Browsing by Subject "Physiology"

Severe constipation can be life-threatening and disproportionately affects patients who may not benefit from conventional treatments. Sacral nerve stimulation (SNS) is an alternative to laxatives and pharmaceuticals, and it modulates propulsive action in the colon. Conventional SNS failed to treat slow-transit constipation. I hypothesized bursts of nerve stimulation interleaved by quiescent periods increase colonic transit more effectively than continuous nerve stimulation. I electrically stimulated the colon directly in computational models and the isolated mouse colon to characterize properties of the colonic motor complex (CMC), and I used optical and fluorescent imaging, electromyography, and manometry to compare the effect of pelvic and sacral nerve stimulation on colonic motility. I developed a computational model of colonic motility and compared the effects of burst and conventional nerve stimulation on pellet velocity and colonic emptying under normal and slow transit conditions. Burst nerve stimulation evoked more frequent calcium and pressure waves, and increased fecal pellet output than continuous nerve stimulation in the isolated mouse colon, anesthetized rat, and computational model, respectively. Burst nerve stimulation with optimized burst frequency, duration, and interval more effectively produced prokinetic motility than continuous nerve stimulation, suggesting that burst SNS may be a viable clinical treatment for severe and slow transit constipation.

It is currently an exciting time to be doing research at the intersection of sports and engineering. Advances in wearable sensor technology now enable large quantities of physiological and biomechanical data to be collected from athletes with minimal obstruction and cost. These technological advances, combined with an increased public awareness of the relationship between exercise, fitness, and health, has created an environment where engineering principles can be integrated with biomechanics, exercise physiology, and sports science to dramatically improve methods for physiological assessment, injury prevention, and athletic performance.

The first part of this dissertation develops a new method for analyzing heart rate (HR) and oxygen uptake (VO2) dynamics. A dynamical system model was derived based on the equilibria and stability of the HR and VO2 responses. The model accounts for nonlinear phenomena and person-specific physiological characteristics. A heuristic parameter estimation algorithm was developed to determine model parameters from experimental data. An artificial neural network (ANN) was developed to predict VO2 from HR and exercise intensity data. A series of experiments was performed to validate: 1) the ability of the dynamical system model to make accurate time series predictions for HR and VO2; 2) the ability of the dynamical system model to make accurate submaximal predictions for maximum heart rate (HRmax) and maximal oxygen uptake (VO2max); 3) the ability of the ANN to predict VO2 from HR and exercise intensity data; and 4) the ability of a system comprising an ANN, dynamical system model, and heuristic parameter estimation algorithm to make submaximal predictions for VO2max without requiring VO2 data collection. The dynamical system model was successfully validated through comparisons with experimental data. The model produced accurate time series predictions for HR and VO2 and, more importantly, the model was able to accurately predict HRmax and VO2max using data collected during submaximal exercise. The ANN was successfully able to predict VO2 responses using HR and exercise intensity as system inputs. The system comprising an ANN, dynamical system model, and heuristic parameter estimation algorithm was able to make accurate submaximal predictions for VO2max without requiring VO2 data collection.

The second part of this dissertation applies a support vector machine (SVM) to classify lower extremity movement patterns that are associated with increased lower extremity injury risk. Participants for this study each performed a jump-landing task, and experimental data was collected using two video cameras, two force plates and a chest-mounted single-axis accelerometer. The video data was evaluated to classify the lower extremity movement patterns of the participants as either excellent or poor using the Landing Error Scoring System (LESS) assessment method. Two separate linear SVM classifiers were trained using the accelerometer data and the force plate data, respectively, with the LESS assessment providing the classification labels during training and evaluation. The same participants from this study also performed several bouts of treadmill running, and an additional set of linear SVM classifiers were trained using accelerometer data and gyroscope data to classify movement patterns, with the LESS assessment again providing the classification labels during training and evaluation. Both sets of SVM's performed with a high level of accuracy, and the objective and autonomous nature of the SVM screening methodology eliminates the subjective limitations associated with many current clinical assessment tools.

The ocean environment poses several adversities to usual mammalian function. Perhaps most consequential to life is the lack of air underwater. For marine mammals, like whales and dolphins, that are required to perform breath-hold dives to forage for prey, this necessitates a unique set of adaptations to efficiently manage oxygen resources while diving. In an era of global environmental change, this hostile habitat is expected to become increasingly challenging for air-breathing mammals; warming waters will necessitate deeper foraging trips and noisier oceans may compel unplanned dives to evade perceived threats. An understanding of marine mammals’ solutions to the physiological challenges of a dually-constrained lifestyle is therefore important not only to reveal how marine mammals are built to thrive where other mammals, particularly humans, falter but also the extent to which these adaptations may scale in a changing ocean environment. In this dissertation, I explore the physiological adaptations, particularly those of the cardiovascular and respiratory systems, that this taxon has evolved to mediate the challenges associated with breath-hold diving. I take a multi-scale approach to investigating these physiological traits, exploring hypotheses at the molecular, tissue-specific, and organismal scales. Accordingly, I leverage both familiar and emerging methodologies in the field of marine mammal physiology to examine adaptations that support the extended dive capacities of whales and dolphins. Cellular and molecular responses to environmental stimuli influence tissue-specific and organismal physiological responses. Despite the inextricable link between molecular and organismal physiology, studies of the molecular adaptations of marine mammals for diving are limited, in part due to the logistical complexity of obtaining molecular samples from this difficult-to-study group. To fill this gap, my collaborators and I deployed RNA-seq and enzymatic assays to examine the molecular-level changes that occur in bottlenose dolphins (Tursiops truncatus) performing extended breath-holds (Chapter 1). We demonstrated that dolphins exhibit transcriptomic and proteomic changes that occur in a time-dependent fashion during breath-holding that could support their ability to maintain selective perfusion during diving. The upregulation of ALOX5, a gene targeted for the treatment of eosinophilic asthma in humans, and lipoxygenase suggest a mechanism by which differential gene regulation could contribute to sustained vasoconstriction during the dive response. These findings illustrate the importance of responses at the molecular level for supporting the unique physiology of marine mammals. Coordinated, tissue-specific physiological changes are central to the mammalian dive response. During dives marine mammals drastically reduce their heart rate (fH) while narrowing the blood vessels that supply their peripheral tissues, thereby slowing oxygen consumption of the heart itself as well as reducing the supply of oxygen-rich blood to non-essential tissues. The factors that modulate fH and contribute to diving bradycardia are complex, largely because they are numerous and often linked, but are crucial to understanding oxygen consumption patterns and, ultimately, whole-organism physiology and behavior. Using simultaneous electrocardiographic (ECG) recordings and respirometry, I show that whales and dolphins exhibit a strong cardiorespiratory coupling that may support the conservation of blood oxygen for hypoxia-intolerant tissues during a breath-hold. This variation in fH with breathing, or respiratory sinus arrhythmia (RSA), is modulated by breathing rate (fR) in bottlenose dolphins such that slow breathing results in larger fluctuations in fH (Chapter 2). Following a breath, fH increases rapidly to a maximum and then decreases through the end of the inter-breath interval (IBI). Notably, some of the minimum fH’s of the RSA were comparable to reported diving fH’s for this species suggesting the importance of apnea alone in modulating the fH of a diving marine mammal. I also demonstrate that this cardiorespiratory coupling scales with body size and fR across five cetacean species suggesting both physical scaling laws and dynamic physiological needs play a role in determining the magnitude of the RSA (Chapter 3). These studies highlight the complexity of tissue-specific responses and the need to contextualize physiological rates. Ultimately, it is the interactions of tissues that determine organismal physiology – the fundamental constraint on an organism’s behavior. To investigate the connection between organismal physiology and behavior, I developed a novel method for extracting fR from free-ranging whale biologging tag data (Chapter 4). I found that the high-flow rate and large tidal volume breaths of cetaceans generate movement signals which are captured by the accelerometers of biologging tags, enabling respiration event detection from historical biologging tag datasets. I applied this tool to movement data collected from short-finned pilot whales in Cape Hatteras, North Carolina using digital acoustic recording tags (DTAGs) and examined variation in respiratory patterns associated with diving (Chapter 5). I found that whales vary their pre- and post-dive surface duration and post-dive fR in proportion to the duration and activity of upcoming dives illustrating the physiological challenge of preparing for and recovering from breath-hold diving and highlighting optimization of surface behavior required to support breath-holds. Physiological responses are coordinated across multiple levels of biological organization necessitating the use of various tools and techniques to fully elucidate the adaptations that support marine mammals’ capacity to dive for minutes to hours without a breath. The findings of this dissertation underscore that the physiological function of breath-holding whales and dolphins is coordinated across scales, the physiological responses of cardiovascular and respiratory systems are linked, and sensing vital rates can provide insights into the physiological demands of a dive. Future studies should continue to focus on integrating methods across scales to better understand the physiological function of these animals and its plasticity in a changing ocean.

An understanding of sensory ecology, how animals receive and respond to their environment, can be a powerful tool for the conservation of endangered species because it can allow us to assess the potential success of actions designed to mitigate particular threats. We have a general understanding of how sea turtles perceive and respond to certain visual, magnetic, and chemical cues, but we understand very little about how they perceive and respond to acoustic cues. This dissertation explores the acoustic ecology of sea turtles, focusing on their auditory capabilities, responses to acoustic stimuli and the implications of this knowledge for their conservation. I measured the underwater and aerial hearing sensitivities of juvenile green (Chelonia mydas), hatchling leatherback (Dermochelys coriacea), and hatchling hawksbill (Eretmochelys imbricata) sea turtles by recording auditory evoked potential responses to tonal stimuli. Green turtles detected tonal stimuli between 50 and 1,600 Hz underwater (maximum sensitivity: 200-400 Hz) and 50 and 800 Hz in air (maximum sensitivity: 300-400 Hz), leatherbacks detected tonal stimuli between 50 and 1,200 Hz underwater (maximum sensitivity: 100-400 Hz) and 50 and 1,600 Hz in air (maximum sensitivity: 50-400Hz), and hawksbills detected tonal stimuli between 50 and 1,600 Hz in both media (maximum sensitivity: 200-400 Hz). Sea turtles were more sensitive to aerial than underwater stimuli when audiograms were compared in terms of sound pressure, but they were more sensitive to underwater stimuli when audiograms were compared in terms of sound intensity. I also examined the behavioral responses of loggerhead sea turtle (Caretta caretta) to simulated low frequency acoustic deterrent devices (ADDs) and found that these turtles exhibited a mild, aversive response to these sounds. This finding indicates that low frequency tonal ADDs have the potential to warn sea turtles of the presence of fishing gear and suggest that field tests of ADDs are warranted. Finally, I conducted a comprehensive review of our knowledge of the acoustic ecology of sea turtles, examined the sources of marine anthropogenic sound sea turtles are able to detect, evaluated the potential physiological and behavioral effects of anthropogenic sound, identified data gaps, and made recommendations for future research.

Brain-machine interfaces (BMIs) use neuronal activity to control external actuators. As such, they show great promise for restoring motor and communication abilities in persons with paralysis or debilitating neurological disorders.

While BMIs aim to enact normal sensorimotor functions, so far they have lacked afferent feedback in the form of somatic sensation. This deficiency limits the utility of current BMI designs and may hinder the translation of future clinical BMIs, which will need a means of delivering sensory signals from prosthetic devices back to the user.

This dissertation describes the development of brain-machine-brain interfaces (BMBIs) capable of bidirectional communication with the brain. The interfaces consisted of efferent and afferent modules. The efferent modules decoded motor intentions from the activity of populations of cortical neurons recorded with chronic multielectrode recording arrays. The activity of these ensembles was used to drive the movements of a computer cursor and a realistic upper-limb avatar. The afferent modules encoded tactile feedback about the interactions of the avatar with virtual objects through patterns of intracortical microstimulation (ICMS).

I first show that a direct intracortical signal can be used to instruct rhesus monkeys about the direction of a reach to make with a BMI. Rhesus monkeys placed an actuator over an instruction target and obtained, from the target's artificial texture, information about the correct reach path. Initially these somatosensory instructions took the form of vibrotactile stimulation of the hands. Next, ICMS of primary somatosensory cortex (S1) in one monkey and posterior parietal cortex (PPC) in another was substituted for this peripheral somatosensory signal. Finally, the monkeys made direct brain-controlled reaches using the activity of ensembles of primary motor cortex (M1) cells, conditional on the ICMS cues. The monkey receiving ICMS of S1 was able to achieve the same level of proficiency with ICMS as with the stimulus delivered to the skin of the hand. The monkey receiving ICMS of PPC was unable to perform the task above chance. This experiment indicates that ICMS of S1 can form the basis of an afferent prosthetic input to the brain for guiding brain-controlled prostheses.

I next show that ICMS of S1 can provide feedback about the interactions of a virtual-reality upper-limb avatar and virtual objects, enabling active touch. Rhesus monkeys initially controlled the avatar with the movements of their arms and used it to search through sets of up to three objects. Feedback in the form of temporal patterns of ICMS occurred whenever the avatar touched a virtual object. Monkeys learned to use this feedback to find the objects with particular artificial textures, as encoded by the ICMS patterns, and select those associated with reward while avoiding selecting the non-rewarded objects. Next, the control of the avatar was switched to direct brain-control and the monkeys continued to move the avatar with motor commands derived from the extracellular neuronal activity of M1 cells. The afferent and efferent modules of this BMBI were temporally interleaved, and as such did not interfere with each other, yet allowed effectively concurrent operation. Cortical motor neurons were measured while the monkey passively observed the movements of the avatar and were found to be modulated, a result that suggests that concurrent visual and artificial somatosensory feedback lead to the incorporation of the avatar into the monkey's internal brain representation.

Finally, I probed the sensitivity of S1 to precise temporal patterns of ICMS. Monkeys were trained to discriminate between periodic and aperiodic ICMS pulse trains. The periodic pulse-trains consisted of 200 Hz bursts at a 10 Hz secondary frequency. The aperiodic pulse trains had a distorted periodicity and consisted of 200 Hz bursts at a variable instantaneous secondary frequency. The statistics of the aperiodic pulse trains were drawn from a gamma distribution with equal mean inter-burst intervals to the periodic pulse trains. The monkeys were able to distinguish periodic pulse trains from aperiodic pulse trains with coefficients of variation of 0.25 or greater. This places an upper-bounds on the communication bandwidth that can be achieved with a single channel of temporal ICMS in S1.

In summary, rhesus monkeys were augmented with a bidirectional neural interface that allowed them to make reaches to objects and discriminate them by their textures--all without making actual movements and without relying on somatic sensation from their real bodies. Both action and perception were mediated by the brain-machine-brain interface. I probed the sensitivity of the afferent leg of the interface to precise temporal patterns of ICMS. Moreover, I describe evidence that the BMBI controlled avatar was incorporated into the monkey's internal brain representation. These results suggest that future clinical neuroprostheses could implement realistic feedback about object-actuator interactions through patterns of ICMS, and that these artificial somatic sensations could lead to the incorporation of the prostheses into the user's body schema.

Cardiac arrhythmias such as atrial fibrillation and ventricular tachycardia are closely associated with microscopic fibrotic changes in cardiac structure that result in a heterogeneous myocardium. While the incidence of fibrosis is correlated with arrhythmia burden and recurrence, the mechanisms linking the two remain poorly understood. Previous experimental and simulation studies have identified changes in local conduction due to micron-scale structural heterogeneities. However, because of the limited ability to simultaneously study conduction over a range of spatial scales, it remains unclear how numerous microheterogeneities act in aggregate to alter conduction on the macroscopic scale. The overall objective of this dissertation is to elucidate and characterize the effect of microfibrosis on cardiac conduction, through the use of computational models and directly paired experimental studies.

The impact of fibrotic collagen deposition on reentrant conduction was first examined in a model of cardiac tissue. The presence of collagenous septa was shown to prolong the cycle length of reentry; the magnitude of reentry prolongation is correlated with the overall degree of fibrosis and the length of individual collagenous septa. Mechanistically, cycle length prolongation is caused by lengthening of the reentrant tip trajectory and alteration of restitution properties. An equivalent homogenized model of fibrosis is unable to recapitulate the observed cycle length prolongation, suggesting that the details of the microstructure greatly impact the observed macroscale behavior. A hybrid model generated by adding discrete heterogeneities to the coarse, homogenized model is able to partially reproduce cycle length prolongation by replicating the lengthened tip trajectory.

In order to examine the mechanisms by which cardiac microstructure influences global conduction, a new framework for paired computational and experimental studies using the engineered-excitable Ex293 cell line was developed. The Ex293 mathematical model incorporates several measures of variation in cellular and tissue electrophysiological properties, and is novel in its use of stochastic variation in a multidimensional model of tissue. Replicating the range of experimentally observed single-cell and macro-scale behavior requires introducing ionic conductance variation between individual cells and between tissues, as well as conductivity variation between tissues.

This framework was then utilized for paired studies in a geometry of idealized fibrosis to examine fibrosis-induced changes in micro- and macro-scale behavior. The presence of microscopic heterogeneities slows conduction and alters the curvature of the macroscopic wavefront. On the microscale, branching of tissue around heterogeneities leads to conduction slowing due to imbalances of electrical source and load, while wavefront collisions at sites of tissue convergence lead to acceleration of propagation. The observed macroscopic behavior is directly attributable to the combination of these microscopic effects and the tortuosity of propagation around heterogeneities. Under diseased conditions involving reduced excitability, alteration of these microscale behaviors leads to reversal of changes in wavefront curvature.

These findings advance our knowledge of the role of myocardial micro-heterogeneities in conduction. Further application of these techniques to examine how the effects of microstructure are dynamically modulated may help improve our understanding of the factors giving rise to cardiac arrhythmia.

Temperature is an omnipresent environmental factor that shapes the growth, development and survival of plants. However, global warming has been an inevitable process and caused unusual temperature patterns across the world. As a consequence, forestry as well as agricultural plants are reportedly facing challenges from their environment. Several temperature responses in plant have been described, including short-term responses (such as acclimation) that increase tolerance towards sudden temperature stresses; as well as long-term responses (for example vernalization and flowering) that adjust growth and development to cope with seasonal temperature changes. However, the molecular mechanisms of how plants perceive temperature changes remain poorly understood. It has been observed for decades that one earliest response of plants towards low temperature is a transient increase of the cytosolic free Ca2+ concentration ([Ca2+]i). Considering the highly conserved role of [Ca2+]i increases in mediating thermal perception in animals, it has been speculated that [Ca2+]i increases may also play a role in thermal perception in plants. Nevertheless, despite intensive efforts, the molecular components responsible for cold-induced [Ca2+]i increases remain elusive. In this study, we carried out Ca2+-imaging-based forward genetic screen in Arabidopsis thaliana, isolated mutants defective in cold-induced [Ca2+]i increases (coca) and identified corresponding genes responsible for the coca phenotype through physical mapping. One of the mutants, named coca1, is highly specific to low temperature perception versus other stimuli, including osmotic, ionic and oxidative stimuli. coca1 displays compromised cold-induced [Ca2+] increases in both cotyledons and roots, as well as reduced growth fitness under ambient cool temperature. COCA1 encodes the dynamin-related protein 1A (DRP1A) and is localized on the plasma membrane. Our pharmacological studies showed that DRP1A acts upstream of plasma membrane rigidification and may mediate temperature perception by modification of membrane curvature which in turn opens Ca2+ channels. Alternatively, DRP1A may regulate endocytosis and channel activity through endocytosis signaling. Identification of coca1 as the first Arabidopsis mutant defective in cold-induced [Ca2+]i increases and DRP1A as a key player in thermal perception will greatly extend our understanding of plant adaptation to temperature changes, open up new avenues for studying Ca2+ signaling towards other stimuli and provide potential molecular genetic targets for engineering cold-resistant crops.

Acetyl-CoA holds a prominent position as the common metabolic intermediate of glucose, amino acid and fatty acid oxidation. Because acetyl-CoA fuels the tricarboxylic acid (TCA) cycle, the primary source of reducing equivalents that drives mitochondrial oxidative phosphorylation, understanding acetyl-CoA pool regulation becomes imperative to understanding mitochondrial energetics. Carnitine acetyltransferase (CrAT), a muscle-enriched mitochondrial enzyme, catalyzes the freely reversible conversion of acetyl-CoA to its membrane permeant carnitine ester, acetylcarnitine. Because CrAT has long been thought to regulate the acetyl-CoA metabolite pool, we investigated the role of CrAT in acetyl-CoA regulation. Although the biochemistry and enzymology of the CrAT reaction has been well studied, its physiological role remains unknown. Investigations herein suggest that CrAT-mediated maintenance of the mitochondrial acetyl-CoA pool is imperative for preservation of energy homeostasis. We provide compelling evidence that CrAT is critical for fine-tuning acetyl-CoA balance during the fasted to fed transition and during exercise. These studies suggest that compromised CrAT activity results in derangements in mitochondrial homeostasis.

In chapter 3, we examined the effects of obesity and lipid exposure on CrAT activity. Recent studies have shown that acetyl-CoA-mediated inhibition of pyruvate dehydrogenase (PDH), the committed step in glucose oxidation, is modulated by the CrAT enzyme. Because PDH and glucose oxidation are negatively regulated by high fat feeding and obesity, we reasoned that nutritional conditions that promote lipid availability and fat oxidation might likewise compromise CrAT activity. We report an accumulation of long chain acylcarnitines and acyl-CoAs but a decline in the acetylcarnitine/acetyl-CoA ratio in obese and diabetic rodents. This reduction in the skeletal muscle acetylcarnitine/acetyl-CoA ratio was accompanied by a decrease in CrAT specific activity, despite increased protein abundance. Exposure to long chain acyl-CoAs in vitro demonstrated that palmitoyl-CoA acts as a mixed model inhibitor of CrAT. Furthermore, primary human skeletal muscle myocytes exposed to fatty acid and or CPT1b overexpression had elevated long chain acylcarnitines but decreased production and efflux of CrAT-derived short chain acylcarnitines. These data suggest that exposure to fatty acids in obesity and diabetes can counter-regulate the CrAT enzyme leading to decreased activity.

Alternatively, chapter 4 addresses the importance of acetyl-CoA buffering during exercise and suggests that a deficit in CrAT activity leads to fatigue. Because CrAT is highly expressed in tissues specifically designed for work and because acetylcarnitine, the primary product of the CrAT reaction, is increased during contraction, we reasoned that CrAT could play an important role in exercise. To investigate this possibility, we employed exercise intervention and ex-vivo analysis on a genetically novel mouse model of skeletal muscle CrAT deficiency (CrATSM-/-). Though resting acetyl-CoA levels were elevated in CrATSM-/- mice, these levels dropped significantly after intense exercise while acetylcarnitine content followed the opposite pattern. This contraction-induced acetyl-CoA deficit in CrATSM-/- mice was coupled with compromised performance and diminished whole body glucose oxidation during high intensity exercise. These results imply that working muscles clear and consume acetylcarnitine in order to maintain acetyl-CoA buffering during exercise. Importantly, provision of acetylcarnitine enhanced force generation, delayed fatigue and improved mitochondrial energetics in muscles from CrATfl/fl controls but not CrATSM-/- littermates, emphasizing the importance of acetyl-CoA maintenance. In aggregate, these data demonstrate a critical role for CrAT-mediated acetyl-CoA buffering in exercise tolerance and suggest its involvement in energy metabolism during skeletal muscle contraction and fatigue. These findings could have important clinical implications for individuals with muscle weakness and fatigue due to multiple conditions, such as peripheral vascular or cardiometabolic disease.

In summary, data herein emphasize the role of CrAT in regulation of mitochondrial acetyl-CoA pool. We demonstrate that CrAT is critical for fine-tuning acetyl-CoA balance both during the fasted to fed transition and during exercise. These data suggest that a deficit in CrAT activity leads to glucose intolerance and exercise fatigue. We examine these studies and suggest future areas of study.

Insulin secreting β-cells within the pancreatic islets of Langerhans are vital to maintaining glycemic control. β-cell functional mass is lost during the progression to both Type 1 and Type 2 diabetes mellitus, resulting in hyperglycemia. Therefore, a major goal of diabetes research is to uncover pathways that can be exploited to induce β-cell replication while simultaneously maintaining β-cell function.

We previously reported that adenovirus-mediated overexpression of the transcription factor PDX1 is sufficient to induce β-cell replication, but underlying mechanisms remain to be resolved. Using statistical modeling, we identified the miR-17 family, a member of the miR17~92 miRNA cluster, as a candidate regulator of the PDX1-gene network. We show that PDX1 can directly regulate the MIR17HG promoter, the first example of β-cell specific regulation for this important miRNA cluster. Furthermore, the miR17~92 target PTEN is reduced in PDX1-overexpressing β-cells, and chemical inhibition of PTEN potentiates PDX1-mediated β-cell replication, supportive of the presence of a PDX1/miR17~92/PTEN regulatory node.

Recombinant adenovirus approaches pioneered by our laboratory have been the main method of genetic manipulation of primary islets in culture since 1994. Whereas adenovirus vectors have proved useful in an otherwise difficult model system, virus construction, especially for cell-type specific applications, is still laborious and time-consuming. To overcome this, we have created a new modular cloning system (pMVP) that allows a gene of interest to be rapidly recombined in the context of an array of promoters, N- or C-terminal epitope tags, inducible gene expression modalities, and/or fluorescent reporters, into 18 custom destination vectors, including adenovirus, expression plasmid, lentivirus, and Sleeping Beauty transposon, thus, permitting the creation of > 8000 unique vector permutations. Multiple features of this new vector platform, including cell type-specific and inducible control of gene expression, were validated in the setting of pancreatic islets and other cellular contexts. Furthermore, using pMVP as a foundation, we also developed an S. aureus dCas9 epigenetic engineering platform, pMAGIC, that enables the packaging of 3 guide RNAs with Sa-dCas9 fused to one of five epigenetic modifiers into a single vector. Using pMAGIC-derived adenoviruses, we functionally validated the regulation of PDX1 by Area IV, a cross-species conserved enhancer, through LSD1-mediated epigenetic modification in both INS1 832/13 cells and primary rat pancreatic islets.

In sum, my work has uncovered novel information about the role of PDX1 in regulation of the miR17~92 miRNA cluster in pancreatic islet cells. In an effort to contribute more broadly to our laboratory’s pancreatic islet research efforts, I also designed and built the pMVP and pMAGIC systems for efficient generation of purpose-built, customized vectors for manipulation of gene expression in islets and other cell types, including via targeted epigenetic modification of putative regulatory elements within their native chromatin context. Development of this novel vector platform facilitated additional discoveries about the role of Area IV in control of PDX1 expression in islet β-cells.

Apoptosis is a conserved mode of cell death executed by a group of proteases named caspases, which collectively ensure tissue homeostasis in multicellular organisms by triggering a program of cellular "suicide" in response to developmental cues or cellular damage.

Accumulating evidence suggests that cellular metabolism impinges directly upon the decision to initiate cell death. Several links between apoptosis and metabolism have been biochemically characterized. Using Xenopus oocyte extracts, our laboratory previously discovered that caspase-2 is suppressed by NADPH metabolism through an inhibitory phosphorylation at S164. However, the physiological relevance of these findings has not been investigated at the whole organism level. Studies presented in this dissertation utilize both Schneider's Drosophila S2 (S2) cells and transgenic animals to untangle the influence of metabolic status on fly apoptosis.

We first demonstrate a novel link between Drosophila apoptosis and metabolism by showing that cellular NADPH levels modulate the fly initiator caspase Dronc through its phosphorylation at S130. Biochemically and genetically blocking NADPH production removed this inhibitory phosphorylation, resulting in the activation of Dronc and the subsequent apoptotic cascade in cultured S2 cells and specific neuronal cells in transgenic animals. Similarly, non-phosphorylatable Dronc was found to be more potent than wild-type in triggering neuronal apoptosis. Moreover, upregulation of NADPH prevented Dronc-mediated apoptosis upon abrogation of Drosophila Inhibitor of Apoptosis (IAP) protein 1 (DIAP1) by double-stranded RNA (dsRNA) or cycloheximide (CHX) treatment, revealing a novel mechanism of DIAP1-independent apoptotic regulation in Drosophila. Mechanistically, the CaMKII-mediated phosphorylation of Dronc hindered its activation, but not its catalytic activity. As NADPH levels have been implicated in the regulation of oocyte death, we demonstrate here that a conserved regulatory circuit also coordinates somatic apoptosis and NADPH levels in Drosophila.

Given the regulatory role of NADPH in the activation of Dronc in Drosophila and caspase-2 in vertebrates, we then attempted to further elucidate the underlying signaling pathways. By tracking the catabolic fate of NADPH, we revealed that fatty acid synthase (FASN) activity was required for the metabolic suppression of Dronc, as both the chemical inhibitor orlistat and FASN dsRNA abrogated NADPH-mediated protection against CHX-induced apoptosis in S2 cells. Interestingly, it has been previously demonstrated that blocking FASN induces cell death in numerous cancers, including ovarian cancer; however, the mechanism is still obscure. As our results predict that suppression of FASN activity may prevent the inhibitory phosphorylation of Dronc and caspase 2 (at S130 and S164 respectively), we examined the contribution of caspase-2 to cell death induced by orlistat using ovarian cancer cells. Indeed, caspase-2 S164 was dephosphorylated upon orlistat treatment, initiating the cleavage and activation of caspase-2 and its downstream target, Bid. Knockdown of caspase-2 significantly alleviated orlistat-induced cell death, further illustrating its involvement.

Lastly, we developed an assay based on bimolecular fluorescence complementation (BiFC) to monitor the oligomerization of Dronc in S2 cells, a crucial step in its activation. The sensitivity of this assay has been validated with several apoptotic stimuli. A future whole-genome screen employing this assay is planned to provide new insights into this complex apoptotic regulatory network by unbiasedly identifying novel apoptotic regulators.

Estimates of the energetic costs of locomotion (COL) are necessary to understand one of the potential impacts of anthropogenic disturbance on marine mammals. A new generation of biologging devices has enabled the measurement of fine-scale behavioral responses to disturbance, but calibration experiments are required to convert these measured changes in activity level into energy expenditure. Such calibrations have been conducted in many terrestrial and avian taxa but, due to logistical constraints, have been performed with only a few marine mammals. Very few studies have tested these calibrations against independent estimates of energy expenditure, such as measurements of caloric intake and the doubly labeled water (DLW) method. Calibration studies will help us to better understand how best to estimate energy expenditure from activity measurements. In my dissertation, I ask whether short-term increases in activity caused by disturbance may impact marine mammal energy budgets. I address this question with the long-term resident community of common bottlenose dolphins (Tursiops truncatus) living in Sarasota Bay, Florida, which experiences very high levels of traffic from small vessels. I first correlated overall dynamic body acceleration (ODBA) and energy expenditure with bottlenose dolphins in human care. I combined measurements of ODBA derived from accelerometry tags with respirometry during submerged swim trials. I then subtracted measured resting metabolic rate (RMR) from the energy expenditure of each trial to estimate COL. I found a linear relationship between ODBA and COL. Next, I deployed tags on the same dolphins for longer periods (24 hours) and combined COL, RMR, and specific dynamic action (SDA; energy expenditure associated with digestion) to estimate total daily energy expenditure. I compared this estimate of total daily expenditure with estimates derived from measurements of caloric intake records and DLW. The COL+RMR+SDA values largely agreed with the calories ingested, but the smaller DLW sample was considerably more variable. I then used the correlation between ODBA and COL to estimate the cumulative energetic costs associated with responses to vessels by wild dolphins in Sarasota. I analyzed 12 digital acoustic tag (DTAG) records for the presence or absence of vessels. I used periods without vessels as controls to calculate baseline estimates of COL for each animal. I then subtracted this baseline from total COL to derive the cumulative COL attributable to vessels. The overall increase in COL attributable to the response to vessels was less than 0.3% of estimated daily energy expenditure, suggesting that avoidance, while necessary to prevent injury or death, does not contribute significantly to the daily energy budgets of these dolphins. The methods I developed can be applied to a variety of other marine mammals to study the fitness consequences of anthropogenic disturbance. Future studies should focus on sensitive species that are likely to exhibit significant avoidance responses to acoustic stimuli.

The striatum is a key brain region for learning and producing movement. Little is known about the molecular mechanisms in the early postnatal period that regulate how medium spiny neurons (MSNs), the predominant cell type in this region, mature. Using electrophysiology in acute brain slices in combination with pharmacological and genetic manipulations of the metabotropic glutamate receptor, mGluR5, I present evidence that mGluR5 may regulate synapse unsilencing. This developmental effect of mGluR5 signaling appears to be modulated by other processes, which I was unable to fully elucidate. However, activation of mGluR5 signaling later in postnatal development is sufficient to reduce excitatory glutamatergic transmission. These data indicate that mGluR5 has important roles in regulating striatal transmission that may be differentially regulated over development.

The non-secreted fibroblast growth factor (FGF) homologous factor (FHF) FGF13 is a noncanonical FGF with identified roles in neuronal development, pain sensation, and cardiac physiology, but recent reports suggest broader roles. The in vivo functions of FGF13 have not been widely studied. In this study, we have generated a global heterozygous Fgf13 knockout mouse model. In these animals, we observed hyperactivity and accompanying reduced core body temperature in mice housed at 22 °C. In mice housed at 30 °C (thermoneutrality) we observed development of a pronounced obesity. Defects in thermogenesis and metabolism were found to be due to impaired central nervous system regulation of sympathetic activation of brown fat. Neuronal and hypothalamic specific ablation of Fgf13 recapitulated weight gain at 30 °C. In global heterozygous animals, norepinephrine turnover in brown fat was reduced at both housing temperatures, while direct activation of brown fat by a β3 agonist showed an intact response. Further, we found that FGF13 is a direct regulator of NaV1.7, a hypothalamic Na+ channel associated with regulation of body weight. Our data expand the physiologic roles for FGF13, and enhance the understanding of the multifunctional FHFs.

Fibroblast growth factor (FGF) homologous factors (FHFs, FGF11-14) are a family of FGFs that are not secreted, nor activate FGF receptors. Instead, they remain intracellular and bind to the voltage-gated Na+ channel C-terminus and modulate function. FGF14 is a locus for the neurodegenerative disease spinocerebellar ataxia 27 and the disease has been attributed to decreased neuronal excitability from changes in Na+ channel function. However, several lines of evidence, including data from heterologous expression systems and the distribution of FGF13 within the ventricular cardiomyocyte suggested that it also modulates the Ca_V1.2 voltage-gated Ca2+ channel. The central hypothesis to this study is that FHFs modulate both voltage-gated Na+ and Ca2+ channel channels in the ventricular cardiomyocyte and therefore are loci for cardiac arrhythmia. Using an adult ventricular cardiomyocyte system with adenoviral gene transfer, we manipulated the levels of FGF13 in the cell and performed electrophysiology, biochemistry and immunocytochemistry to analyze the effects on voltage-gated Ca2+ channel channel localization and function. We showed that FGF13 is in complex with Junctophilin-2 and modulates Ca_V1.2 current density and localization to the t-tubule, leading to changes in Ca2+ channel-induced Ca2+ channel release and ultimately a shortened ventricular action potential. Through collaboration with the Mayo Clinic, a mutation in FGF12, the most highly expressed FHF in human ventricle was found in a patient with Brugada syndrome. Using similar methodology, we determined that this mutation results specifically in a Na_V1.5 loss of function without affecting Ca_V1.2 function, resulting in a Brugada-like ventricular action potential. This data shows that FHFs are potent modulators of multiple ion channels and novel arrhythmogenic loci.

This study takes an interdisciplinary approach to examine the relationship

between sport and spiritual formation. By inviting to the conversation contributions

from sociological research, personal narratives, biblical themes and philosophical

arguments, it aims to examine how voluntary suffering in sport could provide a context

conducive to spiritual growth. Rather than look at physical engagement in sport and

spiritual formation as unrelated domains of pursuit, we will map the contours where the

two converge and even stimulate one another. We will analyze courage as a unique

quality fit for cultivation in suffering, and positions it as an integral part of living out

faith, hope and love. This study seeks to address the rigidity that is prevalent in the way

Christians think of spirituality and deepen the conversation as it relates to formative

frameworks in athletics.

Vertebrate animals undergo a constellation of physiological responses when they experience submersion. These responses, collectively known as the dive response, include apnea (breath-hold), bradycardia (a reduction in heart rate), and peripheral vasoconstriction (the restriction of oxygenated blood to organs critical to life). Cetaceans, the order of mammals that includes whales, dolphins, and porpoise, are obligate air-breathing mammals and one of the few mammalian taxa to become fully aquatic. Given this evolutionary trajectory, cetaceans are an excellent model for investigating the physiological extremity of the dive response.

One limiting factor in dive response research involving cetaceans is the relative lack of non-invasive physio-logging devices that can be attached in free-swimming animal contexts. To address this gap, my collaborators and I invented a new multi-sensor, suction cup-attached device called the FaunaTag. The FaunaTag was custom-built to enable non-invasive collection of cardiovascular physiological data in cetacean species. Equipped with a novel contact sensor column that interfaces with the body surface of the tagged animal, the FaunaTag's near-infrared spatially-resolved diffuse reflectance bio-optical sensor and its accelerometer and gyroscope sensors were used to investigate aspects of the dive response in bottlenose dolphins (Tursiops truncatus), the most accessible and well-studied member of the cetacean order.

In the first set of experimental trials, I used the FaunaTag and a new methodological approach to investigate the extent to which dolphin cardiac heart rate changes during alternating bouts of stationary surface free-breathing and submerged apnea. In these trials, the FaunaTag and its unique contact sensor measured the vibrations associated with the cardiac cycle at the dolphin's chest wall. These vibrations were used to compute instantaneous heart rate and instantaneous kinetic energy associated with cardiac contractility. During these trials, we also tested the efficacy of the FaunaTag's near-infrared bio-optical sensor to measure dolphin heart rate before, during, and after apnea, with the FaunaTag placed at a variety of body locations, and the extent to which optically-computed heart rate estimates matched the cardiac frequency estimates calculated from cardiac vibrations.

I found that instantaneous heart rate estimates measured in this study were consistent with the heart rates computed using electrocardiography in previous studies involving these same animals. I also observed expected patterns of bradycardia during extended apneas, respiratory sinus arrhythmias following respiration events, and a return to a baseline heart rate shortly after respiration. I also found that instantaneous kinetic energy of cardiac contraction varies between free-breathing and breath-holding trial phases, with a decline to a stable apneic baseline during submerged breath-holds, followed by a steep rise following cessation of apnea and an eventual return to a variable but reduced post-apnea baseline. The FaunaTag's near-infrared spectroscopy performed poorly at dorsal body locations, detected 60% of the matched heartbeats while attached to the cardiac window of the bottlenose dolphin, and achieved a match rate exceeding 90% in the best trial. Future efforts involving the FaunaTag will feature an improved bio-optical sensing module which may resolve poor optical cardiography at the dorsal surfaces of the dolphin body and other cetacean species.

Seasonal heterothermy—an orchestrated set of extreme physiological responses—is directly responsible for the over-winter survival of many mammalian groups living in seasonal environments. Historically, it was thought that the use of seasonal heterothermy (i.e. daily torpor and hibernation) was restricted to cold-adapted species; it is now known that such thermoregulatory strategies are used by more species than previously appreciated, including many tropical species. The dwarf and mouse lemurs (family Cheirogaleidae) are among the few primates known to use seasonal heterothermy to avoid Madagascar’s harsh and unpredictable environments. These primates provide an ideal study system for investigating a common mechanism of mammalian seasonal heterothermy. The overarching theme of this dissertation is to understand both the intrinsic and extrinsic drivers of heterothermy in three species of the family Cheirogaleidae. By using transcriptome sequencing to characterize gene expression in both captive and natural settings, we identify unique patterns of differential gene expression that are correlated with extreme changes in physiology in two species of dwarf lemurs: C. medius under captive conditions at the Duke Lemur Center and C. crossleyi studied under field conditions in Madagascar. Genes that are differentially expressed appear to be critical for maintaining the health of these animals when they undergo prolonged periods of metabolic depression concurrent with the hibernation phenotype. Further, a comparative analysis of previously studied mammalian heterotherms identifies shared genetic mechanisms underlying the hibernation phenotype across the phylogeny of mammals. Lastly, conducting a diet manipulation study with a captive colony of mouse lemurs (Microcebus murinus) at the Duke Lemur Center, we investigated the degree to which dietary effects influence torpor patterns. We find that tropical primate heterotherms may be exempt from the traditional paradigms governing cold-adapted heterothermy, having evolved different dietary strategies to tolerate circadian changes in body temperature.

The kidney's major role in filtration depends on its high blood flow, concentrating mechanisms, and biochemical activation. The kidney's greatest strengths also lead to vulnerability for drug-induced nephrotoxicity and other renal injuries. The current standard to diagnose renal injuries is with a percutaneous renal biopsy, which can be biased and insufficient. In one particular case, biopsy of a kidney with renal cell carcinoma can actually initiate metastasis. Tools that are sensitive and specific to detect renal disease early are essential, especially noninvasive diagnostic imaging. While other imaging modalities (ultrasound and x-ray/CT) have their unique advantages and disadvantages, MRI has superb soft tissue contrast without ionizing radiation. More importantly, there is a richness of contrast mechanisms in MRI that has yet to be explored and applied to study renal disease.

The focus of this work is to advance preclinical imaging tools to study the structure and function of the renal system. Studies were conducted in normal and disease models to understand general renal physiology as well as pathophysiology. This dissertation is separated into two parts--the first is the identification of renal architecture with ex vivo MRI; the second is the characterization of renal dynamics and function with in vivo MRI. High resolution ex vivo imaging provided several opportunities including: 1) identification of fine renal structures, 2) implementation of different contrast mechanisms with several pulse sequences and reconstruction methods, 3) development of image-processing tools to extract regions and structures, and 4) understanding of the nephron structures that create MR contrast and that are important for renal physiology. The ex vivo studies allowed for understanding and translation to in vivo studies. While the structure of this dissertation is organized by individual projects, the goal is singular: to develop magnetic resonance imaging biomarkers for renal system.

The work presented here includes three ex vivo studies and two in vivo studies:

1) Magnetic resonance histology of age-related nephropathy in sprague dawley.

2) Quantitative susceptibility mapping of kidney inflammation and fibrosis in type 1 angiotensin receptor-deficient mice.

3) Susceptibility tensor imaging of the kidney and its microstructural underpinnings.

4) 4D MRI of renal function in the developing mouse.

5) 4D MRI of polycystic kidneys in rapamycin treated Glis3-deficient mice.