characterize microglial podosomes, and show that they associate with Iba1, Orai1 and calmodulin, proteins not yet observed in podosomes of other cell types. I also present evidence that podosome formation depends on Ca2+ and its entry through store-operated Ca2+ channels. The findings in this thesis contribute to a better understanding of podosome dynamics and their probable roles in microglia migration.

Cystic Fibrosis (CF) is a life-threatening autosomal recessive disease affecting 1:3600 children born in Canada. CF is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channel. The most common disease causing mutation is a deletion of residue F508, resulting in a structurally compromised protein which is retained in the endoplasmic reticulum and targeted for proteasomal degradation. Therapeutic strategies currently being pursued to alleviate the afflictions caused by this and other mutants include the use of corrector compounds to modify the surface expression of the channel, and potentiator compounds to increase cAMP-mediated channel activity. Despite the discovery of a number of small molecules affecting CFTR, much is still unknown about the nature of these interactions. This thesis contains the investigation of two potentiators: VRT-532 and VX-770, and two correctors VX-809 and C18. We assessed the consequences of interactions with these drugs on CFTR channel activity, ATPase activity and phosphorylation. We demonstrated that VRT-532 binds directly to mutant CFTR to modify its channel and ATPase activity. VX-770, known to bind directly to CFTR, can stimulate channel activity in the absence of cAMP stimulation in baby hamster kidney (BHK) cells. Correctors VX-809 and C18, based off the same molecular scaffold, are both capable of acutely augmenting cAMP-stimulated channel activity, providing evidence for potentiator activities in these compounds. Quantitative mass spectrometry (MS) techniques demonstrate a defect in phosphorylation at Ser-660 in the regulatory (R) domain in the major mutant. Treatment with C18 was unable to repair this defect. These novel findings regarding interactions between several small molecules and CFTR contributes to the understanding of the mechanism of action of these compounds, and will help identify how they may be modified for greater efficacy to improve the treatment of CF disease.

Cystic Fibrosis (CF) is a life-threatening autosomal recessive disease affecting 1:3600 children born in Canada. CF is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channel. The most common disease causing mutation is a deletion of residue F508, resulting in a structurally compromised protein which is retained in the endoplasmic reticulum and targeted for proteasomal degradation. Therapeutic strategies currently being pursued to alleviate the afflictions caused by this and other mutants include the use of corrector compounds to modify the surface expression of the channel, and potentiator compounds to increase cAMP-mediated channel activity. Despite the discovery of a number of small molecules affecting CFTR, much is still unknown about the nature of these interactions. This thesis contains the investigation of two potentiators: VRT-532 and VX-770, and two correctors VX-809 and C18. We assessed the consequences of interactions with these drugs on CFTR channel activity, ATPase activity and phosphorylation. We demonstrated that VRT-532 binds directly to mutant CFTR to modify its channel and ATPase activity. VX-770, known to bind directly to CFTR, can stimulate channel activity in the absence of cAMP stimulation in baby hamster kidney (BHK) cells. Correctors VX-809 and C18, based off the same molecular scaffold, are both capable of acutely augmenting cAMP-stimulated channel activity, providing evidence for potentiator activities in these compounds. Quantitative mass spectrometry (MS) techniques demonstrate a defect in phosphorylation at Ser-660 in the regulatory (R) domain in the major mutant. Treatment with C18 was unable to repair this defect. These novel findings regarding interactions between several small molecules and CFTR contributes to the understanding of the mechanism of action of these compounds, and will help identify how they may be modified for greater efficacy to improve the treatment of CF disease.

Eccentric exercise causes skeletal muscle damage, yet the acute cellular responses post-exercise have yet to be fully elucidated. To better understand the post-exercise response, heat shock protein (HSP) 72 content and nuclear factor-κB (NF-κB) activation where examined in Adult (A; 6 month) and Late middle-aged (LMA; 24 month) Fischer 344xBrown Norway rats. Animals were randomly divided into five groups (n=6): non-exercising controls (C), level (L) or eccentric (ECC) (-16°) running at 16m.m-1 and killed immediately post-exercise (0), 48 hours post-exercise (48). Following ECC, vastus intermedius (VI) from A and LMA showed more damage compared to L exercise. Neither age-group had significantly increased VI HSP72 content compared to C. Pooled results founded increased HSP72 content in ECC-48 compared to C (p<0.02). NF-κB activation in the VI was lower in LMA (p<0.001) and unchanged in WG when compared to AC. These findings suggest HSP72 is increased following eccentric exercise in the VI.

Left ventricular (LV) filling increases during exercise, but left atrial (LA) phasic function and its contribution to LV filling is poorly understood. Sixteen endurance-trained middle-aged males were studied at rest and during light (LE) and moderate (ME) intensity cycle-ergometry. Atrioventricular-plane displacement (AVPD) increased from rest to LE (from 14±2 mm to 18±2 mm, p<0.01), but did not increase further at ME. LA reservoir volume increased from rest to LE (from 32±8 mL to 40±10 mL, p<0.01). LA passive contribution increased at LE (from 21±5 mL to 27±8 mL, p<0.01), while LA active contribution increased from rest only at ME (from 12±5 mL to 23±9 mL, p<0.01). AVPD, and thus the longitudinal shortening of LV systole, contributes to LA filling primarily during LE, but is a limited mechanism beyond LE. These data suggest that LV filling appears to shift to a reliance on conduit function to increase LV filling at ME.

Oxygen is a necessary part of our everyday lives and is important for normal eye function. Blood flow through the retinal vasculature supplies oxygen to the inner retina. The resistance of the retinal vessels can change, increasing and decreasing blood flow by dilation and constriction of the vessel. The response of retinal hemodynamics to vasoactive stimuli is termed vascular reactivity. To investigate vascular reactivity characteristics, a system that prospectively targets a certain level of oxygen is employed. We characterize how the retinal vessels respond over time to hypoxia as well as define vascular reactivity to different oxygen concentrations in healthy participants. We demonstrate that the vessels increase diameter fully after 6 minutes and flow after 10 minutes. The relationship between retinal hemodynamics and arterial partial pressure of oxygen (PaO2) is demonstrated in healthy humans. Future studies should investigate these changes in diseased models to better understand when the retinal vasculature response may be insufficient.

The present study examined the hemodynamic response to recreational hockey (n= 22) in middle-aged men (53±6 yrs). Study participants were equipped with ambulatory blood pressure and heart rate monitoring equipment prior to a weekly hockey games. Participants were monitored throughout the duration of their hockey game for “On-Ice” responses and during seated bench time (“Bench”), and for a brief period afterwards. On-Ice HR’s and blood pressures were significantly higher than values obtained during maximal cycle exercise (HR 174±8.9 vs. 163±11.0 bpm) (SBP 17%; DBP 15%) (p<0.05), Blood pressures decreased throughout the duration of the game while HR increased significantly. The On-Ice endocardial viability ratio (EVR), an index of myocardial oxygen supply and demand, did not change from early (1.56±0.05) to late (1.44± 0.06) in the game. In conclusion, recreational hockey is an extremely vigorous form of interval exercise that produces cardiovascular responses exceeding intensities commonly recommended for continuous training.

Cerebral ischemia and intracerebral hemorrhage (ICH) are both characterized by a prolonged inflammatory response and secondary injury phase, yet the spatial/temporal relationships between inflammation and white matter (WM) damage were largely unknown. Thus, I quantified the development of WM damage and inflammation over 7 days after ischemia, and 14 days after ICH. Following ischemia, myelin and axons were progressively damaged, and myelin damage coincided with neutrophil infiltration. Activated microglia/macrophages increased dramatically in the lesion core and edge, and selectively infiltrated damaged WM tracts while surrounding undamaged ones. To investigate the involvement of neutrophils in WM damage and inflammation after ICH, rats were rendered neutropenic before performing ICH. Neutrophil depletion reduced peri-hematomal axonal damage, BBB breakdown, and MMP-9 production at early times, and lessened microglia/macrophage and astrocyte responses at later times. Activated microglia/macrophages infiltrated peri-hematomal WM tracts, correlating with myelin fragmentation and axonal loss, and this was reduced with neutrophil depletion.