Inhibitor of differentiation 4 (ID4), a member of the helix-loop-helix family of transcriptional regulators has emerged as a tumor suppressor in prostate cancer (PCa). Recent studies have shown that Id4 is highly expressed in the normal prostate and decreases in prostate cancer due to epigenetic silencing. Id4 knockdown in androgen sensitive LNCaP cells has been shown to lead to castration resistant prostate cancer (CRPC) in vitro and in vivo. Id4-/- mice leads to underdeveloped prostate with PIN like lesions without the loss of Androgen Receptor (AR) expression. In this study we demonstrate that the loss of ID4 expression in PCa cell line LNCaP and DU145 may promote tumorigenesis by promoting stemness. LNCaP cells with stably silenced ID4 ((-)ID4) using retroviral based shRNA and LNCaP transfected with non-specific shRNA were used to perform colony forming assay and prostatosphere formation using matrigel. Expression of cancer stem cell markers was determined using western blotting and immunocytochemistry (ICC). FACS analysis was used to sort stem cells and determine the ID4 expression. Xenograft study was performed on SCID mice using CD133 positive LNCaP cells. LNCaP(-)ID4 and DU145 cells lacking ID4 showed increased holoclone as well as decreased paraclone formation, which are believed to be derived from stem cells and differentiated cells respectively, as compared to non-silencing control in the colony forming assay. There was also an increase in prostatosphere development in the LNCaP (-) ID4 cells indicating that the loss of ID4 is responsible for promoting the LNCaP cells towards cancer stem cells. The results were further validated via western blotting and ICC using known cancer stem cell markers on the holoclones and paraclones formed by these cells. Xenograft study showed that 10,000 cells from CD133 positive LNCaP cells developed tumor on SCID mice. This study reports for the first time that loss of ID4 increases holoclone and prostatosphere formation indicating that Id4 may contribute to promoting stemness in prostate cancer cells.

Plants are in a perpetual evolutionary arms race with a wide range of pathogens. Their sessile nature has led plants to evolve defense mechanisms that can quickly recognize a unique stressor and deploy a resistance tailored for a specific attack. The salicylic acid (SA) mediated defense pathway has been shown to be one of the major defense tactics plants can initiate to defend themselves against microbial pathogens. Following a pathogen attack high levels of methyl salicylate (MeSA) are produced that can be converted to SA by the enzyme salicylic acid binding protein 2 (SABP2). A yeast two-hybrid screening was performed to identify protein interactions with SABP2 to better understand the regulation of the enzyme on a cellular level. SBIP-436 is an interacting protein of tobacco SABP2 which showed high homology to phospholipase D-δ (PLD- δ). With an abundance of stimulators PLD- δ may be a lipid signaling modulator developed to perform various functions in different situations. PLD- δ may be a key player in a lipid signaling cascade in the SA mediated defense pathway. We present a novel Nicotiana tabacum PLD- δ putative gene construct. We demonstrate that the putative PLD- δ is subject to alternative splicing and its expression is differentially modulated under biotic and abiotic stress. Our results indicate that this putative PLD- δ may play a role in the SA mediated defense pathway.

Vibratory communication has evolved in numerous animal groups, including insects, spiders, fishes, mammals, and was recently discovered in veiled chameleons (Chamaeleo calyptratus). I examined the mechanism by which C. calyptratus produce these biotremors. Muscle activity data were gathered during simulated anti-predator responses via electromyography (EMG) with simultaneous recordings of biotremor production using an accelerometer. I correlated EMG data with the accelerometer data to implicate the muscles responsible for the production of the biotremors. Mixed-effect linear regression models described the mechanism, and a model selection framework determined which model fit the data best. I then used an analysis of variance to partition the variance to each variable to determine which muscles were most important in the biotremor producing mechanism. The Mm. sternohyoideus superficialis et profundus, Mm. mandibulohyoideus, and M. levator scapulae were active during the production of biotremors. Mean latency calculations revealed that the M. levator scapulae and Mm. mandibulohyoideus activated prior to the vibration onset, and the Mm. sternohyoideus superficialis et profundus activated after the vibration onset. The M. sternohyoideus superficialis then ceased activity prior to vibration cessation, and the M. sternohyoideus profundus, Mm. mandibulohyoideus, and M. levator scapulae ceased activity after the vibration had ended. The description of the biotremor producing mechanism further supports that C. calyptratus can produce biotremors, possibly for communication.

With advancements in CRISPR-cas9 broadening the potential paths for clinical usage of genetic editing, conversations about genetic editing have grown to outside simply scientific communities and into mainstream conversations. This study focuses specifically on Christian discourse of genetic editing and locates four major tensions for many Christians when they think about genetic editing: beginning of life, Creator-human relationship, imago Dei, and stewardship. With these major concerns in mind, I identify epigenetics, somatic cell genetic editing, and in vivo genetic editing research as important research paths to pursue as they can potentially produce techniques that more Christian individuals would feel comfortable using. I pursue one of these paths and conclude with an experimental proposal for an analysis of in vivo and in vitro CIRSPR-Cas9 efficiency in regards to on- and off-target rates.

To understand the function and origins of sleep, sleep needs to be studied across many different species. Although it is well conserved throughout mammals, 95% of papers are restricted to just three species, Homo sapiens, Mus musculus, and Rattus norvegicus. We aimed to characterize sleep and wake in a Murid rodent Acomys cahirinus in greater detail alongside the well-studied laboratory house mouse (Mus musculus) and wild M. musculus using a well validated, non-invasive, piezoelectric system for sleep and activity monitoring. We confirmed A. cahirinus, M. musculus, and wild M. musculus to be primarily nocturnal, but with clearly distinct behavioral patterns. Specifically, the activity of A. cahirinus sharply increases right at dark onset, which is common in nocturnal species, but surprisingly, decreases sharply just one hour later. Using infra-red camera recordings in single and group cage conditions, we found that A. cahirinus is more active early in the night period than late night period in single and group cages, and this decreased activity in the latter half of the night is much greater compared to M. musculus. In order to better understand these differences in activity, we investigated the sleep architecture of A. cahirinus using electroencephalogram (EEG) recordings. Our data show that A. cahirinus have a few key differences in sleep from M. musculus. A. cahirinus have significantly longer daily sleep periods and exhibit a much higher amount of REM sleep. A. cahirinus are awake at dark onset, but sleep more than M. musculus after the middle of the night. Most strikingly, A. cahirinus do not close their eyes virtually at all while sleeping, day or night. In order to test whether the sleep patterns of A. cahirinus are affected by or responsive to different light input, we set up a light flashing experiment during the daytime. While sleep amount did not change significantly during light flashing, A. cahirinus spent significantly less time in REM compared to baseline. In contrast, M. musculus had no difference in REM sleep percentage. Histological studies showed A. cahirinus have thinner retinal layers, but much thicker corneas than M. musculus. Electroretinography (ERG) results, specifically b-wave amplitudes, are significantly different between these two species. While eye closure and sleep have not been systematically studied across mammals, our observation is clearly a rare behavior. This raises further questions about A. cahirinus sleep architecture, the adaptive value of eyes open sleep to A. cahirius and whether they may have limited visual processing even during normal sleep.

Transient ablation of regulatory T cells has been shown to be effective at hindering tumor growth and metastasis in murine breast cancer model. Based on our lab’s previous work, we have demonstrated that NK cells and CD8+ cytotoxic T cells were not required for the protective effect of Treg cell ablation. However, we also reported that CD4+ helper T cells and IFN-γ were required for the protective effect of Treg cell ablation. Furthermore, we observed that CD11B+ cells responded to Treg ablation therapy by up-regulating target genes of IFN-γ. Therefore, this study aimed to investigate the connection between the myeloid cell compartment and IFN-γ signaling after regulatory T cell ablation therapy. Through a combination of conditional knockout mouse models, cellular fate mapping experiments, adoptive transfers, and co-injection experiments, we demonstrated that tumor-associated macrophages (TAMs), derived from the bone marrow via CCR2/CCL2 axis, were responsible for the therapeutic effect of regulatory T cell ablation. In addition, we determined that IFN-γ signaling was required for the TAMs to mediate the protective phenotype seen after regulatory T cell ablation. Furthermore, based on our findings, we developed a genetic signature based on TAMs from treated or untreated tumors that had a predictive value for patient survival. Thus, our findings indicated a strong connection between IFN-γ release, classical activation of TAMs, and depletion of regulatory T cells, and taken together, our data could offer potential clinical strategies to mimic regulatory T cell ablation.

The low-lying topographic nature of salt marshes makes plants in these communities particularly vulnerable to increased salinity and inundation exposure associated with sea level rise. Both increased salinity and inundation have been cited as major causes of reduced plant performance and survival in marsh and areas fringing marsh. In addition to limitations imposed by physical stress, interspecific interactions have also been shown to mediate the performance and survival of salt marsh and salt marsh fringing species. The Stress Gradient Hypothesis (SGH) postulates that species interactions shift from competitive to facilitative as stress levels increase and predicts that (a) the frequency and intensity of facilitative interactions increase as conditions become more stressful for plants and (b) the strength of competitive interactions increases as abiotic stress levels diminish. The SGH has been rigorously tested to examine how both the frequency and intensity of species interactions change under varying physical stress levels. Studies conducted in salt marsh systems have shown facilitation to be as strong of a driving force as competition in influencing plant performance and survival and have shown that while competition appears to be the pervasive force in the less physically stressful terrestrial zones fringing salt marshes, facilitation influences the performance and survival of species in harsher marsh areas. Under conditions of sea level rise, it remains unclear if the nature of interspecific interactions would shift as stress levels change. This research endeavors to examine the interplay between abiotic stresses and biotic interactions under conditions of increased salinity and inundation exposure. The first study presented here investigated the effects of increased inundation and soil salinity associated with sea level rise on four salt marsh fringing species, and assesses how competition and facilitation impact survival of salt marsh fringing plant survival under these changing conditions. All plant species experienced reduced growth and photosynthetic inhibition below their current distributional positions, both in the presence and absence of neighboring above ground vegetation. The findings also signal a potential shift in the nature of interspecific interactions from competition to facilitation to neutral as plants begin to experience increased salt and inundation exposure. The second study aimed to disentangle the effects of increased soil salinity and increased soil moisture on four salt marsh fringing species, and to examine the effects of plant neighbors. The results showed that fringe plants exposed to increased inundation experienced a two-fold reduction in performance and survival over 750 g pure salt addition, suggesting that inundation may be a more important limiting factor than salinity with rising sea levels. Landward transplants at the forest-fringe margin exposed to lower soil salinity and decreased inundation exhibited a three-fold increase in performance and survival when compared to controls. Neighbor manipulation studies, which consisted of trimming neighboring vegetation to ground level, again suggested that interspecific interactions in salt marsh fringing species may shift from competitive to facilitative with climate-induced sea level rise. Overall, our findings suggest that salt marsh fringing species may not be able to tolerate changing conditions associated with sea level rise and their survival may hinge on their ability to migrate towards higher elevations. The final experiment tested the Stress Gradient Hypothesis and investigated the relative importance of facilitation and competition in a salt marsh system under varying stress levels. This study also ascertained whether salt or inundation exposure is the primary influence on salt marsh plant performance and survival. As in previous studies, our findings suggest that many salt marsh plants don't require, but merely tolerate harsher abiotic conditions. The results showed that plants at higher elevations were depressed by strong competitive pressure from neighboring fringe species while plants at lower elevations benefited from the presence of neighbors. Collectively, the results of these studies indicate that species interactions are an integral driver of plant distribution in salt marsh communities. Furthermore, our findings indicate that changing stress levels may not always result in a shift in the nature of interspecific interactions. These studies have endeavored to show that the interplay between competition and facilitation interacts with physical processes to determine the growth and performance of both fringe and marsh plant species. The paucity of studies examining the roles of species interactions and changing abiotic stress levels on multiple salt marsh and salt marsh fringing species warrants the need for additional research. The responses of salt marsh and salt marsh fringing species to sea level rise can not only serve as very valuable and sensitive indictors of climate change, but will also aid in predicting the future location of the marsh-fringe-forest ecotone, which is predicted to shift inland as sea levels continue to rise.

This work describes the fascinating interplay between two tumor suppressors Prostate apoptosis response-4 (Par-4) and p53. The guardian of the genome, p53, is frequently mutated in human cancers, and may contribute to therapeutic resistance. However, p53 is intact and functional in normal tissues, and we observed that specific activation of p53 in normal fibroblasts could induce apoptosis selectively in p53-deficient cancer cells. This paracrine apoptotic effect was executed by Par-4 secreted in response to p53 activation. Accordingly, activation of p53 in wild-type mice, but not in p53-/- or Par-4-/- mice, caused systemic elevation of Par-4 that induced apoptosis of p53-deficient tumor cells. Mechanistically, p53 induced Par-4 secretion by suppressing the expression of UACA, a binding partner of Par-4, and thereby releasing Par-4 from sequestration by UACA. Thus, normal cells can be empowered by p53 activation to induce Par-4 secretion for inhibition of therapy-resistant tumors. Conversely, our studies have also revealed a definite role for Par-4 in regulating p53 expression. The pro-apoptotic tumor suppressor Par-4 is lost, down-regulated, inactivated or mutated in a number of cancers. Loss of Par-4 is associated with therapeutic resistance and poor disease prognosis, yet the mechanism for resistance is not clearly understood. Using genetically matched cells, we show that Par-4 expression is required for stabilization and function of the tumor suppressor p53, which constitutes the hub of signaling networks controlling important cellular and organismal phenotypes. In particular, the expression of p53 protein and its stabilization in response to genotoxic stress were remarkably attenuated in response to Par-4 loss. Accordingly, Par-4-null or -knockdown cells demonstrated increased resistance to apoptosis induced by genotoxic stress. Par-4 loss resulted in elevated Mdm2 activity, which is known to cause p53 degradation. Our findings suggest that Par-4 stabilizes p53 by inhibiting Akt-mediated phosphorylation of Mdm2 that is known to prevent translocation of Mdm2 into the nucleus for p53 ubiquitination and degradation. These studies identify a novel regulatory relationship between two tumor suppressors and may provide a better understanding of therapeutic resistance in tumors with p53 wild type status.

The global human population is expected to exceed 9 billion individuals by 2050, putting greater strain on the natural resources needed to sustain such a population. To feed this many people, some expect agricultural production will have to double and agrochemical use will have to increase anywhere from two- to five-fold relative to the turn of the century. Although industrial agriculture has provided many benefits to society, it has caused declines in biodiversity, both directly (e.g., through conversion of habitat) and indirectly (e.g., through contamination of adjacent natural habitats). Agricultural activity has also been linked to increased prevalence and intensity of trematode infections in wildlife and humans - directly by increasing available aquatic habitat for the snail intermediate hosts of trematode parasites and indirectly by altering the biological composition of aquatic habitats in ways that increase snail density. While the effects of single agrochemical contaminants on aquatic communities and trematode disease risk have been examined, agrochemical pollution typically occurs as mixtures of multiple chemical types in surface waters and the effects of mixtures on aquatic communities have received less attention. Moreover, given the high number of chemicals approved for agricultural use, the number of potential combinations of agrochemicals renders testing all possible combinations implausible. Thus, there is a critical need to develop better risk assessment tools in the face of this complexity. I developed and tested a theoretical framework that posits that the net effects of agrochemical mixtures on aquatic communities can be predicted by integrating knowledge of each functional group's 1) sensitivity to the chemicals (direct effects), 2) reproductive rates (recovery rates), 3) interaction strength with other functional groups (indirect effects), and 4) links to ecosystem properties. I conducted a freshwater mesocosm experiment to quantify community- and ecosystem-level responses to pairwise mixtures of four major agrochemical types (fertilizer, herbicide, insecticide, and fungicide) and single chemical treatments. The responses of biodiversity and ecosystem properties to agrochemicals alone and in mixtures were indeed predictable. Moreover, these results show that community ecology theory holds promise for predicting the effects of contaminant mixtures and offer recommendations on which types of agrochemicals to apply together and separately to reduce their impacts on aquatic ecosystems. I extended this framework to test if the direct effects of pesticides can be predicted by chemical class and/or mode of action. I performed standard toxicity trials on two invertebrate predators of snails (crayfish and giant water bugs) exposed to six insecticides belonging to two chemical classes (organophosphates and pyrethroids) to determine if environmental risk can be generalized to either insecticide class or insecticide exposure. Survival analyses demonstrated that insecticide class accounted for 55.7% and 91.1% of explained variance in crayfish and water bug survival, respectively. Simulated environmental exposures using US EPA software suggested that organophosphate insecticides present relatively low risk (as defined by the US EPA) to both crayfish and water bugs, while pyrethroid insecticides present consistently high risk to crayfish but not to water bugs, where only λ-cyhalothrin produced consistently high-risk exposure scenarios. Thus, risk to non-target organisms is well predicted by pesticide class. Furthermore, identifying insecticides that pose low risk to aquatic macroarthropods might help meet increased demands for food while mitigating against potential negative effects on ecosystem functions. Because evidence from field data and manipulated experiments demonstrated both top-down and bottom-up effects of agrochemical pollution that increased snail densities and trematode infections in wildlife, I conducted an additional agrochemical mixture experiment with freshwater communities containing the snail hosts of schistosomiasis, which has also been linked to agriculture. As expected, top-down and bottom-up effects of insecticide, herbicide, and fertilizer exposure indirectly increased snail densities, individually and as mixtures. Agrochemical exposure and snail density together accounted for 88% of the variation in the density of infected snails. Thus, agrochemical pollution has great potential to increase human exposure to schistosome parasites, and underscores the importance of identifying low-risk alternative pesticides. A subsequent mesocosm experiment with the same six insecticides used previously in laboratory trials confirmed that insecticide exposure indirectly mediates the densities of snail hosts that can transmit schistosomiasis through the direct effects of insecticides on crayfish mortality. Importantly, crayfish mortality in semi-natural mesocosm trials closely matched mortality from controlled laboratory trials. Thus, standard laboratory toxicity tests can be a useful tool for identifying alternative insecticides that might pose lower environmental risks to important predators that regulate snail densities. Identifying practices or agrochemicals that minimize this risk is critical to sustainably improving human health in schistosome-endemic regions. The theoretical framework presented here demonstrates the feasibility of predicting the effects of contaminant mixtures and highlights consistent effects of major agrochemical types (e.g. fertilizers, insecticides, etc.) on freshwater aquatic community composition. Furthermore, the strong top-down effects of invertebrate snail predators highlight that managing for high snail predator densities in might be a particularly effective strategy for reducing the burden of schistosomiasis in tropical countries.

Advancements in molecular sequencing have revealed unexpected cryptic genetic diversity and contrasting evolutionary histories within genes and between genomes of many organisms; often in disagreement with recognized taxonomy. Incongruent patterns between the mitochondrial and nuclear evolutionary history can have several plausible explanations, but widespread systematic conflict inevitably challenges our conceptions of species boundaries when there is discordance between coevolving and coinherited genomes. It is unknown to what degree mitonuclear conflict drives the process of divergence, or how ubiquitous these patterns are across the tree of life. To understand the evolutionary relevance of intergenomic discordance we must identify the conflicting patterns that exist in natural systems by generating robust estimates of the underlying species history, quantify support for alternative hypotheses of lineage formation, and describe patterns of genetic variation present in robust nuclear genomic datasets. Empirically testing correlations between mitonuclear genomic conflict and reduced gene flow at the organism level will contribute toward a better understanding of lineage boundaries and how intergenomic interactions shape the process of divergence. Mitochondrial introgression has been inferred in many salamander systems with limited perspective from nuclear sequence data. Within dusky salamanders (Desmognathus), these patterns have been observed between morphologically and geographically disparate populations. I sequenced regions throughout the nuclear genome to reconstruct species trees, performed population-level analyses testing concordance between the mitochondrial, nuclear datasets, and nuclear genes with mitochondrial functions with the expectation that coevolutionary interactions among genomes are more likely to manifest in these regions. I also estimated migration rates between populations that may have experienced historical mitochondrial introgression to evaluate phylogeographic patterns. Using these data we definitively reject species models in which genetic boundaries are based solely on mitochondrial data, favoring geographic models instead. Furthermore, analyses soundly reject current taxonomic models based on morphological characteristics, suggesting there is greater lineage diversity than is currently recognized. I also used empirical assays of pre-zygotic reproductive mating behavior within and among populations containing diverse mitochondrial lineages to test metrics of reproductive isolation, and to determine if introgression shapes the evolution of complex traits directly influencing rates of divergence. These results may explain incongruent patterns observed between the mitochondrial and nuclear data as a function of inheritance and population dynamics rather than directly functioning to suppress nuclear gene flow. This research builds upon recent studies suggesting that speciation is a highly complex and often non-bifurcating process in which introgression can have a profound and lasting signature on the nuclear evolutionary history. Mechanisms responsible for divergence with gene flow challenge evolutionary biologists to reevaluate our notions and definitions of species boundaries to accommodate seemingly conflicted genomic patterns within and between genomes.