Predation pressure is a powerful agent of natural selection and is responsible for the evolution of various antipredator defence strategies in animals. Sensory thresholds for predator detection could play an important role in the evolution of defence strategies. I tested the hypothesis that the ability of animals to detect predators is correlated with defence strategy, with the prediction that animals with poor predator detection abilities rely more on preventative (primary) defence strategies than animals with low predator detection thresholds. Bats and their insect prey were used as a simple study system for these experiments due to the reliance on a single modality (hearing) for both predator and prey detection. Many insects have ears tuned to the ultrasonic echolocation calls of bats and can use this predator cue to initiate evasive action, but variation in auditory thresholds exists among species. In moths, a group in which the only known function of hearing is predator detection, a clear relationship was found between auditory thresholds for predatory ultrasound and a risky behaviour, nocturnal flight time. A more complicated situation exists when the sensory system serves more than one purpose, as with the ears of orthopteran insects used for both predator detection and mate localization. Some gleaning bats use calling song as a cue to locate these insects as prey, and both primary (reduced calling) and secondary (song cessation in response to ultrasound) defences have been identified in orthopterans. The auditory interneurons considered the “bat-detectors” in katydids and crickets most likely have context dependent functions in several groups, as a predator-detector in flight and mate-detector on the ground. The relationship between reliance on primary over secondary defence and auditory sensitivity in these insects appears to be influenced by the nature of the calling song of the species and their mating strategy.

The overall aim of my thesis is to gain insight into the cellular and molecular basis of the hierarchical organization of the human blood system, and how these normal development processes are subverted into leukemogenesis. To date, the major cellular classes that comprise human blood remain ill defined as rigorous clonal analysis required to define the self-renewal and lineage potential of single cells has not yet been performed. Here, identification CD49f as a novel marker of human HSC led to the ability to transplant single human HSC in NOD-scid IL2Rgc-/- mice. Loss of CD49f and Thy1 uniquely demarcated multi-potent progenitors (MPP) from HSC. The classical model of hematopoiesis posits the segregation of lymphoid and myeloid lineages as the earliest fate decision during lineage restriction from HSC. The validity of this model in the mouse has been questioned; however, little is known about the lineage potential of human progenitors. By clonally mapping the developmental potential of seven progenitor classes from neonatal cord blood and adult bone marrow, human multi-lymphoid progenitors (MLP) were identified as a distinct population of Thy1-/loCD45RA+ cells in the CD34+CD38- stem cell compartment that can give rise to all lymphoid cell types, as well as monocytes, macrophages and dendritic cells. This indicates that these myeloid lineages arise in early lymphoid lineage specification. Thus, as in the mouse, human hematopoiesis does not follow a rigid model of myeloid-lymphoid segregation. While non-genetic mechanisms govern cell-fate commitment and lineage specification, hematopoietic malignancies are often initiated by aberrant gene rearrangements that can subvert normal cellular processes. Full transformation requires the accumulation of multiple genetic lesions. Most tumours exhibit dramatic genetic heterogeneity downstream of the initiating oncogenic event and are composed of pockets of genetically distinct clonal subpopulations. However little is known of how diversity evolves or the impact diversity has on functional properties. Here, using xenografting and DNA copy number alteration (CNA) profiling of human BCR-ABL1 lymphoblastic leukaemia, it was demonstrated that genetic diversity occurs in functionally defined leukaemia-initiating cells (L-IC) and that many diagnostic patient samples contain multiple genetically distinct L-IC subclones. Reconstructing the subclonal genetic ancestry of several samples by CNA profiling demonstrated a branching multi-clonal evolution model of leukaemogenesis, rather than linear succession. For some patient samples, the predominant diagnostic clone repopulated xenografts, while in others it was outcompeted by minor subclones. Reconstitution with the predominant diagnosis clone was associated with more aggressive growth properties in xenografts, deletion of CDKN2A/B, and a trend to poor patient outcome. Our findings link clonal diversity with L-IC function and underscore the importance of developing therapies that eradicate all intratumoural subclones.

In a previous study, three determinants of fitness were identified as mutant alleles (each designated "e") that arose in yeast populations propagated in divergent environments. In a low-glucose environment, MDS3e and MKT1e interacted positively to confer a fitness advantage. PMA1e from a high-salt environment interacted negatively with MKT1e in low glucose, indicating a mechanism of reproductive isolation. In this thesis, I demonstrated that the negative interaction between PMA1e and MKT1e is mediated by alteration in intracellular pH and likely by a delay of the cell division cycle, while the positive interaction between MDS3e and MKT1e is mediated by changes in gene expression affecting glucose transporter genes. I also confirmed the evolutionary significance of the positive interaction by showing that an MDS3e genetic background is required for the recapitulation of the MKT1e mutation. Collectively, these results illustrate how epistasis can play a central role in both adaptation and speciation.

Studies of symbiotic bacteria have demonstrated that they provide multiple benefits to their hosts. These studies, however, have overlooked the importance of interactions with other bacteria and environmental factors that affect bacterial assemblages. To understand what shapes bacterial assemblages, I manipulated the diet of ants from the genus Cephalotes and disturbed their gut microbiome. I found that a deficit of nitrogen reduces bacterial densities. Furthermore, the data suggest that bacterial abundance may influence ant survival. I followed this experiment up by manipulating a putative protein source in the field. Our lab assigned Allomerus octoarticulatus ant colonies to treatments in which potential prey were present or absent. I collected data on foraging behaviour, colony performance, and composition of the bacterial community. The absence of prey increased ant recruitment to protein-rich baits; these ants were also less fit than ants that had insect prey but their bacterial assemblages were not affected.

Master of Science / Department of Biology / Jack Cully, Jr. / Disturbance is defined as any discrete event that disrupts ecosystem, community, or population structure and changes resources, substrate availability, or the physical environment. Habitat use by an organism is based on its perception of where to maximize its own fitness, and can be altered in response to disturbance-induced changes in resources, substrate, or physical features modified by disturbance. Disturbance-induced changes to vegetation structure reshape a small mammal’s surrounding physical environment and/or resources, and may influence its utilization of an area. Effective wildlife and resource management is dependent on a thorough understanding of how individual species and communities utilize their surroundings and how disturbance affects a species’ response to changes in its surroundings. We investigated seasonal habitat associations of three small mammal species and for overall species diversity across a gradient of military combat-vehicle disturbance intensities at the Fort Riley Military Reservation, Kansas. Deer mouse (Peromyscus maniculatus) abundance did not vary across a categorical gradient of disturbance created by military-combat vehicles, regardless of season. Western harvest mouse (Reithrodontomys megalotis) abundance was associated with more highly disturbed areas irrespective of season. Prairie vole (Microtus ochrogaster) abundance was associated with habitat that was less disturbed in the spring but more highly disturbed in the fall. Shannon diversity of the small mammal community was higher in the more highly disturbed areas regardless of season. This research shows that small mammals respond to disturbances created by military training with combat vehicles in a species-specific manner, and indicates that there may be differences in the effects of military training versus natural or agricultural disturbances on the abundance and diversity of small mammals. This is an important consideration given that the Department of Defense manages more than 12 million ha of land in the United States, and is charged under the Sikes Act with conserving natural resources on these lands, including biological diversity. Thus, the findings of other ecological research on the effects of disturbance on small mammals may not be directly applicable to the types of disturbances that occur on military lands, which underscores the need for further research on the specific effects of military-training activities on species’ responses.

The SH2D5 signaling molecule is a previously uncharacterized adaptor-like protein, containing an N-terminal phosphotyrosine binding (PTB) domain and a noncanonical Src Homology-2 (SH2) domain. With an antibody that I developed, I have shown that SH2D5 is highly enriched throughout adult brain regions. Furthermore, SH2D5 is localized to purkinjie cells in the cerebellum, the cornu ammonis (CA) of the hippocampus and pyramidal cells in the cortex. Despite harbouring two potential phosphotyrosine (pTyr) recognition domains, SH2D5 binds minimally to pTyr ligands. To discover the interaction partners of SH2D5 I conducted an immunoprecipitation/ mass spectrometry (IP/MS) screen from cultured Human Embryonic Kidney (HEK) 293T and Neuro2A cells along with murine brain lysates. These experiments revealed novel binding partners to SH2D5 including a prominent association with the RacGAP protein, Breakpoint Cluster Region protein (BCR), which is also highly expressed in brain. I have defined the interaction between SH2D5 and BCR and show that the PTB domain of SH2D5 engages an NxxF motif located within the N-terminal region of BCR. To address the biological significance of SH2D5, I utilized an siRNA approach to deplete the neuroblastoma cell-line, B35, of iii SH2D5. In these assays, B35 cells display a cell rounding phenotype and grow in a lattice formation. Furthermore, upon SH2D5 depletion these cells display low levels of activated Rac, associated with cell rounding. Taken together, these data reveal the first characterization of the SH2D5 signaling protein, its novel interaction with BCR and phenotype in neuronal-like cells. These data signify a biological function for SH2D5 in neurobiologic signaling perhaps applicable to learning and memory.

Short-chain fatty acids (SCFAs) are anaerobic bacterial metabolites. Cystic fibrosis (CF) lung disease is a condition caused by mutations in the cystic fibrosis transmembrane conductange regulator (CFTR) gene and is characterized by persistent lung inflammation and bacterial colonization. We measured the concentrations of SCFAs in sputum of patients with CF and tested the effect of these compounds on bacterial growth. Furthermore we found that SCFAs can influence the inflammatory protein expression and cytokine release in airway epithelial cells. SCFAs differentially alter cytokine release in CF bronchial epithelial cells (CFBE) compared to CFBE expressing wild-type CFTR. We also studied the effect of SCFAs in an acute lung injury model in BALB/cJ mice and found that intratracheally administered SCFAs can affect the inflammatory environment of the airways in vivo. We conclude that SCFAs may be important in the airways and that further investigation is warranted to understand their effects on inflammation and infection.

Short-chain fatty acids (SCFAs) are anaerobic bacterial metabolites. Cystic fibrosis (CF) lung disease is a condition caused by mutations in the cystic fibrosis transmembrane conductange regulator (CFTR) gene and is characterized by persistent lung inflammation and bacterial colonization. We measured the concentrations of SCFAs in sputum of patients with CF and tested the effect of these compounds on bacterial growth. Furthermore we found that SCFAs can influence the inflammatory protein expression and cytokine release in airway epithelial cells. SCFAs differentially alter cytokine release in CF bronchial epithelial cells (CFBE) compared to CFBE expressing wild-type CFTR. We also studied the effect of SCFAs in an acute lung injury model in BALB/cJ mice and found that intratracheally administered SCFAs can affect the inflammatory environment of the airways in vivo. We conclude that SCFAs may be important in the airways and that further investigation is warranted to understand their effects on inflammation and infection.

The SH2D5 signaling molecule is a previously uncharacterized adaptor-like protein, containing an N-terminal phosphotyrosine binding (PTB) domain and a noncanonical Src Homology-2 (SH2) domain. With an antibody that I developed, I have shown that SH2D5 is highly enriched throughout adult brain regions. Furthermore, SH2D5 is localized to purkinjie cells in the cerebellum, the cornu ammonis (CA) of the hippocampus and pyramidal cells in the cortex. Despite harbouring two potential phosphotyrosine (pTyr) recognition domains, SH2D5 binds minimally to pTyr ligands. To discover the interaction partners of SH2D5 I conducted an immunoprecipitation/ mass spectrometry (IP/MS) screen from cultured Human Embryonic Kidney (HEK) 293T and Neuro2A cells along with murine brain lysates. These experiments revealed novel binding partners to SH2D5 including a prominent association with the RacGAP protein, Breakpoint Cluster Region protein (BCR), which is also highly expressed in brain. I have defined the interaction between SH2D5 and BCR and show that the PTB domain of SH2D5 engages an NxxF motif located within the N-terminal region of BCR. To address the biological significance of SH2D5, I utilized an siRNA approach to deplete the neuroblastoma cell-line, B35, of iii SH2D5. In these assays, B35 cells display a cell rounding phenotype and grow in a lattice formation. Furthermore, upon SH2D5 depletion these cells display low levels of activated Rac, associated with cell rounding. Taken together, these data reveal the first characterization of the SH2D5 signaling protein, its novel interaction with BCR and phenotype in neuronal-like cells. These data signify a biological function for SH2D5 in neurobiologic signaling perhaps applicable to learning and memory.

Doctor of Philosophy / Department of Biology / Walter K. Dodds / Much of the North American tallgrass prairie ecosystem has been converted to cropland or urbanized. One threat to the remaining prairie ecosystems, and the streams within, is woody vegetation encroachment. Stream productivity, measured as metabolism, is a fundamental process comprised of gross primary production (GPP) and (CR) community respiration. Understanding GPP and CR is important because these processes are vital to ecosystem function and can be impacted by a change in canopy cover. First, I investigated improvements in existing methods for estimating whole-stream metabolism as estimated from diel patterns of oxygen (O2). I compared measured and modeled O2 and aeration (a physical parameter required for measurement of metabolism) rates to determine if direct measurement of aeration is necessary and the importance of temperature correction of metabolism. Modeling was moderately successful in determining aeration rates, and temperature correction of GPP and CR substantially improved model fits. Second, effects of woody vegetation encroachment on prairie stream function were investigated. Stream metabolism was measured for four years in duplicate reaches with varying canopy cover (closed canopy, naturally open canopy, and vegetation removal reaches). The removal reaches had closed canopy for the first two years and open canopy for the last two years. Canopy cover increased CR rates and had minimal effects on GPP. Third, the same experiment was used to determine the effects of woody vegetation encroachment on prairie stream ecosystem structure and food web interactions. Chlorophyll a and filamentous algal biomass were greater in naturally open and vegetation removal reaches, although the effects were stronger on filamentous algal biomass. As canopy cover decreased, the filamentous algal biomass to chlorophyll ratio increased, indicating a shift in algal community structure. Stable isotope analysis indicated some shift in pathways of nitrogen and carbon flux into the food web related to degree of canopy cover, but overlap in the signature of food sources made distinct food sources difficult to identify. The data indicate that riparian encroachment can influence ecosystem structure and function in prairie streams and restoration to remove woody riparian cover may restore some ecosystem features of naturally open canopy streams.