Increasing evidence suggests that normal pulmonary vascular morphogenesis is critical for the formation and maintenance of the lung parenchyma, both pre- and postnatally. Indeed, the disruption of angiogenic pathways, whether through inherent genetic predisposition or as a consequence of life-saving interventions, may underlie many pulmonary diseases of infancy, including alveolar capillary dysplasia (ACD) and bronchopulmonary dysplasia (BPD). To understand the etiology of – and advance treatments for – such diseases, we must first identify the fundamental genetic regulators that orchestrate normal parenchymal development. Neuropilin-1 (NRP1) is a transmembrane receptor that plays essential roles in normal and pathological vascular development, and binds two distinct ligand families: Vascular endothelial growth factor (VEGF) and Class 3 Semaphorins (SEMA3). Although VEGF-NRP1 interactions in systemic vascular development have been described, the importance of SEMA3-NRP1 signalling in systemic or pulmonary vascular morphogenesis is uncertain. We hypothesized that SEMA3-NRP1 and VEGF-NRP1 interactions are fundamental pathways in the orchestration of pulmonary vascular development in both the embryo and neonate. Disruption of these pathways would therefore lead to significant interruption of normal angiogenic and vascular maturation processes that are relevant to the pathogenesis of pulmonary diseases. Using extensive histopathological analyses of NRP1 loss-of-function mice, we show evidence of a significant role for SEMA3-NRP1 signalling in fetal microvascular development: congenital loss of SEMA3-NRP1 signalling resulted in severely attenuated development of the distal vasculature and alveolar-capillary interface, leading to fatal respiratory distress at birth that is reminiscent of clinical ACD. By contrast, VEGF-NRP1 and SEMA3-NRP1 signalling appear unessential for normal alveolar and vascular development in the postnatal period, per se, despite increased mortality. Our results demonstrate the critical involvement of SEMA3-NRP1 signalling in endothelial development and substantiate the idea that NRP1 mediates opposing and cooperative functions between SEMA3 and VEGF ligands.

Sound source localization depends on the auditory system to identify, recognize, and segregate elements of salient sources over distracting noise. My research investigates sensory mechanisms involved in these auditory processing tasks of an insect hearing specialist, to isolate individual sound sources of interest over noise. I first developed quantitative methods to determine signal features that the acoustic parasitoid fly Ormia ochracea (Diptera: Tachinidae) evaluate for host cricket song recognition. With flies subjected to a no-choice paradigm and forced to track a switch in the broadcast location of test songs, I describe several response features (distance, steering velocity, and angular orientation) that vary with song pulse rate preferences. I incorporate these response measures in a phonotaxis performance index that is sensitive to capturing response variation that may underlie song recognition. I demonstrate that Floridian O. ochracea exhibit phonotaxis to a combination of pulse durations and interpulse intervals that combine to a range of accepted pulse periods. Under complex acoustic conditions of multiple coherent cricket songs that overlap in time and space, O. ochracea may experience a phantom source illusion and localize a direction between actual source locations. By varying the temporal overlap between competing sources, I demonstrate that O. ochracea are able to resolve this illusion via the precedence effect: exploitation of small time differences between competing sources to selectively localize the leading over lagging sources. An increase in spatial separation between cricket song and masking noise does not reduce song detection thresholds nor improve song localization accuracy. Instead, walking responses are diverted away from both song and noise. My findings support the idea that the ears of O. ochracea function as bilateral symmetry detectors to balance sound intensity, sound arrive time differences, and temporal pattern input to both sides of the auditory system. Asymmetric acoustic input result in corrective turning behaviour to re-establish balance for successful source localization.

Bladder obstruction (neurogenic or anatomic) induces strain injury in detrusor smooth muscle cells. Signaling via strain injury in other systems has been highly studied, while in bladder obstruction, it has been quite limited to a small number of pathways. In our study we have examined the effects of strain injury using a combination of in vivo, ex vivo and in vitro models, with the aim of understanding disease pathogenesis in the bladder. Using a combination of literature searches, phospho-protein screens and pathway analysis, we uncovered three pathways activated by mechanical strain, ERK, STAT3 and mTOR, with potential for changing not only the way we understand but also the way we treat obstructive myopathies of the bladder. We found that not only were these pathways activated in response to strain and distension injury of BSMC, but they were also responsible for proliferation and sometimes de-differentiation. Included herein are three chapters, published in 2006 and 2010, on the role of ERK, STAT3 and mTOR pathways in bladder smooth muscle cell proliferation and differentiation, 8 Appendices containing the first pages of other papers and reviews published during the course of my studies.

Doctor of Philosophy / Biology / Michael A. Herman / Nematodes play an important role in various habitats where numerous factors serve to shape their communities. One such factor is the potentially pathogenic nematode-prey interaction. This project is focused on the elucidation of the genes that the bacterivorous nematode Caenorhabditis elegans employs to respond to the emerging nosocomial bacterial pathogen Stenotrophomonas maltophilia. A virulent S. maltophilia strain JCMS requires the action of several C. elegans conserved innate immune pathways that serve to protect the nematode from other pathogenic bacteria. However, insulin-like DAF-2/16 signaling pathway mutants that are typically pathogen resistant are susceptible to JCMS, and several DAF-2/16 regulated genes are not significantly differentially expressed between JCMS and avirulent E. coli OP50. We have determined the complete set of mRNA transcripts under different bacterial treatments to identify genes that might explain this JCMS specific DAF-2/16 pathway evasion. The identified set included 438 differentially expressed transcripts among pairwise comparisons of wild-type nematodes fed OP50, JCMS or avirulent S. maltophilia K279a. Candidate genes were nominated from this list of differentially expressed genes using a probabilistic functional connection model. Six of seven genes that were highly connected within a gene network generated from this model showed a significant effect on nematode survival by mutation. Of these genes, C48B4.1, mpk-2, cpr-4, clec-67 and lys-6 are needed for combating JCMS, while dod-22 was solely involved in K279a response. Only dod-22 had a documented role in innate immunity, which merits our approach in the identification of gene candidates. To a lesser extent, we have also focused on the identification of virulence factors and the mode of action employed by S. maltophilia. JCMS virulence requires rpfF, xps and involves living bacteria that accumulate in the intestinal lumen. Additionally, the bacterial secretion encoding genes cs, p773, p1176, pi1y1 and xdi are involved in JCMS evasion of daf-2. In summary, we have discovered a novel host-pathogen interaction between C. elegans and S. maltophilia JCMS, revealed genes that are involved in each partner of the interaction, and established a new animal model for the study of S. maltophilia mode of action.

Master of Science / Department of Biology / Stella Y. Lee / CLN5 is one of several proteins that when mutated result in the lysosomal storage disorder (LSD) Neuronal Ceroid Lipofuscinosis (NCL). CLN5 is a soluble lysosomal protein that has no known function at this time. Previously we showed that eight asparagine residues in CLN5 are N-glycosylated, and that this modification is important for the protein’s transport and function. Now, we have identified a link between the activation of autophagy and CLN5 deficiency. The autophagy-lysosomal protein degradation system is one of the major pathways the cell uses to degrade intracellular material and recycle cellular building blocks. It was recently shown that other CLN proteins affect the relative level of autophagy, indicating a potential link between the autophagy pathway and the NCLs. By knocking down endogenous CLN5 in HeLa we showed that, upon stress induction, cells responded with higher levels of autophagy activation. Consistent with these knockdown experiments, there is a higher level of the autophagy marker protein, LC3-II, in CLN5 patient cells that are naturally deficient for the CLN5 protein. Pharmaceutical induction of autophagy through different means also showed higher LC3-II levels compared to control, though patterns differed in the type of autophagy induced. In summary, we discovered that the autophagy pathway is altered in CLN5 deficient cells, indicating a potential role for CLN5 in autophagy. Further analyses of the autophagy pathway will shed light on where CLN5 is acting and the mechanism by which defective CLN5 causes NCL.

Master of Science / Biology / Keith B. Gido / Negative interactions with nonnative fish are often cited as a leading cause of declining native fish populations, but quantifying these interactions is difficult. Movement ecology and consumptive demand estimates of nonnative fish predators is needed to better understand potential impacts these organisms are having on native species. The objective of this thesis were to estimate the consumptive demand of Flathead Catfish Pylodictis olivaris on native fishes across an elevational gradient, and characterize the movement at hourly, daily, and seasonal scales of this introduced predator. This research was conducted in the upper Gila River basin of southwestern New Mexico. Bioenergetics modeling was used to estimate consumptive demand; model results were coupled with measured densities and size structure of Flathead Catfish populations, and water temperatures, to predict its predatory threat. Potential consumption was highest at lower elevation sites because of higher water temperatures, but actual consumption was highest at mid-elevation sites because of the prevalence of large-bodied individuals. Potential annual consumptive demand of Flathead Catfish on native fish across our nine sampling sites ranged from 0.0 to 3.1 g/m²/yr, which exceeded native fish productivity at one site. To characterize the movement of Flathead Catfish, we used radio telemetry and tracked individuals from May 2014 to June 2015. Movement behaviors varied among individuals with a majority moving <150 m from capture location and some more mobile, moving substantial distances (692-42,840 m). During the course of the study, activity was greatest in summer and fall, and individuals moving substantial distances moved downstream to warmer river reaches before the winter. Nightly movements only involved short distances (5 m) and no fish exceeded a single movement >80 m. Daily activity was greatest during evening but late afternoon activity was observed in summer and fall. Results from this study identify areas within the upper Gila River where introduced Flathead Catfish consumption is likely to negatively impact native fish populations and managers can use this information to understand potential overlap with native species, target future removal efforts in areas where these fish are concentrated, and avoid stocking native fishes in reaches where Flathead Catfish tend to aggregate.

Doctor of Philosophy / Department of Biology / Brett K. Sandercock / Samantha Wisely / Anthropogenic activities and climate change have dramatically altered landscapes worldwide. The ability of species to cope and adapt to ongoing changes is likely a function of their behavior, movements, and sensitivity to fragmentation. Greater Prairie-Chickens (GPC) are a lek mating grouse native to the Great Plains Landscape Conservation Cooperative (GPLCC), for which inbreeding depression and anthropogenic avoidance are a concern. The goals of my dissertation were to: 1) identify genetic correlates of male performance which may influence population viability under current land use practices, 2) identify GPC habitat characteristics and delineate areas of critical GPC habitat necessary for GPC conservation, and 3) identify the relative importance of distance and habitat quality for maintaining genetic connectivity among spatially structured populations. First, I found male reproductive success and survival to be positively associated with genetic diversity. Using multistate modeling in Program Mark, male survival across the observed range of variation in number of alleles (15-22) increased more than fourfold from 0.17 to 0.77. Second, I found 35-40% of Kansas, and 1.5 % (11,000 Km squared) of the GPLCC, were considered high-quality lek habitats. Top performing logistic models predicting lek presence (wi=0.95) included strong effects of grassland cover and avoidance of anthropogenic disturbance. When this model was applied to putative future landscapes based on climate change and current land use trends over a 70-year period, I found a 27-40% reduction in habitat area and a 137 Km southeast shift in habitat distribution. Under equilibrium conditions we expect isolation by distance (IBD) to explain the distribution of genetic diversity. However, if the landscape restricts dispersal, then we might observe isolation by resistance (IBR). I used model selection procedures to choose among competing IBR or IBD models to explain the distribution of genetic diversity among GPC populations across Kansas and the GPLCC. IBD was never supported (R-square<0.02, P>0.09). The best models for Kansas (R2=0.69, P<0.02) and for the GPLCC (R-square=0.46, P<0.02) indicated that human-mediated landscape changes have influenced landscape permeability for dispersal. The integration of behavioral, landscape, and genetic data provided new insights on prairie-chicken ecology, and is a powerful approach for developing conservation strategies for sensitive species.

Master of Science / Department of Biology / Jesse B. Nippert / Jesse B. Nippert / Anthropogenic climate change is likely to alter the function and composition of ecosystems worldwide through increased precipitation variability and temperatures. To predict ecosystem responses, a greater understanding of the physiological and growth responses of plants is required. Dominant species drive ecosystem responses, and it is essential to understand how they respond to understand potential ecosystem changes. Dominant species, such as switchgrass (Panicum virgatum L.), posses large genotypic and phenotypic variability, which will impact the degree of responses to projected climate changes. I studied the physiological and growth responses of switchgrass, a common perennial warm-season C4 grass that is native to the tallgrass prairie, to alterations in precipitation amount and temperature. The first experiment I conducted focused on the responses of three ecotypes of P. virgatum to three precipitation regimes (average, 25% below, 25% above). I concluded that the physiological responses of photosynthesis, stomatal conductance, transpiration, dark-adapted fluorescence, and mid-day water potential in P. virgatum were explained by ecotypic differences. Robust responses to altered precipitation were seen in the water use efficiency, mid-day water potential, and aboveground biomass. Ecotypic differences were also seen in several aboveground biomass variables, and most strikingly in flowering times and rates. There were few interactions between ecotype and precipitation, suggesting precipitation is a strong driver of biomass production, whereas adaption of ecotypes to their local environment affects physiological processes. A second experiment studied the response of local populations of P. virgatum to nocturnal warming. Results showed significant differences in daytime E, daytime gs, and flowering phenology between treatments. Differences in aboveground biomass were between topographic positions. I concluded that water availability, based on topographic position, is a strong driver of P. virgatum aboveground biomass production, but nocturnal warming has the potential to impact flowering phenology, physiological responses, and exacerbate plant water stress. I also reviewed the literature on the ecological effects of implementing switchgrass cultivation for biofuel. From the literature review, I concluded that large-scale switchgrass cultivation will have widespread ecological impacts. If landscape heterogeneity is maintained through harvest rotations, no till farming, and mixed species composition, ecosystem services can be maintained while providing economic value.

Master of Science / Department of Entomology / John C. Reese / The soybean aphid, Aphis glycines is documented to have arrived in North America in mid 2000 and has ever since established itself as a formidable pest of soybeans, with the capacity to cause immense crop losses. This formidable pest with its complex life cycle and habits represents a current threat to soybean production. Host plant resistance is a promising avenue that can offer considerable control over the soybean aphid problem. Antibiosis being the most effective host plant resistance category, this study was aimed at attempting to understand the effects of induction on the antibiosis levels of soybeans. In the first set of experiments, different soybean genotypes and two soybean aphid biotypes were tested to comprehend if and how the genotypes and biotypes affected the survival and reproduction of the aphid. The experiments revealed mixed results that can be attributed to the genotypes tested and the biotypes used. While some genotypes showed no significant changes due to previous infestation, K1621 suggested signs of induced resistance to biotype 1 and PI567301B showed induced resistance to biotype 2, while K1639 pointed towards induced susceptibility to biotype 2. A follow up feeding behavior study with Electrical Penetration Graph (EPG) technique was carried out on PI567301B to elucidate if the induced resistance was tissue-specific, which could affect the feeding behavior of the aphid (biotype 2); but the results showed no appreciable differences in the feeding behavior of the aphids on clean vs. infested plants. Induced response studies shed light on how plants respond to herbivory and help us identify how changes in plant physiology affect the various herbivores that visit it for food and shelter. This knowledge can thus be applied to the development of superior varieties of crops that can defend themselves better against recurring infestations.

Master of Science / Department of Biology / Keith B. Gido / Recognizing habitat needs of fishes across space and time is increasingly important for managing altered stream networks, such as in the Colorado River basin. Recent work on warm-water fishes suggest they might benefit from access to tributaries and their confluences. Fish movements or distributions within tributaries relative to distance from mainstem confluences in two streams with different network types (linear versus dendritic) were investigated in the San Juan River basin, USA. Upstream distance from the San Juan River resulted in species declines (Chaco Wash, linear network) or turnover (McElmo Creek, dendritic network). McElmo Creek movement patterns were likely attributed to spring spawning migrations of flannelmouth sucker (Catostomus latipinnis), spawning aggregations of razorback sucker (Xyrauchen texanus), foraging or refuge seeking by Colorado pikeminnow (Ptychocheilus lucius), and monsoon-related movements for channel catfish (Ictalurus punctatus) and razorback sucker. Razorback sucker and Colorado pikeminnow dominated movements at Chaco Wash, suggesting this backwater-like tributary supplied thermal or current refuge, foraging habitat, or both. Within McElmo Creek, a second study explored the importance of confluences by characterizing habitat use and movements of fishes at the junction of McElmo and Yellow Jacket creeks. Native fish dominated the confluence community composition. The reach downstream of the confluence had consistently higher abundances, species richness, and more frequent detections of tagged fishes relative to upstream reaches. Movement behaviors inferred by detection frequency of tagged fish among reaches surrounding the confluence differed among species. Small flannelmouth sucker (< 300 mm) and roundtail chub (Gila robusta) were commonly detected in Yellow Jacket Creek whereas large flannelmouth sucker (> 300 mm), bluehead sucker (C. discobolus), and channel catfish used McElmo Creek reaches. Monsoons increased McElmo Creek discharge which triggered upstream movements of channel catfish and displaced large flannelmouth sucker and bluehead sucker. Monsoons increased movements between McElmo and Yellow Jacket creeks by roundtail chub, small flannelmouth sucker, and black bullhead (Ameiurus melas). Combined, these two field studies emphasized using links between patterns and processes of tributary fish communities. Conservation, rehabilitation, and maintenance of connectivity and habitat heterogeneity at confluence zones likely can be a localized management strategy with expansive ecosystem effects.