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Alterations in macronutrient intake can have profound effects on energy intake and whole-body metabolism. For example, reducing protein intake increases energy expenditure, increases insulin sensitivity and decreases body weight in rodents. Fibroblast growth factor 21 (FGF21) signaling in the brain is necessary for the metabolic effects of dietary protein restriction and has more recently been proposed to promote protein preference. However, the neuron populations through which FGF21 elicits these effects are unknown. Here, we demonstrate that deletion of β-klotho in glutamatergic, but not GABAergic, neurons abrogated the effects of dietary protein restriction on reducing body weight, but not on improving insulin sensitivity in both diet-induced obese and lean mice. Specifically, FGF21 signaling in glutamatergic neurons is necessary for protection against body weight gain and induction of UCP1 in adipose tissues associated with dietary protein restriction. However, β-klotho expression in glutamatergic neurons was dispensable for the effects of dietary protein restriction to increase insulin sensitivity. In addition, we report that FGF21 administration does not alter protein preference, but instead promotes the foraging of other macronutrients primarily by suppressing simple sugar consumption. This work provides important new insights into the neural substrates and mechanisms behind the endocrine control of metabolism during dietary protein dilution.

Abstract Rationale Classical interpretation of cystic fibrosis (CF) lung disease pathogenesis suggests that infection initiates disease progression, leading to an exuberant inflammatory response, excessive mucus, and ultimately bronchiectasis. Although symptomatic antibiotic treatment controls lung infections early in disease, lifelong bacterial residence typically ensues. Processes that control the establishment of persistent bacteria in the CF lung, and the contribution of noninfectious components to disease pathogenesis, are poorly understood. Objectives To evaluate whether continuous antibiotic therapy protects the CF lung from disease using a ferret model that rapidly acquires lethal bacterial lung infections in the absence of antibiotics. Methods CFTR (cystic fibrosis transmembrane conductance regulator)–knockout ferrets were treated with three antibiotics from birth to several years of age and lung disease was followed by quantitative computed tomography, BAL, and histopathology. Lung disease was compared with CFTR-knockout ferrets treated symptomatically with antibiotics. Measurements and Main Results Bronchiectasis was quantified from computed tomography images. BAL was evaluated for cellular differential and features of inflammatory cellular activation, bacteria, fungi, and quantitative proteomics. Semiquantitative histopathology was compared across experimental groups. We demonstrate that lifelong antibiotics can protect the CF ferret lung from infections for several years. Surprisingly, CF animals still developed hallmarks of structural bronchiectasis, neutrophil-mediated inflammation, and mucus accumulation, despite the lack of infection. Quantitative proteomics of BAL from CF and non-CF pairs demonstrated a mucoinflammatory signature in the CF lung dominated by Muc5B and neutrophil chemoattractants and products. Conclusions These findings implicate mucoinflammatory processes in the CF lung as pathogenic in the absence of clinically apparent bacterial and fungal infections.

We used a functional magnetic resonance imaging motor activation paradigm for both hands and functional connectivity analyses to investigate motor deactivation. These analyses revealed ipsilateral (to the task) postcentral gyrus connectivity with the ipsilateral primary motor cortex as well as contralateral cerebellum for both hands. Analyses using default-mode network nodes as seed regions revealed connectivity patterns similar to previous studies of the default network and therefore provide evidence that this network is demonstrable using a synchronized motor activation paradigm. We did not find evidence suggesting that motor deactivation represents modulation of the default mode network. Therefore, motor deactivation is likely a motor-specific process. Finally, we found no evidence of basal ganglia circuit deactivation, which suggests that the two-pathway hypothesis of frontal–subcortical circuit function may be incomplete.

The epithelial cells of the mammalian airways protect the lung from damage and infection. When these airways become damaged following injury or disease, specialized epithelial stem cells (SCs) are activated to regenerate new cells. SC populations reside all along the epithelial layers of the lung. In the proximal airways, basal cells (BCs) of the surface airway epithelium (SAE) serve as the primary SCs of the region. However, other cell types residing within the submucosal glands (SMGs) are also capable of regenerating lost epithelia following injury. The processes that control the proliferation and differentiation of these different SCs in response to injury and disease remain incompletely understood. In this thesis, we discuss the signals and factors that regulate proximal airway epithelial SC function. We provide evidence that the Wnt signaling transcription factor Lef-1 controls BC proliferation and differentiation, and is necessary for proper BC-mediated SAE repair following injury. Furthermore, we demonstrate that SMG-localized SCs can be lineage-traced by their expression of Sox9. In addition, we show that deletion of Sox9 from cultured SMG epithelial SCs reduces clonogenicity and influences lineage fate, suggesting that Sox9 is an important regulator of SMG SC function. Overall, these findings provide novel insights into the function and regulation of airway SCs.

Metabolic syndrome is a major health risk in the United States and imbalances in macronutrient intake can contribute to the development of this disease. While there is evidence in humans for independent appetites for macronutrients (fats, proteins, and carbohydrates), the mechanisms that determine appetites for specific macronutrients is lacking. Preferences for carbohydrates in particular can be strong, as sugars contribute a major source of energy. Little is known, though, about central integration of peripheral nutrient signals. Our studies seek to understand how fibroblast growth factor 21 (FGF21), a hepatic endocrine hormone, signals to the brain to regulate macronutrient consumption. FGF21 signals through a receptor complex comprised of FGF receptor complex 1 (FGFR1c), and its co-receptor, β-klotho (KLB). While FGFR1c is ubiquitously expressed, KLB is more selectively expressed, conferring FGF21’s specificity.In this work, we characterize KLB expression both centrally and in peripheral tissues to uncover potential target sites of FGF21 signaling. Furthermore, we show that FGF21 decreases sugar intake by signaling to glucose-sensing glutamatergic neurons within the ventromedial hypothalamus (VMH), while acting on glutamatergic neurons outside of the VMH to mediate sweet-taste preference. FGF21 is also required for the metabolic effects of protein deficient diets in lowering body weight and improving insulin sensitivity. Herein, we demonstrate that FGF21 signaling to glutamatergic neurons is required to facilitate low-protein diet-mediated body weight loss but is not required to mediate increases in insulin sensitivity. Through these studies, we have identified new mechanistic information into this liver-to-brain hormonal axis that regulate central pathways controlling energy homeostasis.

Photocatalysis is being explored as a possible alternative to current disinfection treatment methods. Switching to photocatalysis could remove issues with disinfection byproducts and lower the operating costs. However, the activity of photocatalysts is too low to be used commercially. The most common photocatalyst is titanium dioxide, and while the activity of titanium dioxide is high when compared to other photocatalysts, improvements need to be made before commercialization. Increasing the activity of TiO 2 can be done in a variety of methods, but this dissertation will focus only on a single method. Previous research has found that TiO2 has a range of activities based on the different phases and orientations. Many groups are focusing on growing crystals that are mainly covered with the most active facet. However, photocatalysis is about balancing reactions, and combining the most active oxidation site with poor reduction site will lower the activity. It is believe that by combining a site that is good for oxidation with a site that is good for reduction can outperform a catalyst with random orientations or even those with the expression of a single orientation. Controlling the phase and orientation of the titanium dioxide will be done by depositing TiO2 onto patterned substrates. The patterns were created through block copolymer lithography,providing sub-50 nm features over the surface of the substrate. The pattern will then be expressed through the film, creating a film that has controlled texture based upon the size scale of the patterned surface. These films will then be characterized by measuring the photoactivity through methylene blue degradation experiments. In addition to the textured films, an experiment was carried out in effort to help identify and quantify reactivity of different orientations of TiO 2. This was done by photodepositing metal ions onto different TiO 2 films and measuring the metal deposition over time. These experiments showed the range in activities for oxidation and reduction reactions for the phases and orientations tested.

Configuring clinical linear accelerators (linacs) for ultra-high dose rate (UHDR) electron experiments typically requires invasive hardware manipulation and/or irreversible manufacturer modifications, limiting broader implementation. We present an independently developed UHDR electron configuration of a clinical TrueBeam linac that allows reversible switching between preclinical UHDR and conventional (CONV) modes using only non-invasive software settings. UHDR mode was achieved via service mode software with RF and beam current settings typical of a photon beam, the photon target and monitor chamber retracted, and a clinically unused low-energy scattering foil inserted. An external AC current transformer (ACCT) for beam monitoring, anatomy-specific collimator, and sample holder were mounted on the accessory tray, with external ion chamber in solid water for exit dose monitoring. Percent depth dose (PDD) was measured for UHDR and CONV beams. Dose-per-pulse (DPP) was varied by adjusting gun voltage and quantified with radiochromic film at different source-to-surface distances (SSD). Beam profiles assessed dose uniformity and usable field size. Dose calibration was established between film, ACCT, and ion chamber, and day-to-day reproducibility was tested. PDD confirmed similar energies for UHDR (12.8MeV) and CONV (11.9MeV) beams with matching profiles through mouse thickness. Maximum DPP exceeded 0.5Gy, reaching ~1.5Gy for collimated in vivo setups and ~0.7Gy at extended SSD for tissue culture. Field flatness and symmetry were maintained, supporting organ-specific irradiations and up to 5cm fields for culture. Calibration showed strong linearity across detectors, and output variation was <4%. We demonstrated accurate, reproducible UHDR delivery on a widely available clinical linac with no invasive hardware manipulation, enabling preclinical FLASH research on a clinical treatment machine.

The frontal-subcortical skeletomotor circuit is thought to be a motor processing network. However, the exact function of the circuit is poorly characterized. This fMRI study utilized a motor activation paradigm for both hands to probe circuit engagement and connectivity. Activation of the circuit decreased over time for the right hand, which suggests circuit engagement can vary during task execution. Changes in activation of the right dorsolateral prefrontal cortex were highly correlated with changes in activation of right skeletomotor circuit input, output and intrinsic nuclei for both the left and right-hand tasks, which indicates significant functional connectivity between these brain regions during motor activity. This finding suggests circuit involvement in motor execution may be more complex than predicted by the two-pathway hypothesis of circuit function. Finally, patterns of activation suggested that the two-pathway hypothesis does not completely explain activation in response to a synchronized motor task.[PUBLICATION ABSTRACT]

Configuring clinical linear accelerators (linacs) for ultra-high dose rate (UHDR) electron experiments typically requires invasive hardware manipulation and/or irreversible manufacturer modifications, limiting broader implementation. We present an independently developed UHDR electron configuration of a clinical TrueBeam linac that allows reversible switching between preclinical UHDR and conventional (CONV) modes using only non-invasive software settings. UHDR mode was achieved via service mode software with RF and beam current settings typical of a photon beam, the photon target and monitor chamber retracted, and a clinically unused low-energy scattering foil inserted. An external AC current transformer (ACCT) for beam monitoring, anatomy-specific collimator, and sample holder were mounted on the accessory tray, with external ion chamber in solid water for exit dose monitoring. Percent depth dose (PDD) was measured for UHDR and CONV beams. Dose-per-pulse (DPP) was varied by adjusting gun voltage and quantified with radiochromic film at different source-to-surface distances (SSD). Beam profiles assessed dose uniformity and usable field size. Dose calibration was established between film, ACCT, and ion chamber, and day-to-day reproducibility was tested. PDD confirmed similar energies for UHDR (12.8MeV) and CONV (11.9MeV) beams with matching profiles through mouse thickness. Maximum DPP exceeded 0.5Gy, reaching ~1.5Gy for collimated in vivo setups and ~0.7Gy at extended SSD for tissue culture. Field flatness and symmetry were maintained, supporting organ-specific irradiations and up to 5cm fields for culture. Calibration showed strong linearity across detectors, and output variation was <4%. We demonstrated accurate, reproducible UHDR delivery on a widely available clinical linac with no invasive hardware manipulation, enabling preclinical FLASH research on a clinical treatment machine.