Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3281. Adviser: Christopher A. Phillips. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2009. / Title from PDF t.p. (viewed on Jul 8, 2010). Source: Dissertation Abstracts International, Volume: 70-10, Section: B, page: 5920. Adviser: Gregory E. Demas.

Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2006. / Source: Dissertation Abstracts International, Volume: 67-04, Section: B, page: 1801. Adviser: Wayne C. Forrester. "Title from dissertation home page (viewed June 20, 2007)."

abstract: The impact of urbanization on wildlife is becoming an important topic in conservation. However little is known concerning the proximate mechanisms involved which enable some species to persist in cities, while others perish. Adapting to novel city environments requires individuals to maintain a functional physiological response to stressful stimuli, while concurrently using the necessary resources (food) needed to persist. A primary function of the stress response is the mobilization of intrinsic energy resources, and thus both requirements (energy and stress) are explicably linked. This dissertation investigates the interaction of energetic reserves and the physiological stress response in a native bird species, the Curve-billed Thrasher, within the context of this species' colonization of Phoenix, Arizona. This research uses a combination of comparative studies, statistical modeling, and experimental approaches conducted in field and captive settings to demonstrate how urban and desert populations of these species differ in energetic state and stress physiology. These studies reveal that the current energetic status of an individual bird influences the secretion of glucocorticoids (primary stress hormones) and can alter how energy reserves are used for gluconeogenesis to produce energy during acute stress. In addition, this research also identifies how differing levels of a hypothalamic neuropeptide (vasotocin) may play a role in mediating differences in stress physiology between populations. The quantity of food available and even temporal variability in its abundance may alter how native birds respond to stress. Increased body condition offsets the costs of maintaining the stress response in urban areas. / Dissertation/Thesis / Ph.D. Biology 2010

Este trabalho consiste na realização experimental de uma Curva de Resposta de Fases (PRC) de ritmos biológicos infradianos observados em insetos cavernícolas da espécie Folsomia candida. Sendo os insetos cegos, utilizamos pulsos de temperatura para provocar os deslocamentos de fase. Obtivemos PRCs com apenas atrasos, com a característica topológica do Tipo 1, para as três durações diferentes dos pulsos utilizados. Essa característica difere das típicas respostas observadas nos osciladores do tipo ciclo-limite, os quais são associadas à maioria dos ritmos biológicos. A dimensão de um oscilador determina quais os tipo de PRCs que o sistema pode apresentar, quando sujeito a perturbações, em suas diferentes fases. Relógios simples são associados a ciclos constituídos por uma sequência discreta de eventos encadeados, estando cada evento dependente do término do anterior, para se iniciar. Esses são sistemas unidimensionais, diferindo dos ciclos-limites pela impossibilidade de apresentar PRCs do Tipo 0 e singularidades de fase. Associamos um modelo de relógios simples para os ritmos infradianos estudados, baseados nas características observadas nas PRCs e por estar de acordo com as bases fisiológicas dos mesmos. / In this work, we constructed Phase Resetting Curves (PRC) of the infradian biological rhythms of the cave insects Folsomia candida. We used temperature pulses to reset the phases of these rhythms, since the insects are blind. We obtained PRCs with delays only, of the topological Type 1, for the three different pulse durations. This is an unusual behavior, considering the typical responses of the limit-cycle oscillators associated to the majority of the biological rhythms. The dimension of an oscillator determines the PRC types that a system, submitted to pulses at different phases, can present. Simple clocks are associated to a sequence of discrete linked events, each event leading to the next and playing a functional role in generating the rhythmicity. They are unidimensional systems and can\'t present Type O PRCs and singularities, as the two-dimensional limit cycles. Based on the properties of the experimental curves and knowing the phisiological bases of these insect rhythms, we propose a simple clock model for the infradian rhythms studied here.

Post-transplant apoptosis is a major obstacle to successful cell replacement therapy for retinitis pigmentosa. Over-expression of the X-linked inhibitor of apoptosis (XIAP) protein could increase transplant survival leaving a greater numbers of cells available to replenish photoreceptors lost during retinal degeneration. Sonic Hedge hog expanded C57BL/6 mouse neonatal retinal progenitor cells (Hh-RPCs) were infected with two lentiviral constructs, encoding XIAP/GFP or GFP only, as well as with the TAT-fusion protein, TAT-eGFP. The optimal delivery conditions and expression patterns were assessed. It was found that lentiviral infection, in conjunction with fluorescence activated cell sorting (FACS) allowed for the creation of a nearly pure line of Hh-RPCs which over-expressed XIAP protein for at least one month. Although the TAT-fusion protein efficiently transduced Hh-RPCs, its nuclear localization made it unsuitable for XIAP protein delivery. These results demonstrated two methods of transducing primary retinal progenitor cells and represent an important first step towards efficient cell replacement therapy in the retina.

Mammals have three members of the small taste receptor gene family responsible for the perception of sweet and savory tastes: two genes (T1R2 and T1R3) comprise the canonical sweet receptor, and a third gene, T1R1, acts with T1R3 to make the savory receptor. Here, in a joint effort with a team of international collaborators, we show that even though birds are missing the taste receptor gene (T1R2) required by other vertebrates to perceive carbohydrates and sweeteners, hummingbirds still detect sugars—but in a novel way. This project spanned multiple fields and field sites, integrating taste tests on wild birds, behavioral analysis of captive animals, bioinformatics, receptor cloning, and cell-based functional assays. The first published avian genome, that of the chicken, revealed a surprising lack of T1R2. Chickens are sweet-insensitive: however, many nectar-feeding birds appear highly attuned to sugars like sucrose, fructose and glucose. Our initial field experiments with a panel of artificial sweeteners as well as high-speed filming and choice tests on captive birds indicated a rapid response to sugars rather than a post-ingestive metabolic sensing of caloric value. As the response appeared sensory, we pursued a candidate gene approach to search for possible taste receptors, and cloned T1R taste receptors from chickens, hummingbirds, and swifts. By analyzing genomes from an additional 10 birds and an alligator, we documented widespread absence of T1R2 and identified signatures of positive selection in the remaining hummingbird T1Rs. Together with Dr. Yasuka Toda at the University of Tokyo, we were able to test the function of these receptors in cell culture. We used a cell-based luminescence assay to measure functional responses. As expected, chicken and swift receptors responded to amino acids, but, surprisingly, the umami receptor in hummingbirds had acquired a new function and was now sensitive to carbohydrates as well. Chimeric studies of receptors containing hummingbird and chicken sequence identified 19 mutations involved in this functional change: since divergence from swifts, the umami receptor underwent extensive re-modeling. Further behavioral tests with wild hummingbirds revealed that most agonists from the cell-based assay were appetitive, while artificial sweeteners which did not activate the receptors were not preferred—a concordance between in vivo and in vitro studies that indicates that this re-purposed receptor guides hummingbird taste behavior. Diet shifts have profound physiological effects and evolutionary ramifications: the radiation of hummingbirds is likely due, at least in part, to their ability to colonize an empty niche. However, much remains to be learned about the roles of taste in changes in diet, and the causes and effects of shifts in diet and perception are often unclear. For instance, birds appear to have lost T1R2 early in their evolutionary history. As they are the descendants of carnivorous dinosaurs, birds may have experienced relaxed selection on the sweet receptor similar to that seen in mammalian carnivores; alternatively, the loss could be due to the overall genome-wide reduction seen in birds. In Chapter 3, we begin to investigate causes and consequences of the changes in hummingbird taste receptors, and present new behavioral studies regarding the ability to detect amino acids. Together, these findings raise questions about the evolution of sensory systems and of neural circuits underlying perception: studying taste receptors in a comparative context yields insight into basic aspects of the evolutionary process as well as molecular mechanisms underlying behavior. / Biology, Organismic and Evolutionary

Temperature plays an important role in shaping the form and function of every species. Ectothermic organisms are particularly sensitive to fluctuations in their thermal environment. Their inability to produce appreciable amounts of heat through physiological mechanisms makes them particularly vulnerable to thermal shifts, and ideal for the study of temperature-mediated evolution. The central goal of this dissertation is to understand how temperature shapes the evolutionary history of terrestrial ectotherms during the colonization of novel environments. Towards this aim, I focus on a single species of lizard, the North American green anole, Anolis carolinensis. In the first chapter of my dissertation I trace the phylogeographic history of A. carolinensis in order to identify the geographic distribution of major genetic lineages within the species and its Cuban relatives, date times of divergence between these lineages, and identify geographic barriers to dispersal. In the second chapter, I use an integrated approach to identify aspects of the environment that may have influenced evolutionary adaptation within the species. I combine geo-referenced climate data, environmental niche modeling, thermal physiology, common garden experiments and genomic techniques to understand phenotypic and genomic response of this historically subtropical Cuban lizard to the more temperate regions of the American Southeast. Finally, in the third chapter I use experimental temperature manipulations and physiological testing to explore the roles of phenotypic plasticity and local adaptation in shaping latitudinal variation in thermal tolerance and identify potential systemic mechanisms involved. As a result, I have identified a Miocene origin of the initial over-water dispersal event leading to the establishment of the green anole in peninsular Florida, followed by a rapid Pleistocene range expansion of the species northward into higher latitudes. Range expansion on the mainland has led to thermal niche expansion, mediated by a combination of local adaptation of cold tolerance and genetic isolation by environment between populations from different thermal habitats. Phenotypic plasticity and canalized differentiation both shape variation in cold tolerance across latitude and energy conservation via metabolic suppression under acute and chronic cold onset may help to extend the limits of cold tolerance in this species at its northernmost latitudes. / Biology, Organismic and Evolutionary

Insulin-Degrading Enzyme (IDE) is a zinc-metalloprotease responsible for the clearance of insulin in peripheral tissues. Despite decades of speculation that inhibiting endogenous insulin degradation might treat Type-2 Diabetes, the functional relationship between IDE and glucose homeostasis remains unclear. IDE inhibitors that are active in vivo are therefore needed to elucidate IDE’s physiological roles and to determine its potential to serve as a target for the treatment of diabetes. In this thesis I describe the development of the first highly specific IDE in vivo probe, identified from a DNA-templated library of macrocycles, which enabled the first study of the physiological consequences of IDE inhibition. An X-ray structure of the macrocycle bound to IDE reveals that it engages a novel binding pocket away from the catalytic site, which explains its remarkable specificity and its suitability to study IDE in vivo. Treatment of lean and obese mice with this inhibitor revealed that IDE regulates multiple metabolic hormones, including glucagon and amylin, in addition to insulin. Under physiological conditions that mimic a meal, such as oral glucose administration, acute IDE inhibition leads to substantial improvement in glucose tolerance, owing to the potentiation of endogenous insulin and amylin levels over glucagon signaling. These studies demonstrated the feasibility of modulating IDE activity as a therapeutic strategy to treat diabetes and expanded our understanding of the roles of IDE in glucose and hormone regulation. Based on these studies we sought to develop substrate-selective inhibitors that block IDE’s ability to degrade insulin but not its ability to degrade glucagon, which would represent a major step forward towards IDE-targeted therapeutics. The first-generation DNA-templated inhibitor was retailored into a fluorescent anisotropy tool for high-throughput screening of diverse small-molecule libraries. We discovered and characterized a family of IDE inhibitors with sub-micromolar potencies that inherited the remarkable specificity for IDE over other metalloproteases, and selectively obstruct IDE-mediated insulin degradation in a way that accommodates for glucagon cleavage. In conclusion, these findings offer new insights into the biological roles of IDE and establish a novel strategy to selectively potentiate the physiological insulin response in order to improve blood sugar control in Type-2 Diabetes. / Chemistry and Chemical Biology

Yolk provides an important source of nutrients during the early development of oviparous (non-platental) organisms. In addition to phosphate and lipids, it is com- posed mainly of vitellogenin proteins packed into membrane-bound compartments called yolk platelets. Catabolism of yolk is initiated by acidification of the yolk platelet, leading to the activation of cathepsin-like proteases that degrade yolk contents, but it is unknown how this process is triggered. Using maternal shRNA technology in Drosophila melanogaster embryos we find that yolk catabolism depends on components of the Tor pathway, a well-characterized regulator of cellular metabolism. Knockdown of Tor also leads to severe nuclear fragmentation, abnormal gastrulation, and an increased ratio of AMP/ATP. This phenotype is more severe than inhibition of Tor in later development, or in cell culture models, suggesting that Tor may have additional functions during early development. Additionally, we identify a downstream target of Tor, Atg1, as necessary for yolk catabolism. Atg1 is responsible for initiation of autophagy, a process that de- grades both protein and organelles within the cell. While Atg1 is required for a burst of spatially-regulated autophagy during late cellularization, autophagy is not required for yolk catabolism. We find that knockdown of Atg1, but not downstream autophagy proteins, can rescue shRNA-Tor embryos, suggesting that Atg1’s role in yolk cataboilism may be through regulation of Tor. Last, we find that Rheb, a GTPase responsible for activation of Tor on the lysosome membrane, is present on Xenopus laevis yolk platelets. Therefore, regulation of yolk catabolism by the Tor pathway may function in a similar manner to Tor’s activity on the lysosome. Together, this work connects the conserved Tor and Atg1 metabolic sensing pathways to yolk catabolism, and may provide insight into the metabolic regulation of lysosomes more generally. / Systems Biology