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This site is home to many hands-on, ready-to-use science lessons and activities, which explore nature and energy. It is aimed at classroom teachers who are more likely to attack one lesson at a time, so the lessons have been rearranged by topic. It includes lesson plans, background knowledge, a materials list, and student handouts. The learning objectives have been tailored to Oregon and Washington curriculum goals. It has many topics: Group Dynamics, Scientific Method, Measurement, Surface tension, Density; Newton's Laws of Motion; States of Matter; Power; Biology, which contains few lessons that familiarize students with hydroponics, microorganisms, and capillary action in plants. Other topics are Bernoulli's Principle; Water, which includes watersheds, ways of testing water quality, water conservation, and the water cycle. Also, topics include Geology, explaining plate tectonics, continental drift, and volcanoes. Ecology explaining types of wildlife, the food web, preservation, plant classification, pollution, recycling, acid rain, and smoking. The site includes Native American philosophies, a summary adapted from the Federation of Saskatchewan Indian Nations Wildlife Curriculum, 1992, that provides an excellent cross-curricular lesson that ties the science to the many issues related to rivers, energy, wildlife, and Native American rights and philosophies. The site contains a glossary of terms that are used throughout these science lessons. Educational levels: Intermediate elementary, Middle school

En este trabajo aportamos información sobre las especies de bivalvos de agua dulce de las Superfamilias Sphaerioidea Deshayes, 1855 y Unionoidea Rafinesque, 1820 en siete yacimientos arqueológicos del sur de Iberia analizados por los autores. Así mismo, se ha reunido toda la información existente en la bibliografía sobre estas especies, aportando datos de otras 33 localidades. En conjunto se han identificado cuatro especies: Pisidium casertanum, Margaritifera margaritifera, Margaritifera auricularia y Potomida littoralis en los sitios estudiados directamente a las que hay que añadir la presencia de Unio mancus, Unio delphinus y Unio tumidiformis, identificadas en colecciones depositadas en distintos museos. Los ejemplares proceden de un segmento cronocultural que abarca desde el Paleolítico Medio hasta la Edad Media. Por otra parte, aportamos información sobre la ecología y biología de las distintas especies, así como de su relación con otras (interespecíficas) dentro del ecosistema., This paper provides information on freshwater bivalve species of the Superfamily Sphaerioidea Deshayes, 1855 and Unionoidea Rafinesque, 1820 from seven archaeological sites in southern Iberia analyzed by the authors. All the information available in the literature on these species has been compiled, providing data from a further thirty-three localities. Overall, four species have been identified: Pisidium casertanum, Margaritifera margaritifera, Margaritifera auricularia, and Potomida littoralis in the sites studied directly, to which must be added the presence of Unio mancus, Unio delphinus and Unio tumidiformis as well as from collections deposited in museums. The specimens come from a chronocultural segment ranging from the Middle Palaeolithic to the Middle Ages. Furthermore, we provide information on the ecology and biology of the different species, as well as their relationship with other (interspecific) species within the ecosystem.

Recent advances in DNA sequencing have revolutionized evolutionary biology by allowing for genome-scale studies of non-model organisms. We can now readily connect genotype with phenotype, i.e., identify the genetic basis of particular traits, a key goal in the study of evolution. In addition, genome-scale sequence analysis has shifted our understanding of the frequency and magnitude of gene flow in nature. Once viewed as important only for its role preventing divergence, the common view now is that in many taxa gene flow occurs among many lineages in the early stages of divergence. My dissertation focuses on gene flow in bearded manakins (genus Manacus), which are notable for their intense tandem courtship display, high degree of reproductive skew among males, and bright male plumage. In western Panama, yellow-collared M. vitellinus and white-collared M. candei interbreed in a narrow hybrid zone. Male vitellinus secondary sexual traits, including the yellow collar, have introgressed roughly 50 km west across the hybrid zone into candei populations and then stalled at the east bank of the Río Changuinola, the region’s largest river. Evidence from studies of male-male interaction and female choice implicate positive sexual selection for yellow collars as a driver of introgression. For thirty years since this situation was first described in detail, several key issues have remained unresolved, including why introgression has not continued across the river and what gene or genes are responsible for yellow coloration. In the first chapter, I reviewed the current state of knowledge of the Manacus hybrid zone system and proposed new hypotheses for some of the patterns exhibited by these populations. In the second chapter, I used reduced-representation genome sequencing to investigate whether reduced gene flow across the Río Changuinola alone can explain stalled trait introgression. I found that, although advantageous plumage traits have not introgressed far beyond the rive

The overall theme of this dissertation is the application of mass spectrometry approaches to investigate infectious diseases. Chapter 1 contains background information regarding host-pathogen interactions, importance of clinical samples, and advances in mass spectrometry sample preparation, database curation, and data analysis techniques that familiarize the reader with the tools used during these studies. The following chapters describe primary author works completed by the author of this dissertation.Chapter 2 describes the discovery and characterization of microproteins produced by Staphylococcus aureus. The microproteins were discovered using a peptidogenomic approach and further validated with in-vitro and in-vivo assays. One of the novel microproteins is chemically and structurally similar to previously described virulence factors but appears to have a distinct mechanism of action. The other novel microprotein drives a dissociation phenotype reminiscent of exfoliative toxins. Elucidating the structural and biophysical mechanisms driving the interplay of these microproteins may provide a deeper understanding of host-pathogen interaction. Chapter 3 describes the application of multi-omics approaches to differentiate two similarly presenting enterococcal bacteremia subtypes and provides a global level view of the host immune and metabolic dysregulation. Using high resolution proteomics and metabolomics profiling of clinical serum samples we define a panel of over twenty biomarkers for further validation while also providing a number of targets of clinical importance.

Group I introns are a class of catalytic RNAs that can perform a self-splicing reaction. This thesis will examine the large-scale conformational changes of three Tetrahymena group I intron (TET) states. A combination of mutational analysis and cryogenic electron microscopy (cryo-EM) was used to determine the dynamics associated with the L9/P5 mutation (∆L9) in which a long-range tertiary contact that affects the structure-function of TET is knocked out. This mutation allowed for the visualization of dynamics that were not seen in the previously published models of the wild-type (wt) construct. There are three different states of TET modeled: an intermediate form in which the intron is connected to the 5` exon, but the 3` exon is spliced; a second step (S2) state where the intron is base pairing to the ligated exons, and finally, a post-second step (post-S2) state in which the intron is fully spliced. Understanding the structural changes during the splicing of ∆L9 TET provides specific insights into the structure-function relationships of group I introns and general mechanisms of RNA catalysis and dynamics. The structural biology methods developed to facilitate these experiments have also expanded the use of cryo-EM as a general tool for studying many different RNAs. Furthermore, by increasing our knowledge of RNA catalysis, we gain a more comprehensive grasp of these essential biological macromolecules and their role in the origins of life on Earth.

Glioblastoma multiforme, a form of malignant glioma, is a central nervous system (CNS) tumor associated with the worst prognosis in both adults and children. Effective treatments for this deadly cancer have continued to be elusive to this day. Several strategies have been employed to better understand malignant gliomas including genomics, cell survival, and cell fate determination. Previously we found that a glial cell determining factor, Nuclear Factor I-A (NFIA), is expressed in gliomas and has functional roles during glioma tumorigenesis. During CNS development, NFIA expression is regulated by distinct chromatin conformations that involve the transcription factors SOX9 and BRN2. The glial cell specific chromatin architecture is also present in glioma and modulation of the architecture has profound effects on tumor growth. Interestingly, the formation of this glial loop is not mediated by SOX9 and BRN2. A candidate factor for mediating formation of this chromatin architecture is the Mediator complex subunit 12 (MED12). Here we test whether MED12 is a component of the SOX9/BRN2complex, affects NFIA expression, and begin to determine what role MED12 plays in glioma tumorigenesis. Using dual gRNAs in a CRISPR/Cas9 system to delete MED12 expression from human glioma cell lines U87 and LN229, we found that MED12 is important for tumor growth, migration, and proliferation.

Antibiotic resistance is a prominent global health challenge as more bacteria are gaining resistance, making antibiotic obsolete to a growing number of strains. To combat the antibiotic resistant microbes, bacteriophage (phage) therapy, using viruses that target bacteria, became a viable option for aiding patients fighting resistant microbes. In performing phage therapy for these patients, antibiotics are often administered with the phages to provide the best chance for the patients’ survival, but these chemical and viral interactions or even synergistic effects have not been thoroughly studied due to the difficulty and inaccessibility of the current liquid assay methods. Development of solid media assays to test for antibiotic and phage interactions on bacterial lawns allows for easier and accessible testing methods for phage-antibiotic synergy (PAS) using materials readily available in clinical labs worldwide, lowering the entry barrier in international communities to further investigate PAS or to consider synergy in phage therapy testing.

High-stress levels have become a large part of physicians' lives in today's medical workforce, increasing only more so since the COVID-19 pandemic. Few efforts have been made to decrease these high-stress levels, leading to not only negative impacts on physicians' physical and mental well-being but also on their ability to care for patients. Prior research has shown that self-compassion and mindfulness are powerful tools in preventing burnout, but there are few resources available that incorporate these ideals effectively and are time-efficient. Through an online application, participants in our study used a digital mindfulness meditation application to train compassion and practice mindfulness. Participants also took part in an interoceptive breath monitoring task recorded pre- and post-mindfulness training. Through this breathing task, we investigated neural activity in core brain networks through electrophysiological source imaging. We discovered that mindfulness practices can increase personality attributes of self-compassion and state-mindfulness with improvements in self-compassion sustained at follow-up, overall leading to better self-management of stressors. Additionally, longer mindfulness training duration was found to be positively correlated with a greater magnitude of improvement in self-compassion across subjects. Neural results indicated plasticity specific to the default mode network (DMN) region with network suppression in the experimental group at post-intervention. Neurobehavioral correlations revealed the extent of DMN suppression related to self-compassion improvements during post- versus pre-intervention. These findings implicate that brief digital mindfulness practices may improve stress management in physicians.

Schwann cells (SCs) are known for maintaining axonal integrity and promoting regeneration of injured axons in the peripheral nervous system (PNS). Studies have investigated different SC components that contribute to normal SC functionality. Low-density lipoprotein receptor-related protein-1, LRP1, is a transmembrane protein that regulates SC phenotypic modulation and is vital to SC survival. Mitochondria, the powerhouses of the cell, are crucial for ATP production and regulation of inflammatory responses, especially in SCs. Mitochondrial function and LRP1 have been linked in hepatocytes, but it is not known how LRP1 modulates SC mitochondria. To investigate the relationship between LRP1 and SC mitochondria, we utilized Transmission Electron Microscopy (TEM) on sciatic nerves of mice containing a conditional knockout of scLRP1 (scLRP1-/-) and their floxed LRP1 littermates (scLRP1+/+). In adult scLRP1-/- mice, we observed an increase in mitochondrial number in myelinated SCs and the axoplasm of associated axons compared to that of scLRP1+/+ mice. When we separated the data by sex, our findings were consistent in male scLRP1-/- mice, yet female scLRP1-/- mice demonstrated no difference in mitochondrial number in myelinated fiber axoplasm. However, in non-myelinated SCs, a decrease in SC mitochondria number was observed in female scLRP1-/- mice. An aging analysis revealed decreased mitochondrial numbers in myelinated SCs of aged scLRP1+/+ mice, however, loss of LRP1 increased mitochondrial numbers, similar to adults. Together, we hypothesize that LRP1 regulates mitochondria in myelinated and non-myelinated SCs. Other factors, such as sex and age, influence the number of SC and axoplasm mitochondria present in peripheral nerves.

Coastal marine wrack acts as a valuable resource for organisms in rocky and sandy intertidal, but the extent of how important it is to organisms that live primarily in terrestrial systems is less understood. In this study we use stable isotope analysis on, Argentine ants (Linepithema humile), to uncover the extent of how important a pulsed resource subsidy such as marine wrack is to organisms that don’t live traditionally coastal lives. Because Argentine ants themselves pose widespread ecological risk as an effective invader which commonly feeds in intertidal beach habitats in southern and central California, we also sought to learn more about when and how Argentine ants act as consumers in intertidal systems. We wanted to see how this introduced ant might seasonally invade intertidal habitats to take advantage of abundant resources provided by coastal marine wrack. We assessed whether novel marine wrack derived resources could act as an attractive food source to ants in their more preferred riparian ranges, how Argentine ant and potential prey abundance changed in response to a different time of year and whether marine wrack was a significant part of the diet of Argentine ants living alongside the coast. Our findings suggest that Argentine ants that live alongside the coast indeed subsidize a significant part of their diet with marine based resources. These findings also show a potential trend in increased Argentine ant coastal foraging when resources inland become more relatively scarce showing the importance of marine wrack to organisms that live further inland a potential repository of food when food becomes relatively scarce elsewhere.

Hematopoietic stem cells (HSCs) are a specialized cell population capable of self-renewal and differentiation into various lineages of the vertebrate blood system. In vertebrates, Notch signaling has been implicated to contribute to hematopoiesis through arterial specification and sclerotome formation. The involvement of several Notch ligands such as Delta-like ligand 4 (dll4), DeltaC (dlc), and Jagged 1a (jag1a) have been identified in regulating hematopoietic stem cell and progenitor cell (HSPC) development and differentiation. Using the zebrafish as a model, this study aimed to elucidate the roles of Notch ligands DeltaA (dla), DeltaB (dlb), and Jagged1b (jag1b) in the context of HSC development, which have not yet been studied extensively. To minimize the concern of transcriptional adaptation in indel mutants, we utilized a CRISPR/Cas9 system to successfully generate dla, dlb, and jag1b knockout mutants for loss-of-function (LOF) studies. Phenotypic analyses suggested that loss of dla alone does not significantly impair HSC development. Interestingly, jag1b mutants and dlb promoter-less mutants did not display a significant reduction of HSCs. In contrast, jag1b and dlb morphants exhibited a notable decrease in HSC numbers, necessitating the use of multiple LOF models to validate our findings. The results from this detailed approach will establish the foundation for further research to unravel the precise mechanisms governing hematopoiesis. This advancement will bring us one step closer to translating these in vivo findings to the engineering of HSCs in vitro, a currently unattainable feat.

Marine bacteria are responsible for a significant portion of decomposition and recycling of the organic matter produced by primary production. Moreover, these bacteria can impact the distribution of organic matter within the water column by driving aggregation and sinking of various particles in a pathway known as the biological carbon pump. The process of aggregation can be driven by bacteria which are able to stick to one another as well as to particles. While bacteria within the ocean experience a nutrient depleted environment with occasional encounters of nutrient hotspots, we know little about how this may impact their aggregative behaviors. After examining eleven strains derived from nine species of marine bacteria cultured in minimal medium with twenty-nine distinct carbon sources, distinct patterns in aggregation and growth behaviors of these bacteria have emerged. We observed many cases in which cell-cell aggregation can be triggered during growth by the presence of certain dissolved nutrients. Each strain has a unique combination of nutrients which support growth or aggregation. We categorize the strains into three types of responses: constitutive-aggregator, selective-aggregator, and non-aggregators. Constitutive and selective aggregators appear to be metabolic generalists, while non-aggregators show a preference for growth on acids. In a few cases aggregation occurred without bacterial growth. Certain carbon sources had a bias towards triggering aggregation in strains which grew with them, while others demonstrated an even split between growth with and without aggregation. The findings indicate a trend in aggregation behaviors which appears to be nutrient and strain specific.

The Remote Sensing and Signaling Theory predicts inter-organ and inter-organismal small-molecule communication mediated by a network of “drug” transporters. Organic Anion Transporter 1 (OAT1), SLC22A6, is a multi-specific transporter localized to the proximal tubule of the kidney and serves as a member of this sensing network. Here we demonstrate OAT1 in mice is an important regulator of systemic levels of several gut-derived compounds. Genetic deletion of OAT1 leads to elevation of 40 gut-derived compounds in the serum. Following 16S and whole genome sequencing of the gut bacteria we find that loss of OAT1 leads to compositional shifts in the microbiome, favoring members of Prevotellaceae and Lactobacillus. Functional pathway analysis of the gut-microbiome shows enrichment of long chain fatty acid synthesis, tyrosine and tryptophan degradation, which are all pathways associated with OAT1 function. We find that functional enrichment in the gut bacteria explain serum metabolomic changes of gut-derived compounds. These results demonstrate remote communication between host and gut via small molecule communication mediated by OAT1. We believe other drug-transporters handling gut-derived compounds may also have a similar effect on microbiome composition.

Fall-related injuries, stemming from dizziness and imbalance caused by dysfunction of gravity receptors in the vestibular system, are the leading cause of traumatic death in senior citizens. Yet, little is known about the genetic architecture of vestibular function variation and how aging affects the vestibular system. Thus, we initiated the first complete genome-wide association studies (GWAS) of mouse vestibular functional variation using the hybrid mouse diversity panel (HMDP); balance beam and vestibular evoked potentials (VsEP) data from up to 84 strains of young-aged mice were used to identify candidate genes associated with vestibular dysfunction. We found significant differences in both beam times and VsEP data between strains. While our preliminary GWAS yielded no clear gene candidates, our final GWAS with 84 strains yielded potential novel gene candidates that still need to be verified with more strains’ data. Future research will include further GWAS with not only more mice strains’ (up to 100) but also old-aged mice from all strains utilized. Once clear gene candidates are determined, researchers will need to investigate whether their expression and functions support their involvement in vestibular function. Only then can scientists further elucidate the genetic mechanisms underlying vestibular dysfunction and develop pharmaceutical compounds to target the genes responsible for this disease.

The deep sea, once considered homogeneous and devoid of life, has undergone a transformative shift in understanding since the 19th-century HMS Challenger expedition, with advancements in technology contributing to our growing knowledge of this vast and biodiverse habitat, which comprises 96% of habitable space on Earth and remains largely unexplored. Within this ecosystem, annelids, particularly those inhabiting chemosynthetic environments, play a significant ecological role. Despite the recognized diversity within the order Phyllodocida, which encompasses families such as Siboglinidae, Dorvilleidae, Polynoidae, Serpulidae, and Phyllodocidae, elucidating their intricate relationships has proven challenging due to the scarcity of comprehensive data. Notably, the family Lacydoniidae, found within the order Phyllodocida, stands out. The genus Lacydonia exhibits an extensive bathymetric distribution, occupying a range of deep-sea habitats. However, our understanding of the phylogenetic relationships within Lacydonia as well as aspects such as diet, life cycle, and internal anatomy, remains limited. In this study, we present an updated phylogeny of the genus Lacydonia and describe seven novel species based on meticulous morphological examinations and comprehensive molecular sequence analyses. Specimens were collected from methane seeps in the Eastern Pacific Ocean and Caribbean Sea, resulting from recent research expeditions. By elucidating the taxonomy and evolutionary relationships within Lacydonia, our research provides valuable insights into the remarkable diversity and complex evolutionary history of this enigmatic annelid genus, underscoring the significance of deep-sea exploration for unraveling hidden biodiversity and shedding light on the intricate ecological dynamics of these unique ecosystems.

Cuvier’s beaked whales are a deep-diving cetacean species known to forage in the submarine canyons of the Southern California Bight. Although this species is a top predator in deep-sea ecosystems, little is known about their social or foraging strategies because of their extreme diving behavior. In this analysis, time-difference-of-arrival (TDOA) localization is used to track the position of Cuvier’s beaked whales from echolocation clicks recorded on seafloor-mounted hydrophone arrays. This approach yielded 162 final tracks with estimated swim speeds of diving Cuvier’s beaked whales at one acoustic monitoring site from July 2021 to October 2022. The number of individual whales captured on a single track ranged from 1 to 7, with a mean of 2.24. Tracks including four or more individuals were captured most often during the day, with notable peaks in July 2021, February 2022, and July 2022. Three distinct diving behaviors were observed, differentiated by minimum depth, change in depth, and lateral movement: initial descent dive segments (1.657 ± 0.441 m/s), consistent trajectory dive segments (0.913 ± 0.596 m/s), and variable trajectory segments (1.298 ± 0.438 m/s). This long-term monitoring effort and TDOA localization approach collected data from Cuvier’s beaked whale foraging dives to reveal spatial use, group size, and diving behavior trends at our acoustic monitoring site. This extensive tracking dataset gives valuable insight into the social and foraging behavior at depth of Cuvier’s beaked whales offshore Southern California.

Transfer students typically navigate nontraditional educational pathways in higher education, which often confer institutional barriers to entering science, technology, engineering, and mathematics (STEM) at the university level. The transfer pathway disproportionately consists of underrepresented minority groups, who encounter difficulties in STEM due to intersections of marginalizing experiences across race, class, gender, sexuality, and other identities, given STEM’s historic nature of being built upon white, middle class, masculine, and heteronormative values. Despite their high representation in undergraduate populations and unique identities as university students, transfer students continue to be an understudied population. We interviewed 29 transfer students pursuing STEM degrees who described memorable moments that positively or negatively influenced different dimensions of their STEM identities and identified patterns and variations among their experiences. Our findings highlight three major findings. First, the achievement of good grades emerged as a prominent identity-shaping experience, but students did not report feeling acknowledged or becoming more interested in STEM as a result. Furthermore, students who experience analogous events can interpret them both positively and negatively. Finally, students valued acknowledgment from individuals outside of STEM, such as friends or family, differently than acknowledgment from those in STEM, such as professors or peers. Overall, our findings can be informative to policy makers, faculty, staff, and educational institutions on how to best support transfer students in forming strong STEM identities.

Understanding the diversity, phylogenetic relationships, and evolutionary transitions within Acrocirridae, a clade of polychaete worms, is crucial for unraveling the complexities of benthic-pelagic evolutionary transitions in marine ecosystems. In this study, we describe four new species, erect a new genus to accommodate two of these species, and provide an updated phylogeny for Acrocirridae. Additionally, we assemble complete mitogenomes and present the first mitogenome phylogeny for the broader Cirratuliformia, offering valuable insights into the genetic characteristics and evolutionary history of benthic and pelagic species. The new species described in this study significantly contribute to the known diversity within Acrocirridae.Furthermore, the establishment of a new genus highlights the importance of taxonomic revisions in capturing the true evolutionary relationships and diversification patterns within this clade. Our updated phylogeny for Acrocirridae incorporates both morphological and molecular data, providing a comprehensive understanding of relationships. By assembling complete mitogenomes, we unravel the genetic composition and mitochondrial gene arrangement of key taxa within Cirratuliformia. This dataset serves as a valuable resource for future studies investigating the genomic features and evolutionary dynamics within this group of polychaetes. Importantly, our mitogenome-based phylogeny reveals variation in gene arrangements and codon usage. Overall, this study provides a comprehensive investigation of the diversity, phylogeny, and evolutionary transitions within Acrocirridae. Our findings enhance our understanding of the genetic and ecological factors driving benthic-pelagic transitions in polychaete evolution and provides a framework for future research.

Microalgae are single-celled, free-living aquatic organisms responsible for half of global primary productivity that are used for biofuels as a renewable source of energy. Physiological rates of all ectotherms such as respiration and photosynthesis are affected by temperature differently. Experiments show that algal growth often increases with temperature up to an optimum, ranging from 10°C to 30°C. However, these experiments often maintained constant day and night temperatures. Few studies have focused on the effects of nightly drops in temperature on algal growth. High nightly temperatures may increase respiration and therefore reduce algal growth. To investigate how day and night temperatures affect algal growth, I grew Nannochloropsis oceanica under six different temperature regimes consisting of day and nighttime temperatures of 5°C, 15°C and 25°C. Algae exposed to a daytime temperature of 25°C and nighttime temp of 15°C and the 15°C treatment of a 15°C daytime and nighttime temp had the greatest growth of all. Constant temperatures of 25°C or 5°C during the day and night resulted in slower growth. Therefore, a nightly drop in temperature has a positive effect on the growth curve of the algae if the night temperature is 15°C or greater. This indicates that optimal temperatures for growth of Nannochloropsis are lower at night than during the day. Therefore, to predict the effects of climate warming on primary productivity and identify optimal growth conditions for algae used for natural products, we must consider the effects of different day and night temperatures.

Proteins perform their roles in the cell through interactions -- with DNA, with other proteins, or with other molecular substrates. Protein-protein interactions are especially important during the regulation of transcription, as many transcription factors come together to bind to each other and to DNA to form a transcriptional complex. Mutations can disrupt these interactions, leading to abnormal gene regulation, expression, and possibly disease. Here, we analyze the 3D structure of the core binding factor (CBF) complex to create a library of mutations for its subunits CBFB and RUNX1. We use ScalablE fUnctional Screening by Sequencing (SEUSS) to study the effects of the RUNX1 library in myelogenous leukemia K562 cells using single cell RNA sequencing. We then further investigate the effects of selected RUNX1 mutations on chromatin accessibility and gene expression using bulk ATAC and RNA sequencing. We find that our library design was able to select mutations that perturbed interfaces of the complex, resulting in loss of function-like and hypomorphic phenotypes with effects on distinct cellular pathways. Our bulk ATAC and RNA sequencing analysis reveals enrichment of RUNX1 binding in accessible DNA regions associated with regulation of differentially expressed genes. Our work shows the potential of targeting the binding interfaces of a protein to gain insight into how disruption of its function may cause downstream effects in the cell.

Tumor-associated macrophages (TAMs) create an immunosuppressive tumor microenvironment (TME) that will promote tumor progression. The goal of this project is to understand whether the pattern of TAMs in a tumor is determined by the tumor cells or by the host tissue environment. Knowing how the phenotype of TAMs is determined will help us better target TAMs to improve cancer patient’s outcome. Moreover, another goal of this project is to understand whether the TAMs that develop in a primary tumor have the same phenotype as those that develop in subsequent metastases. Comparing gene expression profiles between TAMs in different tissues can provide future directions to improve cancer therapies so that we can better target TAMs in order to effectively eradicate primary and metastatic tumors. Using flow cytometry to examine like tumors growing in different tissues, we found that subcutaneous, orthotopic, and intravenous LLC tumor models all have similar TAM profiles that are distinct from genetically engineered CC10Cre KrasG12D p53-/- spontaneous lung tumor models. In addition, using single-cell sequencing to investigate the PyMT primary mammary gland tumors and metastatic lung tumor the and CC10Cre KrasG12D p53-/- spontaneous lung tumors, we can conclude that the TAM profile of the primary tumor and subsequent metastasized tumor have similar TAM profiles. As a result, we can conclude that the TAM profile is dependent on the tumor cells themselves instead of the microenvironment.

Vibrio cholerae is the bacteria responsible for causing cholera, a diarrheal disease. Cholera affects around 1.3 to 4 million people globally and impacts countries that lack access to sanitary drinking water such as Bangladesh[1]. Given the burden of V. cholerae induced infection, we investigated the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin, a secreted toxin. Cholera is typically considered a non-inflammatory disease, and this may be explained by the actions of the MARTX toxin [3]. In this present study, we sought to determine if the MARTX toxin modulated host inflammation to limit bacterial clearance and confer a fitness advantage. Additionally, we wanted to determine whether the actions of MARTX toxin are dependent on V. cholerae’s cholera toxin (CT). We found that genetic abrogation of the MARTX toxin was detrimental to the fitness of the pathogen within the gut of the host. At 6 hours post infection we observed a significant reduction in intestinal colonization by the MARTX toxin mutant, rtxA relative to the wild-type (WT) strain. RT-qPCR data reveal that mice infected with the WT and the mice infected with rtxA have non-significant different expression of intestinal inflammation genes, CXCL1, CXCL2 and Nos-2. However, we report that expression of these inflammation markers was greater in rtxA relative to WT after 6 hours and thus, could explain the observed bacterial enumeration data. These results help elucidate the interaction between bacterial toxins and their impact on host innate immunity, which can be further developed into a more effective cholera vaccine or provide therapeutic targets.

Caymanostellidae is a group of rarely collected and morphologically unique sea stars that have been exclusively discovered on wood falls in the deep sea. There are currently 3 genera and 7 species within the family, with a wide distribution across the Atlantic, Pacific and Indian Oceans and a depth range between ~414 and 6780 m. Two new species, Caymanostella scrippscognaticausa n. sp. and Caymanostella n. sp., are here identified and described from specimens collected from wood falls in multiple localities across the Pacific Margin of Costa Rica. These records represent the first evidence of caymanostellids occurring on wood falls found at methane seeps and expand the known geographical distribution of caymanostellids. This study also includes the first descriptions of early-stage juvenile caymanostellid sea stars with several collected from experimentally deployed wood, which shows that traits previously considered useful for diagnosis might represent intra-specific ontogenetic variability, with important consequences for caymanostellid taxonomy.

Organisms endure environmental and internal perturbations throughout their lifetime. Environmental cues are integrated by the sensory neuron endings. Within the nervous system, the conserved MAPKKK DLKs play key roles in response to a variety of cellular stress, physical injury and pathological conditions. Previous studies have shown that regulation of DLK protein expression and activity in axonal compartments is pivotal in neuronal development, maintenance and stress response. However, it remains unclear whether DLKs may function in the sensory neuron endings. Moreover, experimental evidence that examines expression of DLKs is mostly based on overexpression approaches; how endogenously expressed DLKs are regulated remains elusive. The goal of my dissertation research focuses on identifying novel regulators of DLK-1 in C. elegans model, starting with a visual genetic screen for mutations altering the expression of endogenously GFP-tagged DLK-1. The first chapter of this dissertation summarizes the previously characterized cellular pathways that regulate DLKs level or activity in neurons. In the second chapter, I investigate the roles of a major group of mutations that affect the intraflagellar transport (IFT) and show that IFT actively controls DLK-1 levels in sensory cilia. Upon IFT disruption, DLK-1 triggers downstream signaling, which in turn represses dlk-1 transcription in sensory neurons. This IFT-dependent DLK-1 signaling protects cilia from degeneration, revealing a surveillance mechanism at the sensory endings of the organism. In the third chapter, I report the design of the genetic screen and phenotypes of additional mutants that specifically alter DLK-1 level in ciliated sensory neurons and are involved in multiple ciliary processes including anterograde IFT transport, BBSome, MAP kinase and olfactory signaling, and repress DLK-1 expression in a cell type-specific manner. Taken together, my dissertation establishes the methodology to uncover and also iden

Phosphoproteomic analysis is a relatively recently devised method used to quantify a phosphoproteome and identify specific amino acid residues on proteins that are phosphorylated in order to further analyze their role in cellular processes. Our lab studies the role of the gene protein tyrosine phosphatase non-receptor Type 2 (PTPN2) in the intestinal epithelium. This gene codes for T-Cell Protein Tyrosine Phosphatase (TCPTP). Loss-of-function mutations in the PTPN2 gene locus are associated with an increased risk of developing several auto-inflammatory diseases such as Inflammatory Bowel Disease (IBD), Celiac Disease, Rheumatoid Arthritis and Type 1 Diabetes. Considering that our candidate gene codes for a phosphatase, we can hypothesize that this will have effect on the quantity and identity of phosphopeptides present. We hoped to find out how changes in the phosphoproteome in PTPN2 knockout (KO) mice compared to wild type (WT) mice. Surprisingly, we found no significant changes in the quantity of phosphoproteins and their residues, no significant changes in phosphorylation of TCPTP substrates, and no significant changes in the pathways identified as a result of the phosphorylated proteins between the two groups.

The pervasive presence of guanine quadruplex (G4) structures within regulatory regions of the genome has spurred intense research efforts to understand their roles in modulating cellular pathways. Numerous investigations affirm that both DNA and RNA G4s actively partake in pivotal biological processes, including DNA replication, transcription, RNA metabolism, translation, and telomere maintenance. However, further investigation is needed to understand the molecular mechanisms through which these secondary nucleic acid structures modulate biological processes. This dissertation focuses on utilizing bioinformatic analyses of publicly accessible datasets to unravel multifaceted roles of G4 structures in histone modifications, three-dimensional chromatin configurations, RNA metabolism, and telomere maintenance.In chapter 2, we revealed the G4 co-localization pattern with transcription factors and constructed an interaction network of candidate G4-interacting proteins. Moreover, we explored the interplay between G4 structures and histone marks, unveiling G4 structures as active transcription marks and potential regulators for histone modifications. In chapter 3, we conducted an intuitive overlapping analysis of previously published RNAPII ChIA-PET and BG4 ChIP-seq data, and our work revealed a strong positive correlation between RNAPII-linked DNA loops and G4 structures in chromatin. In conjunction with HiChIP-seq and RNA-seq, we unveiled vital role of DNA G4 in RNAPII-associated DNA looping and transcription regulation. In chapter 4, we employed a bioinformatic approach based on the analysis of overlap between RNA G4 (rG4)-seq analysis and eCLIP-seq datasets generated from the ENCODE project. We identified a large number of candidate rG4-binding proteins. We validated that one of these proteins, G3BP1, is a direct binder of rG4 structures, and documented a rG4-dependent function in regulating mRNA stabilities and translation efficiencies. In chapter 5, I proposed a nove

The epidermal microbiome of sharks is hypothesized to be consistent across individuals of the same species, regardless of biogeographical influence, suggesting that there are mechanisms that are recruiting and maintain the microbial community. To explore this hypothesis, I conducted an extensive investigation into the epidermal microbiomes of the leopard shark (Triakis semifasciata) across environments and an acute temperature shift. I also tested phylosymbiotic trends between epidermal microbiomes and respective benthic shark hosts. Initially, my research involved the analysis of T. semifasciata's microbiomes in captive, semi-captive, and wild settings. The associated microbiomes demonstrated comparable taxonomic community structure and maintained biodiversity irrespective of the captivity status of the hosts, therefore suggesting the host maintains a consistent microbiome even when born into captivity, and when the host experiences a change in habitat. For my next research chapter, I explored the influence of their evolutionary lineage and physical traits on their skin microbiomes by investigating three benthic shark species, horn (Heterodontus francisci), leopard (Triakis semifasciata), and swell shark (Cephaloscyllium ventriosum). This phase underscored the strong correlation between the diversity of skin microbiomes and the host's phylogenetic lineage, while the relationship with inter-denticle distance was less robust, affecting individual species within the microbiome. Finally, I examined the impact of thermal shifts on T. semifasciata's epidermal microbiomes to investigate the ability of the microbiome to tolerate the effects of global warming. The microbiomes underwent significant alterations of community compositions and functional pathway profiles following temperature changes, suggesting complex adaptive responses. Despite these compositional shifts, the dispersion of these communities remained consistent across temperatures, suggesting degree of resilie

Defensive behaviors in the presence of predator cues are typical innate behaviors in animals. Although predator signals are best detected with the summation of various sensory modalities in nature, olfactory-specific exposure to a predator specimen is sufficient to yield defensive responses in nocturnal prey animals such as mice. Animals display fixed patterns of defensive behaviors such as freezing, flight, and risk assessment in response to olfactory predator cues. These defensive responses varied between mice of varying age and sex. We found that mice freeze robustly when exposed to freshly collected cat saliva, but with lessened intensity when exposed to saliva that aged several hours. The underlying molecular and neural mechanisms for such shift in behavioral decisions are still not well understood. This freezing behavior was observed only when mice had direct contact with the cat odor and abolished in mice lacking functional Trpc2, indicating that the accessory olfactory system is necessary for this behavioral output. Interestingly, neural activation in some brain areas were with behavioral outputs in the fresh saliva-exposed mice but not in the ones exposed to old saliva. These results suggest that fresh cat saliva contains chemical cues that signal imminence of predator threat, and the signal is processed through a specific population of neurons in the accessory olfactory system. In this study, we reveal the candidate proteins in cat saliva that induces freezing and the mouse sensory receptor that detects them. Our research aims to identify the neural mechanism of imminence of threat detection and shed light on the neural basis underlying the defensive behavioral decision making in prey animals.

Cancer is the second leading cause of global mortality. Accumulating evidence strongly suggests that aberrant alterations in signaling cascades can lead to uncontrolled cell growth, driving the progression of this disease. While the promotion of cancer cell growth by hyperglycemia has been extensively studied, the comprehensive mechanism underlying this relationship remains incompletely elucidated. In the current study, we present findings that high glucose levels (HG) indeed bolster DNA synthesis and cell cycle progression, fostering tumor cell growth, as revealed by genome-wide analyses. Moreover, our investigation identified E2F family as the central transcription factor responsible for the adaptive response induced by HG. The use of the pan-E2F inhibitor HLM006474 and shRNA-mediated E2F1 depletion, effectively counteracted HG-triggered DNA synthesis and cell growth. Furthermore, we establish that HG amplifies Rb1 phosphorylation, directly contributing to the activation of E2F1. Inhibition of Rb1 phosphorylation using the CDK2/4/6 inhibitor compound PF-3600 substantially mitigates the E2F1-mediated transcriptional activation of downstream DNA replication genes. Notably, among the E2F1 target genes induced by HG, RRM2 plays an essential role in the nucleotide synthesis by generating essential dNTPs required for DNA replication. Our investigation reveals that HG promotes intracellular dNTP levels in an E2F1-RRM2-dependent manner, closely associated with increased DNA synthesis and subsequent cancer cell growth. The RNR inhibitor Triapine effectively impedes RRM2-mediated elevation of intracellular dATP and dGTP, and DNA synthesis. To gain more insights into the metabolic alterations occurring in HG-treated cancer cells, we carried out the untargeted metabolomic profiling. This analysis not only reaffirms the activation of glycolysis in HG-treated cells, as evidenced by the elevated levels of lactic acid, but also illustrates noteworthy enhancements in the levels of

Nairoviruses, tick-borne viruses from the Bunyaviridae family, pose persistent and significant threats to human health. While the most notable is Crimean-Congo hemorrhagic fever virus (CCHFV), which possesses a fatality rate pushing 30%, other nairoviruses are establishing themselves in our viromes including Pacific Coast tick nairovirus (PCTN) and Songling virus (SGLV). To thwart host antiviral immune response, specifically reversing the post-translational modifications of interferon-stimulated gene product 15 (ISG15) after infection, nairoviruses encode for and utilize an ovarian tumor domain protease (OTU) capable of deISGylation. The fact that ISG15 structure and sequence is hardly conserved between species, coupled with previous studies demonstrating diverse OTU preferences for certain ISG15s, pinpoints these two as key subjects of disease prevention due to possible variations in interaction. To gain a broader understanding of nairovirus OTU-ISG15 interface and identify possible host carriers, a high-throughput method of obtaining binding kinetic data was developed which employed bio-layer interferometry (BLI). ~72 interactions between vOTUs and ISG15 were examined, which revealed significant differences for nairovirus OTU binding preferences. CCHF and PCTN vOTUs put on robust displays of binding, while SGLV vOTU showcased more specificity for which ISG15s it bound to. These insights serve to be progress and contributions towards building a larger biosurveillance tool ultimately unveiling pivotal animal reservoirs of nairoviruses.

Before the 1960s, phylogenetic data sets consisted only of morphological data. Now, molecular data is the most common data type used to create phylogenetic trees for extant taxa. However, morphological data is still required to include extinct taxa in phylogenetic analyses because molecular data cannot be obtained from most fossils and museum specimens. The accuracy of fossil placement in phylogenetics has been questioned due to morphological data being more prone to convergence, which can confuse hypothesized evolutionary relationships. For example, in extant placental mammals, morphological and molecular data support several clades that are incompatible with one another. Additionally, molecular and morphological data disagree on the timeline of the radiation of the placental mammals. The fossil record and morphological phylogenies mainly support an origin of placental mammals after the Cretaceous-Paleogene (K-Pg) boundary while relaxed molecular clocks support an origin within the Cretaceous. In this dissertation, I expand our understanding of these issues in several ways: 1) exploring the issue of sample size in morphological data sets by using the largest morphological matrix available for placental mammals, 2) utilizing ancestral state reconstructions to determine if hypothetical predicted ancestors, as a proxy for undiscovered fossil taxa, increase the accuracy of morphological phylogenetics, 3) exploring if ordered morphological characters change hypothesized phylogenetic relationships, and 4) testing the hypothesis that supports a Paleogene origin of the placental mammals by adding a new fossil taxon into the largest morphological data set available for placental mammals. I concluded the following: 1) increased sample size did not alleviate the problems of homoplasy in this morphological matrix, 2) the use of hypothetical predicted ancestors resulted in a greater number of monophyletic pseudoextinct orders, but the interordinal positions of these groups were

The kinetochore has been viewed as playing a critical role in many aspects of chromosome dynamics as the cell progresses through mitosis. This includes chromosome congression and alignment on the metaphase plate and sister chromosome separation and segregation to the spindle poles. Defects in kinetochore function result in mitotic errors and aneuploidy. Thus, the finding that chromosome fragments lacking a kinetochore (acentrics) are capable of normal congression, sister separation and segregation is fascinating. My thesis takes advantage of a Drosophila system in which acentrics can be efficiently generated and their behavior analyzed through live fluorescence analysis. In Chapter 1, we review how cells transmit chromosomes through mitosis without canonical kinetochore-microtubule interactions. Decades of research and a collection of studies reveal that microtubule-based mechanisms and DNA-based “tethers” connecting the acentric to the main chromosome mass are responsible for proper segregation of acentrics. In Chapter 2, we discuss the mechanisms by which acentric sister chromatids remain paired and eventually separate from one another during anaphase. Taking advantage of Drosophila transgenic for the I-CreI endonuclease, we efficiently generate broken chromosome fragments lacking centromeres, thereby lacking kinetochores. By using a genetic screen and live analysis, we identify proteins responsible for the separation of acentric chromosomes. We conclude DNA catenations are responsible for keeping acentric sisters paired and Topoisomerase II activity and microtubule plus-end pushing forces are needed to resolve these catenations and separate the acentrics. In Chapter 3, we highlight the remarkable ability of acentrics to congress to the metaphase plate despite the absence of kinetochore-microtubule interactions. Through mutational and live cell analysis, we define the forces acting on chromosome arms and the role of the kinetochore in chromosome congression. Utili

Drug discovery is a costly and time-consuming process. It has four major stages: early drug discovery, pre-clinical phase, clinical trials, and regulatory approval. Early drug discovery is the steps of target validation, hit identification (hit ID), hit-to-lead, and lead optimization for new chemical entities (NCEs) and therapeutic targets. Basic research identifies and validates disease-relevant targets. Hit ID is the process of testing a group of compounds against a target protein. Thermal shift assay (TSA) can detect slight changes in protein melting point and stability, and, therefore, be used to screen for hit ID. X-ray crystallography studies can determine the binding between target protein and hits. In vitro enzymatic assays can confirm the inhibition of hits and measure IC50 (inhibitory concentration 50%). Hit-to-lead can be done by ‘SAR by catalog’, in which the features of the hits are used to identify commercially available compounds. During the hit-to-lead stage, hits are developed to improve the binding affinity to move along as a final drug.Structural-based drug discovery (SBDD) is one of the early-stage drug discovery approaches and has been wildly used in academic and industrial settings since the 1990s. SBDD uses small molecules that follow Lipinski's Rule of Five that ensures that the small molecules can be administered orally once they become drugs. Fragment-based lead discovery (FBLD) evolved from SBDD. Fragments follow the Rule of Three that helps reduce off-target effects and for a broad search of chemical diversity using smaller libraries in the low thousands of compounds. In silico artificial intelligence-computer-aided drug discovery (AI-CADD) is used to identify hits and develop leads using computer tools and the simulation of human intelligence processed by machines. Post-translational modifications (PTMs) are the last stage in protein biosynthesis, being either reversible or irreversible chemical events occurring after protein translation

Manzanitas (Arctostaphylos, Ericaceae) are the most taxonomically diverse genus of woody plants in western North America, with an epicenter of diversity in the California Floristic Province (CFP). For more than a century, botanists have recognized the high diversity of the genus, and have worked to come to a satisfactory understanding of how best to circumscribe taxa in the genus. Additionally, there has been much curiosity about how the genus became so diverse in the CFP. However, addressing these questions using the tools of investigation that have been historically available has proven difficult. I undertook three studies, utilizing updated and modern methods, aimed at addressing these questions. In the first chapter, I present the results of a phylogeographic study using reduced representation genome sequence data to reconstruct the biogeographic history of three widespread species in western North America (A. patula, A. pungens, and A. pringlei). For each species, I tested whether they originated in the CFP, or outside of the CFP. I found a likely origin for A. patula to be within the CFP, a likely origin for A. pungens outside of the CFP, and I found an unclear geographic origin for A. pringlei with regard to the CFP - leaving the question of the origins of diversity in the genus unanswered. In the second chapter, I present the results of a morphological study aimed at testing distinguishability and distinctiveness of manzanita taxa. I found that taxa can largely be distinguished, but also found overlap among many taxa in overall morphological variation. Together, this indicates that distinguishability is accounted for by specific, taxonomically prescribed, diagnostic traits. I also found evidence that morphological variation in the genus is not composed of naturally discrete, separated units, but rather, is relatively continuous.In the third chapter, I present the results of a study investigating genetic differentiation among subspecies of a widespread specie

I studied correlated responses to artificial selection for high voluntary wheel-running behavior. Chapter 1 used meta-analysis to summarize comparisons of four replicate High Runner (HR) lines with four non-selected Control (C) lines. Effect sizes from 34 studies demonstrate that HR mice have evolved larger hearts and smaller bodies, and that plateaus in effect sizes for both traits coincide with selection limits for wheel running. Chapter 2 tested whether HR mice would have a higher maximal rate of oxygen consumption during forced exercise (VO2max), a higher basal metabolic rate (BMR), and increases in the size of relevant organs. Although HR mice ran ~3-fold more revolutions/day and had higher VO2max, they did not have a statistically higher BMR nor differ in relative organ masses (heart, lung, liver, kidney, spleen, brain, calf muscle) or hematocrit. Thus, a large evolutionary increase in activity level has not required proportionally large changes in underlying morphological or physiological traits. Chapter 3 examined effects of maternal exercise on maternal care and offspring body composition and activity behavior. HR females ran more than C prior to breeding and before parturition, but not after. When housed with wheels, HR females had reduced maternal care compared to C during days 1-10 after parturition. HR females with wheels had fewer litters survive to weaning than those without wheels, but the opposite occurred for C females. For both HR and C dams with wheels, surviving offspring had delayed development as indicated by eye opening and lean mass at weaning. However, maternal wheel access did not alter offspring adult body mass, activity behavior or reproductive success. Overall, the evolution of high voluntary exercise did not require large changes in underlying physiology (e.g., VO2max, BMR), but morphological traits (e.g., body mass, relative heart mass) may have imposed constraints on the evolution of activity levels in these mice. Moreover, HR mother

The immune system plays a vital role in protecting our bodies from a wide range of pathogens. As we continue to identify new diseases that challenge our immune system, im- munotherapy has gained prominence as a potential medical solution. It utilizes or enhances the body’s own immune system to detect and destroy cancer cells. Immunotherapy presents advantages over traditional treatments, although its successful application demands a more extensive understanding of the immune system. While progress has been made, our un- derstanding of the immune system, specifically the innate immune component, is still not comprehensive.Some key questions that pique my curiosity include: How do innate immune cells distinguish between self and non-self targets? When transplanted to a different host, how do these cells adapt to their new surroundings? Can this process be elucidated through equations or quantitative models? I have always been deeply interested in exploring these questions. In this thesis, we apply statistical methods to tackle these issues. We develop a quantitative model to elucidate the regulatory mechanisms governing the activity of one type of innate immune cell, namely natural killer cells. We develop a model to simulate the interactions between natural killer (NK) cells and their target cells, illuminating how NK cells learn to identify unhealthy signals from their environment. We apply our model across a range of experimental scenarios, showcasing the algorithm that we have designed to mirror specific experimental settings and delving into the relationship between model and experimental parameters. Further, we provide evidence for the efficacy of our model by demonstrating that it is possible to identify rational values for model parameters that yield accurate estimations of the experimental data. This effectively validates our approach. Progressing from this foundation, we present a high-dimensional extension of our model, contributing insights into immune pro

PURPOSE: Increased awareness of the distinct tumor biology for adolescents and young adults (AYAs) with cancer has led to improvement in outcomes for this population. However, in cholangiocarcinoma (CCA), a paucity of data exist on the AYA population. To our knowledge, we present the largest study to date on AYA disease biology, treatment patterns, and survival outcomes in CCA. METHODS: A multi-institutional cohort of patients with CCA diagnosed with intrahepatic cholangiocarcinoma (ICC) or extrahepatic cholangiocarcinoma (ECC) was used for analysis. Retrospective chart review was conducted on patients who were 50 years old and younger (young; n = 124) and older than 50 years (older; n = 723). RESULTS: Among 1,039 patients screened, 847 patients met eligibility (72% ICC, 28% ECC). Young patients had a larger median tumor size at resection compared with older patients (4.2 v 3.6 cm; P = .048), more commonly had N1 disease (65% v 43%; P = .040), and were more likely to receive adjuvant therapy (odds ratio, 4.0; 95% CI, 1.64 to 9.74). Tumors of young patients were more likely to harbor an FGFR2 fusion, BRAF mutation, or ATM mutation (P < .05 for each). Young patients were more likely to receive palliative systemic therapy (96% v 69%; P < .001), targeted therapy (23% v 8%; P < .001), and treatment on a clinical trial (31% v 19%; P = .004). Among patients who presented with advanced disease, young patients had a higher median overall survival compared with their older counterparts (17.7 v 13.5 months; 95% CI, 12.6 to 22.6 v 11.4 to 14.8; P = .049). CONCLUSION: Young patients with CCA had more advanced disease at resection, more commonly received both adjuvant and palliative therapies, and demonstrated improved survival compared with older patients. Given the low clinical trial enrollment and poor outcomes among some AYA cancer populations, data to the contrary in CCA are highly encouraging.

Our understanding of the molecular mechanisms governing plant traits and development is largely restricted to a few model taxa which represent a small fraction of plant biodiversity. As such, these model systems lack the variation necessary to investigate many traits, especially adaptations to extreme environmental conditions. A suite of traits is common among high alpine plants which allow them to mitigate environmental stressors, including dwarfism and the production of stomata on both sides of a leaf (amphistomaty). The blue limestone columbine (Aquilegia jonesii) exemplifies both of these traits. The ability to cross this species with a non-alpine relative, along with an annotated reference genome, gives us an opportunity to fill a significant gap in our understanding of these adaptations at the genetic level. In addition, A. jonesii is unique among columbine species by having lost an otherwise novel floral organ, staminodes, which allows us to dissect aspects of their development. In this dissertation, I present my research investigating the genetic basis and phenotypic nuance of these three exceptional traits in A. jonesii using Quantitative Trait Locus (QTL) analysis. My results demonstrate that staminode loss is not complete in A. jonesii, and the genetic basis of its partial loss is complex. Dwarfism in A. jonesii is similarly complex, likely involving multiple hormonal pathways, in contrast to model taxa where dwarfism is most often caused by a single gene. In contrast, amphistomaty is controlled primarily by a single major-effect QTL. I will show that this QTL is very likely due to cis-regulatory variation at the ortholog of a master stomatal development gene (AqSPEECHLESS). These studies together demonstrate the varying levels of complexity in the mechanisms by which nature selects for adaptive traits.

Non-viral gene editing strategies have been developed for basic science and therapeutic applications. Major gains in efficiency have come from the optimization of nuclease activity and delivery, with comparatively little focus on the role of the homology-directed repair template (HDRT) in this process. Here, we will describe a strategy to boost the efficiency of gene editing via homology-directed repair (HDR) by covalently modifying the template DNA molecule with interstrand crosslinks. Crosslinked templates (xHDRTs) increase Cas9-mediated editing efficiencies by up to fivefold in K562, HEK293T, U2OS, iPS, and primary T cells. Increased editing from xHDRTs is driven by events on the template molecule and requires ataxia telangiectasia and Rad3-related (ATR) kinase and components of the Fanconi anemia (FA) pathway.

Global climate change will likely impose selective pressures on biodiversity to not only increase limits for environmental stress, but the phenotypic plasticity of those limits. While we know a great deal about natural selection on performance traits such as upper thermal tolerance, our knowledge of the evolution of plastic responses to the environment is limited. Whether and when the plasticity of performance traits like thermal tolerance and body size incur fitness costs and benefits is unclear. Opposing theories and a lack of empirical research have resulted in a poor understanding of genetic variation underpinning adaptive phenotypic plasticity. Here I report the results of my dissertation research, which aimed to study ecological and evolutionary processes shaping (i) fitness effects of plasticity in thermal tolerance, (ii) intraspecific variation in plastic and evolved responses to warming, and (iii) the heritability of plastic responses to the environment. Studying populations and families of two coastal marine invertebrates, the intertidal copepod Tigriopus californicus and the purple sea urchin Strongylocentrotus purpuratus, I demonstrated potential for plastic responses to global change to evolve via natural selection evidenced by fitness effects and heritability of phenotypic plasticity. However, this evolution may be constrained by factors including fitness tradeoffs between performance and its plasticity, low genetic variation for maladaptive plasticity, and countergradient variation in performance reducing genetic variation for plasticity.

Giant unilamellar vesicles (GUVs) are spherical structures composed of an aqueous compartment enclosed by a bilayer membrane. They are often seen as a simplified analog of a cell membrane and can be utilized as minimal cell models for studying cellular systems due to their cell-like sizes and capacity to mediate membrane interactions. A paper-based diffusive loading technique termed, OSM-PAPYRUS, is shown to assemble GUVs in physiologically relevant salt solutions with gentle loading of proteins. Characterization of this loading process reveals cell-like variation of encapsulated protein concentrations and a gamma distribution often cited for protein distributions in the cell. This ability to mimic cellular variability in vitro reveals potential in bridging the gap between in vitro and in vivo experimentation. The highly controlled environment of in vitro experiments can be combined with cell-like volumes, phospholipid bilayer, and cellular variation in GUVs. A practical application is explored, encapsulating the post-translation oscillator (PTO) of the cyanobacteria circadian clock system which shows membrane interactions in vivo. The results showed that cellular variation and membrane binding significantly hampers the fidelity of the clock, in contrast to bulk experiments where concentration did not matter once a critical concentration is met. An increase in concentration to cellular levels helps counteract the effect of variation. Modeling the clock reaction using expected distributions and variation of encapsulated proteins, corroborated with the hypothesis that intercellular variation and membrane binding were responsible for trends in the experimental data. The experimental data and model showed that the PTO by itself was not capable of achieving the near 100% fidelity observed in the native cyanobacteria, instead, other cellular components, like SasA and CikA or transcriptional-translational feedback loop (TTFL) would be necessary to achieve in vivo clock fidelity. The GUV model demonstrated advantages over in vivo studies, particularly in the isolation of the PTO, which allowed for the determination that large period variations seen in vivo cannot be produced by the PTO even under cell-like variability and volumes. This demonstrates the ability of GUV in vitro models to obtain context on behaviors not appreciated by either previous bulk in vitro or in vivo studies.

tRNA genes play a vital role in protein synthesis and are essential components of the translational machinery in all living organisms. Bioinformatic predictions of tRNA functional signatures (features determining tRNA gene function), their evolutionary divergence, and the contributing factors to their evolution is crucial for understanding the accuracy of recognition and interaction-determining features of tRNA molecules with other translational system components. This knowledge has implications for therapeutic interventions and understanding disease mechanisms. However, the species-specific mechanisms of tRNA gene family evolution remain poorly understood, likely due to the small size and high similarity of tRNA genes within the same family. Traditional orthology assignment methods are ineffective, and accurate results require high-quality complete genome assemblies. In addition, the coevolution of tRNA genes is often neglected, and there has been no systematic study of this coevolution that takes into account their spatial organization within the genome. This dissertation provides an analytical tool called tRNA Structure Function Mapper (tSFM) that predicts functional signatures of tRNA genes, calculates their evolutionary divergence with improved efficiency and accuracy of their estimation of statistical significance. The dissertation describes the application of tSFM to bioinformatically prioritize and target specific tRNA-protein interactions as divergent between humans and parasitic Trypanosomes for drug discovery. The custom tRNA gene annotation pipeline undertaken for this work led to more complete and accurate tRNA gene annotations in Trypanosome genomes and the first description of widely conserved tRNA gene arrays in those parasitic genomes. Subsequent work expands our custom annotation and analysis pipelines to tRNA gene arrays in recently published long-read genome assemblies in Drosophila. Furthermore, this research presents a systematic approach to the assignment of orthology of tRNA gene arrays among 89 drosophilid species, reveals the impact of tRNA array composition on the evolution of tRNA genes, and describes the common evolutionary mechanisms acting on multi-copy tRNA gene families.

Lab-on-a-chip (LOC) technologies have revolutionized the fields of quantitative biology, tissue engineering, diagnostics, etc. However, the LOC implementations in the studies of small model organisms remain relatively low and are constrained in a small research community due to the complexity and high cost in the device development. For zebrafish research in particular, similar zebrafish-on-a-chip (ZOC) designs have been repeatedly used and some of the needs in zebrafish studies are far from fulfilled. The goal for this dissertation is to develop novel ZOCs and an overall zebrafish testing system to assist and accelerate zebrafish studies.In this dissertation, I reported the development of novel ZOC systems for automated zebrafish embryo positioning and flowthrough assays. The ZOCs are fabricated by a rapid and inexpensive 3D LCD stereolithography assisted prototyping method which is suitable for the labs with limited budgets. A simple computational fluid dynamic based analytical model was developed and validated to estimate ZOC’s embryo trapping potential as well as to guide future ZOC designs. Moreover, the operational conditions in the ZOC were evaluated and optimized via both simulations and experiments. To examine the ZOC’s feasibility in zebrafish studies, I showed the zebrafish embryo can be cultured in ZOC’s flowthrough environment and the embryonic development as well as embryo behavior can be affected by alternating the fluidic conditions. To fill the gap for the ZOC-based whole mount zebrafish immunohistochemistry, the whole mount zebrafish Caspase-3 antibody staining procedure was streamlined and accelerated using the ZOC. At last, the versatile ZOC design can be easily modified into a concentration gradient generator and was used for high resolution embryo caffeine dose-response screening. I foresee the developments, and applications of ZOCs will be accelerated in zebrafish research due to the growing needs for the laboratory automation as well as the increasing accessibility of LOC and 3D printing technologies. In addition, the continued input efforts from both academia and industry will facilitate the emergence of highly integrated zebrafish testing systems for fully automated zebrafish procedures.

This is an intermediate guide to weight lifting., https://www.librarystack.org/my-fitness-program-syllabus-2023/?ref=unknown

“I felt an urge to look at—and possess—the portraits of my brain that had captivated the attention of so many doctors and served the shareholders of so many drug companies. I wanted to see myself as they had seen me.” An essay on living as a medical specimen and reckoning with the booming market for images of diseased brains., https://www.librarystack.org/regions-of-interest/?ref=unknown

“What keeps you occupied? I do my illness, as you can see.” A story about biologics and telepresence, reconfiguring the self and refining corporate behavior. Written in collaboration with Goldin+Senneby; illustrations by Goldin+Senneby with Johan Hjerpe., https://www.librarystack.org/flare-up/?ref=unknown

A series of blockchain-based artworks that offer views of the Seller’s diseased brain and investments in the drugs targeting such images. With a smart contract by Alberto Granzotto., https://www.librarystack.org/spot-price/?ref=unknown

The mechanical properties of the extracellular matrix dictate tissue behaviour. In epithelial tissues, laminin is a very abundant extracellular matrix component and a key supporting element. Here we show that laminin hinders the mechanoresponses of breast epithelial cells by shielding the nucleus from mechanical deformation. Coating substrates with laminin-111—unlike fibronectin or collagen I—impairs cell response to substrate rigidity and YAP nuclear localization. Blocking the laminin-specific integrin ß4 increases nuclear YAP ratios in a rigidity-dependent manner without affecting the cell forces or focal adhesions. By combining mechanical perturbations and mathematical modelling, we show that ß4 integrins establish a mechanical linkage between the substrate and keratin cytoskeleton, which stiffens the network and shields the nucleus from actomyosin-mediated mechanical deformation. In turn, this affects the nuclear YAP mechanoresponses, chromatin methylation and cell invasion in three dimensions. Our results demonstrate a mechanism by which tissues can regulate their sensitivity to mechanical signals., We thank A. Farré and the other members of IMPETUX OPTICS, S.L., for their help and expertise in the design and implementation of the optical tweezers experiments; R. Sunyer for help and advice with the microprinting experiments; S. Usieto, A. Menéndez, N. Castro, M. Purciolas and W. Haaksma for providing technical support; L. Rosetti and S. Saloustros for providing data analysis tools; and J. de Rooij, A. L. Le Roux, L. Faure, A. Labernadie, R. Oria and J. Abenza, as well as all the members of the groups of P.R.-C. and X.T. for helpful discussion. Finally, we thank G. Wiche, A. Sonnenberg and N. Montserrat for providing plasmids, antibodies or cell lines used for this work. We acknowledge funding from the Spanish Ministry of Science and Innovation (PID2021-128635NB-I00 MCIN/AEI/10.13039/501100011033 and ‘ERDF-EU A way of making Europe’ to X.T., PID2019-110949GB-I00 to M.A. and PID2019-110298GB-I00 to P.R.-C.), the European Commission (H2020-FETPROACT-01-2016-731957), the European Research Council (Adv-883739 to X.T.; CoG-681434 to M.A.; StG- 851055 to A.E.-A.), the Generalitat de Catalunya (2017-SGR-1602 to X.T. and P.R.-C.; 2017-SGR-1278 to M.A. and P.S.) and European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 797621 to M.G.-G. The prize ‘ICREA Academia’ for excellence in research to M.A. and P.R.-C., Fundació la Marató de TV3 (201936-30-31 and 201903-30-31-32), and ‘la Caixa’ Foundation (LCF/PR/HR20/52400004 and ID 100010434 under agreement LCF/PR/HR20/52400004). IBEC and CIMNE are recipients of a Severo Ochoa Award of Excellence from MINCIN. A.E.M.B. was supported by a Sir Henry Wellcome Fellowship (210887/Z/18/Z). A.E.-A. receives funding from the Francis Crick Institute, which receives its core funding from the Cancer Research UK (CC2214), the UK Medical Research Council (CC2214) and the Wellcome Trust (CC2214)., Peer Reviewed, Postprint (published version)