Cell migration is vital to numerous biological processes, and is misregulated in pathological processes, such as cancer metastasis. Cell migration is a tightly, spatiotemporally regulated process that requires the coordination of many molecular components. Because adaptor proteins can serve as integrators of various cellular events, they are being increasingly studied as regulators of cell migration. The adaptor protein containing a pleckstrin-homology (PH) domain, phosphotyrosine binding (PTB) domain, and leucine zipper motif 1 (APPL1) is a 709-amino acid endosomal protein that is known to play a role in cell proliferation and survival as well as endosomal trafficking and signaling. However, its function in modulating cell migration is currently poorly understood. In this project, we show that APPL1 expression alters cell migration speeds, but not directionality. APPL1 mediates migration dependent on engagement with Fibronectin and surface level expression of α5β1 integrin, the major Fibronectin receptor. APPL1 alters trafficking patterns of α5β1 integrin, but not αVβ3 integrin, another Fibronectin receptor. Rab5-induced integrin internalization, Rac activation and signaling, and cell migration are hindered by APPL1 interaction with Rab5. These data indicate a role for APPL1 in regulating migration by modulating integrin trafficking as well as Rac activity.

No description available.

The initial steps of vision - the transduction and encoding of physical light stimuli into neural signals - occur in the retina, a multi-layered sheet of neurons that lines the back of the eye. Retinal dopamine (DA) acts as the principal modulatory neurotransmitter, whose signaling is driven by both light-sensitive and intrinsic circadian mechanisms. DA critically shapes retinal circuits and alters the processing of visual signals by initiating slow and sustained changes in the physiology of retinal neurons and synapses. Here, to achieve a mechanistic understanding of how DA reconfigures retinal circuits according to background illumination, we employed various mouse models, electrophysiological, psychophysical, and pharmacological techniques to answer three fundamental questions: (1) how does dopamine transporter (DAT)-mediated volume transmission contribute to retinal physiology? (2) how are the retinal dopaminergic system and overall visual function shaped by circadian perinatal photoperiod? and lastly, (3) how do DA receptors direct differential signaling pathways and influence ganglion cell function? We found that DAT-dependent anomalous dopamine efflux results in elevated retinal light-adapted responses in male mice, but not in female mice. In addition, we showed that developmental photoperiod imprints retinal function. Short, winter-like light cycles during retinal development and maturation have enduring detrimental effects on photopic retinal light responses and visual contrast sensitivity in mice, likely through developmental programming of retinal DA. Lastly, we uncovered differential effects mediated by D1 and D4 receptors on a specific functional type of retinal ganglion cells, ON-center sustained ganglion cells. These results have therefore provided a mechanistic framework for DAâs role in modulating the multiple dimensions of light-adapted vision. Overall our study supports the indispensable role of DA in high-resolution, light-adapted vision. We have elucidated key underlying cellular and network mechanisms by which DA signals shape retinal function and vision. Dynamic DA signaling is regulated by neurotransmitter reuptake, synthesis shaped by developmental light cycles, and segregated actions of receptors on the output of the retina.

Over evolutionary time, mammalian genomes have accumulated a large number of retrotransposons, making up about half of the genome in any given species. These retrotransposons are typically repressed by epigenetic mechanisms, one of the main ones being DNA methylation. It is well known that improper DNA methylation of retrotransposons can have unwanted consequences on nearby gene expression, and hypomethylation of retrotransposons has been frequently observed in various cancers. Nevertheless, it has been notoriously difficult to study retrotransposon loci individually due to the highly repetitive nature of their sequences. To address this issue, we have developed a novel protocol termed HT-TREBS (High-Throughput Targeted Repeat Element Bisulfite Sequencing), which is designed to survey the DNA methylation levels of a large number of interspersed repeat elements on an individual-locus basis. Here we have used this technique on two mammalian retrotransposon families: IAP LTRs in mouse, and the AluYa5 and AluYb8 subfamilies of Alu elements in humans. According to the results, the majority of retrotransposons (~95%) are heavily methylated in mammalian somatic cells. Moreover, only a fraction of loci appear to be sensitive to cell state in both species. Approximately 25% IAP LTRs showed hypomethylation in mouse ES cells, and ~50% in mouse cancer (Neuro2A) cells. In humans, however, the level of response to tumorigenesis in the breast was much more constrained, with only 1% AluYb8 elements being expected to show hypomethylation at an early stage. Interestingly, our results also revealed extensive (up to 10-fold) inter-individual variation in the level of DNA methylation of AluYa5 and AluYb8 elements in humans, similar to the variation previously noted regarding IAP LTRs in mice. Overall, these results highlight the dynamic nature of DNA methylation at retrotransposons, which further leads us to speculate its unique contribution to mammalian evolution and disease susceptibility by allowing for epigenetic variation within one species. Furthermore, it also suggests the potential utility of some of these elements which are sensitive to cell state, but show less variability between individuals, to be used as epigenetic biomarkers for tracking disease progression.

The southeastern United States has a dynamic geologic history. Mountain range uplift, sea level fluctuations, and river basin evolution have shaped species geographic distributions in the region. My dissertation objectively identifies amphibian species with highly fragmented distributions in the Southeast (Chapter 2) and examines the phylogeographic history of one of those species, Plethodon serratus. This salamander species has a widely disjunct range across five regions: the Appalachians, the Ozarks, the Ouachitas, and two allopatric sites in Louisiana. Analyses of mitochondrial and nuclear loci (Chapter 3) showed that P. serratus is comprised of multiple genetic lineages, and the five regions are not reciprocally monophyletic. Instead, there was evidence of historical gene flow between one of the Louisiana sites and the Ouachitas. Niche and paleodistribution modeling results suggested that P. serratus expanded from the Appalachians during the cooler Last Glacial Maximum and has since been restricted to its current disjunct distribution by a warming climate. These data reject the universal applicability of the glacial contraction model to temperate taxa and reiterate the importance of considering the natural history of individual species. Using a large next-generation sequencing data set of ultraconserved elements (Chapter 4), I estimated a fully resolved species tree that confirmed the non-sister relationship of the two Louisiana populations. I successfully generated this data set without modifying the established laboratory protocols; this is an important note, as large genomes can present challenges for next-generation sequencing. I found that P. serratus has a genome size of 21 pg (Chapter 5) and that this species, along with its closest relatives, has undergone genome size reduction since diverging from the ancestor of Plethodon. This dissertation demonstrates that established methods of next-generation sequencing can be used in studies of large-genome salamander phylogeography. This is especially important, as many salamander populations are declining in the face of habitat loss and climate change. Plethodon serratus is only one of many amphibian species in the Southeast with fragmented ranges. Future research on the biogeography of these organisms will be crucial to conservation.

Amacrine cells are a class of retinal interneurons that process the visual signal in the inner retina. Several subtypes of amacrine cells express nitric oxide synthase and produce nitric oxide (NO), making NO a possible regulator of amacrine cell function.  My dissertation research tests the hypothesis that NO alters amacrine cell GABAergic synaptic output.  To investigate this, I made whole-cell voltage clamp recordings of cultured chick amacrine cells receiving synaptic input from other amacrine cells and Ca²⁺ imaging of amacrine cell dendrites, which can be presynaptic.  I find that NO-dependent increases in GABAergic spontaneous postsynaptic current (sPSC) frequency are independent of soluble guanylate cyclase and action potentials. Removal of extracellular Ca²⁺ and buffering of cytosolic Ca²⁺ both inhibit the response to NO. In Ca²⁺ imaging experiments, I confirm that NO increases dendritic Ca²⁺ by activating a Ca²⁺ influx pathway. Neither NO-dependent dendritic Ca²⁺ elevation nor increase in sPSC frequency are dependent upon Ca²⁺ release from stores. NO also enhances evoked GABAergic responses, and because voltage-gated Ca²⁺ channel function is not altered by NO, the enhanced evoked release is likely due to the combination of voltage-dependent Ca²⁺ influx and the voltage-independent, NO-dependent Ca²⁺ influx. Insight into the identity of the Ca²⁺ channel involved in the NO response was provided by characteristics unique to the transient receptor potential canonical (TRPC) channel subunits 4 and 5: the NO-dependent increase in sPSC frequency was dependent on downstream activity of PLC, blocked by 2 mM La³⁺ and enhanced by 10 µM La³⁺. The TRPC inhibitor ML204, which preferentially blocks TRPC4, had no effect on the NO response at 10 µM, but 20 µM ML204 blocked the NO response. The TRPC inhibitor clemizole, which preferentially blocks TRPC5, blocked NO-dependent dendritic Ca²⁺ elevations and the increase in sPSC frequency. Genetic knockdown of TRPC5 in cultured amacrine cells using the CRISPR/Cas9 system confirms that TRPC5 mediates NO-dependent dendritic Ca²⁺ elevations and the increase in sPSC frequency. These results suggest that NO-dependent activation of TRPC5 at amacrine cell presynaptic sites will enhance vesicular GABA release and increase inhibition onto postsynaptic cells.

The evolution of feathers was a key innovation for birds. The many functions that feathers provide have allowed birds to diversify into myriad habitats and styles of life. However, feathers are lightweight and break down, and must be replaced regularly in a process known as molt. The patterns and timing with which birds molt varies within and between species, and these different strategies are likely a product of the different ways birds use their feathers and the uneven distribution of resources available for molt in time and space. So far, no studies have attempted to place these strategies into an evolutionary framework. In my dissertation research, I used phylogenetic comparative analyses combined with molt, phenotype, ecology, and spatial data to investigate how and why birds have evolved such a diverse array of molt strategies. I studied how molt affects the evolution of flightlessness and seasonal dichromatism in birds, as well as latitudinal gradients in the pace of feather growth. I found that molt strategies have profound effects on the evolutionary trajectories of birds. My major findings are that simultaneous wing molt facilitates and perhaps accelerated the evolution of flightlessness in birds, and that a secondary molt that evolved to replace worn feathers can serve as a preadaptation for phenotype alteration. I also found feather growth rate increases with latitude within bird species. Overall, feathers are integrally important to the biology of birds, and it makes sense that the patterns and strategies that birds use to replace their feathers are guided by and influence their evolutionary histories. This dissertation research provides some of the first evidence into the details of how molt strategies interact with other aspects of avian evolution.

The first part of this work induced T-cell lymphoma in mice by employing a breeding scheme involving mouse strains expressing the KrasG12D oncoprotein and mice expressing cyclic recombinase from the mouse mammary tumor virus promoter. Imprinted domains were then systematically surveyed for DNA methylation changes during tumor progression using combined bisulfite restriction analysis and next-generation-bisulfite-sequencing. Hyper-or hypomethylation was detected at the imprinting control regions (ICRs) of the Dlk1, Peg10, Peg3, Grb10 and Gnas domains. These DNA methylation changes at ICRs were more prevalent and consistent than those observed at the promoter regions of well-known tumor suppressors, such as Mgmt, Fhit and Mlh1. Thus, the changes observed at these imprinted domains are the outcome of isolated incidents affecting DNA methylation settings. Within imprinted domains, DNA methylation changes tend to be restricted to ICRs as nearby somatic differentially methylated regions and promoter regions experience no change. Furthermore, detailed analyses revealed that small cis-regulatory elements within ICRs tend to be resistant to DNA methylation changes, suggesting potential protection by unknown trans-factors. The second part of this work further characterized the epigenetic response of imprinted domains during carcinogenesis. This study compared the stability of DNA methylation at a variety if cis-regulatory elements within imprinted domains in two fundamentally different mouse tumors, benign and malignant. The data suggest that imprinted domains remain quite stable in benign processes, but are highly susceptible to epigenetic alterations in infiltrative lesions. The preservation of DNA methylation within imprinted domains in benign tumors throughout their duration suggests that imprinted genes are not involved with the initiation of carcinogenesis or the growth of tumors. However, the frequent detection of DNA methylation changes at imprinting control regions in infiltrative lesions suggest that imprinted genes are associated with tumor cells that have gained the ability to defy tissue boundaries. Overall, this study demonstrates that imprinted domains are targeted for DNA hypermethylation when benign tumor cells transition to malignant. Thus, monitoring DNA methylation within imprinted domains may be useful in evaluating the progression of neoplasms.

Although bioaerosols in the lower altitudes of the atmosphere have been extensively studied, little is known about the distribution and nature of microorganisms at altitudes above the Earths surface. To examine the vertical distribution of microorganisms in the atmosphere, a helium balloon payload system was developed to sample bioaerosols at stratospheric altitudes of up to 38 km, where temperature, air pressure, relative humidity, and ultraviolet-C (UV-C) radiation conditions are similar to the surface of Mars. Bioaerosol concentrations between 3 and 29 km were similar to each other, ~three-fold lower than those in the convective boundary layer (CBL; 0-3 km; 1 x 106 cells m-3), and decreased to 8 x 104 cells m-3 between 35 and 38 km. From these data we estimate that Earths atmosphere contains at least 100-fold more microorganisms (~1024 cells) than previously thought (Burrows et al., 2009a), ~40% of which may exist of bioaerosols in the stratosphere. Isolates recovered from 6 to 29 km were evaluated for the tolerance to desiccation and UV-C in order to better constrain their ability to survive transport in the stratosphere. Based on the survival data, UV-C was determined to be the limiting factor for microbial survival in the atmosphere. Populations of L6-1, the most UV-C tolerant isolate examined, at the low end of stratospheric cell concentrations (3.8 x 105 cells m-3) would be inactivated in 43 days at 20 km, a value that decrease to ~2 hours at 30 km, above the shielding effects of the ozone layer. Based on these data, we hypothesize the ozone layer may represent the upper limit of the biosphere.

<p> The work in this dissertation represents an attempt to investigate multiple temporal and spatial scales of inquiry relating to the variability of marine resources throughout the Gulf of Mexico large marine ecosystem (Gulf LME). This effort was undertaken over two spatial extents within the greater Gulf LME using two different time-series of fisheries monitoring data. Case studies demonstrating simple frameworks and best practices are presented with the aim of aiding researchers seeking to reduce errors and biases in scientific decision making. Two of the studies focused on three years of groundfish survey data collected across the West Florida Shelf (WFS), an ecosystem that occupies the eastern portion of the Gulf LME and which spans the entire latitudinal extent of the state of Florida. A third study was related to the entire area covered by the Gulf LME, and explored a 30-year dataset containing over 100 long-term monitoring time-series of indicators representing (1) fisheries resource status and structure, (2) human use patterns and resource extractions, and (3) large- and small-scale environmental and climatological characteristics. Finally, a fourth project involved testing the reliability of a popular new clustering algorithm in ecology using data simulation techniques.</p><p> The work in Chapter Two, focused on the WFS, describes a quantitatively defensible technique to define daytime and nighttime groundfish assemblages, based on the nautical twilight starting and ending times at a sampling station. It also describes the differences between these two unique diel communities, the indicator species that comprise them, and environmental drivers that organize them at daily and inter-annual time scales. Finally, the differential responses in the diel, and inter-annual communities were used to provide evidence for a large-scale event that began to show an environmental signal in 2010 and subsided in 2011 and beyond. The event was manifested in the organization of the benthic fishes beginning weakly in 2010, peaking in 2011, and fully dissipating by 2012. The biotic effects of the event appeared to disproportionately affect the nighttime assemblage of fishes sampled on the WFS.</p><p> Chapter Three explores the same WFS ecosystem, using the same fisheries-independent dataset, but also includes explicit modeling of the spatial variability captured by the sampling program undertaking the annual monitoring effort. The results also provided evidence of a disturbance that largely affected the nighttime fish community, and which was operating at spatial scales of variability that were larger than the extent of the shelf system itself. Like the previous study, the timing of this event is coincident with the 2010 <i>Deepwater Horizon</i> oil spill, the subsequent sub-marine dispersal of pollutants, and the cessation of spillage. Furthermore, the spatial models uncovered the influence of known spatial-abiotic gradients within the Gulf LME related to (1) depth, (2) temperature, and (3) salinity on the organization of daytime groundfish communities. Finally, the models developed also described which non-spatially structured abiotic variables were important to the observed beta-diversity. The ultimate results were the decomposition of the biotic response, within years and divided by diel classification, into the (1) pure-spatial, (2) pure-abiotic, (3) spatial-abiotic, and (4) unexplained fractions of variation. </p><p> Chapter Five employs a clustering technique to identify regime states that relies on hypothesis testing and the use of resemblance profiles as decision criteria. This clustering method avoids some of the arbitrary nature of common clustering solutions seen in ecology, however, it had never been rigorously subjected to numerical data simulation studies. Therefore, a formal investigation of the functional limits of the clustering method was undertaken prior to its use on real fisheries monitoring data, and is presented in Chapter Four. The results of this study are a set of recommendations for researchers seeking to utilize the new method, and the advice is applied in a case study in Chapter Five.</p><p> Chapter Five presents the ecosystem-level fisheries indicator selection heuristic (EL-FISH) framework for examining long-term time-series data based on ecological monitoring for resources management. The focus of this study is the Gulf LME, encompassing the period of 1980-2011, and it specifically sought to determine to what extent the natural and anthropogenic induced environmental variability, including fishing extractions, affected the structure, function, and status of marine fisheries resources. The methods encompassed by EL-FISH, and the resulting ecosystem model that accounted for ~73% of the variability in biotic resources, allowed for (1) the identification and description of three fisheries resource regime state phase shifts in time, (2) the determination of the effects of fishing and environmental pressures on resources, and (3) providing context and evidence for trade-offs to be considered by managers and stakeholders when addressing fisheries management concerns. The EL-FISH method is fully transferrable and readily adapts to any set of continuous monitoring data. (Abstract shortened by ProQuest.)</p><p>