Search results

RSS feed for this result

1,284 results

Book
1 online resource.
At the restriction point (R), mammalian cells irreversibly commit to divide. R has been viewed as a point in G1 after growth factor signaling initiates a positive feedback loop of Cdk activity. However, recent studies cast doubt on this model by claiming R occurs prior to positive feedback activation in G1 or even before completion of the previous cell cycle. Here we reconcile these results and show that whereas many commonly used cell lines do not exhibit a G1 R, primary fibroblasts have a G1 R that is defined by a precise Cdk activity threshold and the activation of cell cycle-dependent transcription. A simple threshold model, based solely on Cdk activity, predicted with more than 95% accuracy whether individual cells had passed R. That a single measurement accurately predicted cell fate shows that the state of complex regulatory networks can be classified by a few critical protein activities.
Book
1 online resource.
Cellular state is an old concept. However, scientists have only recently begun the systematic manipulation of cells to characterize and understand the functions of myriad states. As biotechnology advances enable innovative and large-scale measurements on cellular components, new biostatistical tools are required to make sense of the increased data size and complexity, which in turn augment our knowledge of cellular states. In this dissertation, I discuss my contributions to the study of cellular states from the theory and computation angles: 1) modeling and inference of regulatory gene networks with systems of nonlinear deterministic and stochastic differential equations; 2) partition-assisted clustering analysis of high-dimensional single-cell mass cytometry data; and 3) the alignment of subpopulations of cells across cytometry samples by similarity in the associated network structures. These contributions cement a platform that furthers the discussion of cellular states by framing it in both mechanistic and quantitative terms. This platform adds layers of biostatistical knowledge to Biosciences and enhances the discovery of cellular state properties.
Book
1 online resource.
Proteins must achieve their native conformations in order to function and avoid aberrant interactions within the cell. The folded state is formed rapidly for proteins with simple topologies. However, the folding of many large proteins with complex folds is assisted by the diverse array of molecular chaperones. The chaperonins are a unique class of essential protein chaperones found in all domains of life. These complexes are comprised of two 7-9 membered rings that undergo dramatic conforma- tional changes upon ATP binding and hydrolysis. Two classes of chaperonins exist, termed group I and group II. Group I chaperonins exist in bacteria and endosymbiotic organelles, while group II chaperonins are found in all eukaryotes and archaea. Both families promote the folding of substrates in an ATP dependent manner by encapsulating them within discrete central chambers. This thesis focuses on detailing the mechanism of a model group II chaperonin from the archaea Methanococcus maripaludis. Work was performed to define the native folding substrates of the complex as well as to detail the cooperative mechanism that controls all group II chaperonin cycling. A key allosteric interface was identified using a mathematical approach that predicts functionally important residues based on patterns of covariation found in multiple sequence alignments of a protein. Biochemical dissection of mutations at this interface reveal that the chaperonins have evolved to be less cooperative than attainable. Early evidence will be presented that suggests the N- and C-terminal tails of the chaperonin likely serve as coordinators of nucleotide cycling.
Book
1 online resource.
G6PD deficiency, an enzymopathy affecting 7% of the world population, is caused by over 160 different amino acid variants in glucose-6-phosphate dehydrogenase (G6PD). This essential enzyme plays a critical role in maintaining redox homeostasis, and while variants that reduce activity are protective against malaria, complete loss of G6PD activity is lethal. G6PD deficiency is a major risk factor for hyperbilirubinemia and kernicterus, and may contribute to many health conditions including neurodegenerative diseases, heart disease, diabetes, and aging. The clinical presentation of G6PD deficiency is diverse, likely due to the broad distribution of variants across the protein and the potential for multidimensional biochemical effects -- previously characterized G6PD variants have been shown to affect catalytic activity, thermostability, and protein folding. However, the relationship between the structural, biochemical, and phenotypic effects of a G6PD variant remains largely unexplored. Recent developments in the fields of bioinformatics and genome sequencing have allowed us to combine existing phenotypic, biochemical, and genomic information about G6PD to develop new hypotheses about its evolution, structure, and function. In this study, we use existing databases of characterized and uncharacterized G6PD variants to interpret the importance of various structural regions of G6PD. Using biochemical analyses, we identify a trade-off between protein stability and catalytic activity as a major determinant of a G6PD variant's clinical phenotype. Additionally, we examine the evolution of G6PD using a recently developed sequence coevolution analysis method. We identify three coevolving sectors of amino acids that are enriched in different classes of G6PD variants; these three sectors also correspond to functionally characterized structural regions. Based on two sectors that span multiple structural regions, we develop novel hypotheses about conformational and allosteric regulation of G6PD. This work expands the current understanding of the structural and biochemical underpinnings of G6PD variant pathogenicity, and suggests a promising avenue for correcting G6PD deficiency by targeting essential structural features of G6PD.
Book
1 online resource.
The pancreas is a mixed endocrine and exocrine organ associated with the digestive tract. Pancreatic islets are spheroid endocrine micro-organs dispersed throughout the pancreas which secrete hormones into the bloodstream, whereas acinar and ductal cells form a branched epithelial network which transmits digestive enzymes into the intestine. During development, islets, ducts, and acinar cells originate from common progenitors. Islet cells begin to differentiate, exit the ductal epithelium, migrate outward, and cluster to form islets. Genes and signals controlling islet cell differentiation and islet morphogenesis in space, time, and lineage remain to be discovered. Additionally, whether the functions of genes and signals characterized in model organisms are conserved in human pancreas biology is frequently difficult to assess. This thesis presents findings and methods that advance knowledge of mechanisms of islet morphogenesis and differentiation at the cellular and tissue levels. In Chapter 2, we address discordance between mouse and human mutant phenotypes by assessing the functional consequences of human gene mutations associated with clinical disease. In Chapter 3, we define a radial axis in islet morphogenesis encoded by the Semaphorin signaling through Neuropilin receptors. In Chapter 4, we identify the transcriptional corepressor ETO as a regulator of islet progenitor development, and in Chapter 5, we develop tools for genetically labeling human α cells. Along with these findings, we describe methods for single-cell resolution live imaging of developing islet cells, somatic delivery of transgenes to intact developing pancreatic tissue cultured ex vivo, and whole-organ high resolution confocal imaging. Overall, the work here contributes to the study of pancreas organogenesis, defining genes and signals that act at regulatory points in development to enable the formation of pancreatic islets.
Book
1 online resource.
Approximately 30--40% of global CO2 fixation occurs inside a non-membrane-bound organelle called the pyrenoid. Pyrenoids are found in chloroplasts of most eukaryotic algae and some hornworts, and are densely packed with the carbon-fixing enzyme Rubisco. The pyrenoid is a core component of the algal carbon concentrating mechanism (CCM), which enables more efficient inorganic carbon capture than that of most land plants by supplying Rubisco with a high concentration of its substrate, CO2. In this thesis, I show that, in contrast to long-held assumptions of the field, the pyrenoid matrix is not a solid crystal, but is strikingly dynamic, and can likely be considered a phase-separated, liquid-like organelle. The studies detailed in the following chapters focus on the dynamics of fluorescently-tagged pyrenoid proteins in live cells of the model green alga Chlamydomonas reinhardtii, assayed by state-of-the-art quantitative fluorescence microscopy techniques. I tracked the inheritance of fluorescently labeled pyrenoids in live cells for the first time, and observe that pyrenoids are primarily inherited by fission, but that de novo growth is also possible. Strikingly, I have discovered that much of the pyrenoid matrix rapidly disperses into the chloroplast stroma immediately before division and quickly re-aggregates afterwards, suggesting that pyrenoid components undergo a phase transition during division. Furthermore, I demonstrate through Fluorescence Recovery After Photobleaching (FRAP) experiments that the major protein components of the pyrenoid matrix undergo rapid internal mixing. Finally, by targeting a genetically encoded pH fluorescent biosensor to the pyrenoid matrix, I have shown that the pH of the pyrenoid increases during photosynthesis, like the stroma. These findings strongly suggest that the pyrenoid matrix undergoes liquid mixing as a phase-separated compartment in equilibrium with the stroma. This new view of the pyrenoid matrix as a phase-separated compartment resolves the paradox of how the chaperone Rubisco Activase can access Rubisco throughout the pyrenoid matrix. More broadly, my findings provide a new paradigm for understanding the structure, regulation, and inheritance of the pyrenoid, new facts that can be used to guide future attempts to engineer such compartments into higher plants.
Book
1 online resource.
Humans have profoundly altered natural environments across the globe. These changes create challenges for ecosystems, wildlife and humans alike. One challenge of the Anthropocene is altered disease regimes -- whether from introduction of novel pathogens or hosts to ecosystems, perturbations of natural disease dynamics or spillover of infection from one host to another. Factors such as deforestation can catalyze adverse disease events through numerous mechanisms such as stressing organisms, making them more susceptible to infection and prone to shedding infectious particles and forcing novel species interactions with potential for spillover. One group of organisms of particular concern for both conservationists and public health officials are bats. Bats are the second largest order of mammals in the world and one of the most ecologically diverse, ranging from the large, tree roosting, fruit-eating flying foxes of Australasia to the small, cave roosting, insectivorous vespertilionids to the true vampire bats. Bats are also recognized as the reservoirs for a number of highly lethal emerging infectious diseases that infect humans including SARS, Marburg fever, Hendra virus and rabies. However, the majority of bat-associated zoonoses are restricted to the Old World; the Neotropics, despite being the center of bat species diversity, were comparatively understudied. Using a combination of field work, ecological statistics, phylogenetics and molecular genetic and genomic techniques, I sought to understand the ecology and evolution of the interaction of reservoirs with their infections and how these dynamics can change with human intervention. The first portion of my dissertation focuses on bats in an agricultural mosaic landscape in southern Costa Rica, in which I examined: the impact of deforestation on Costa Rican bat communities and the factors that impact bat species persistence in anthropogenic habitats (Ch. 2); the impact of deforestation and community change on ectoparasitism of Costa Rican bats (Ch. 3); and the prevalence, distribution and ecology of bat-associated viral and Bartonella infections in Costa Rican bats (Ch. 4). I then broadly considered bats globally and the impact of bat ecology, behavior and biogeography on the macroevolution of their genes for proteins that interact with pathogens (Ch. 5). Finally, I expanded my view to consider the impact of humans on disease globally by examining the evolution and distribution of Bartonella bacteria and the legacy of human impact on the ecology and evolution of this bacterial genus (Ch. 6). Taken together, these results show that bats and their pathogens have a coevolutionary legacy that restricts disease evolutionarily, geographically and ecologically. However, human interventions can disrupt these relationships by altering vector communities and prevalence as well as the prevalence of natural pathogens and introduced pathogens. Some viral groups are currently uncommon in the Neotropics but could survive in the New World if introduced by humans. Bats are a major source of emerging infectious disease but humans are an ultimate driver of the adverse disease patterns affecting both wildlife and humans.
Book
1 online resource.
This thesis explores the effect on human disease of two evolutionary processes: environmental selection on human disease risk alleles and antibody selection in response to vaccination. Examples of the effect of environment on the genetics of human disease exist, but the relative contributions of environmental selection and neutral forces, such as Out-of-Africa migration, on the genes that increase disease risk are not well understood. Chapter 2 explores the stability of a method commonly used to infer selection due to environmental variables and proposes a method to decrease run-to-run variability. Then, using this method as well as a novel method for determining environmental selection across human populations, chapter 3 examines the effect of Out-of-Africa migration and climate variables on the worldwide frequencies of disease risk alleles. The second half of this thesis focuses on the selection of antibodies, proteins that identify and neutralize foreign molecules in the human body, that occurs within an individual in response to seasonal influenza vaccination. Although the general mechanisms underlying antibody selection have been studied for decades, few quantitative measurements have been made, and the details of this process are not well understood. Using high throughput sequence data, chapter 4 explores the dynamics of the antibody response. Our analyses identify selection on specific sequence characteristics and estimate previously unknown distributions that allow inferences on the biological processes that occur during antibody selection. Chapter 5 examines the similarity of the antibody response across individuals, showing that a much stronger convergence of antibody sequences occurs within individuals than between.
Book
1 online resource.
As the pace of environmental change increases, it becomes more important than ever to understand the processes of natural selection that lead to adaptation to environmental change. Local adaptation, or the differential adaptive response of subpopulations within a species diverging in adaptive traits across an environmental gradient, is thought to be an important mechanism in the preservation of genetic diversity within species, as different alleles will be most fit (and thus under positive selection) in different portions of a species range. It is the maintenance of this standing genetic variation that is the fuel for rapid adaptation to new environmental conditions, but the geographic location of these differently adaptive alleles is a critical component of the adaptability of populations. When environmental conditions change significantly and newly adaptive alleles are located far away, it may take several generations for dispersal to bring significant numbers of these alleles to fill in the newly changed environment. However, if small-scale variability in environmental conditions occurs within a 'local' habitat, then adaptive alleles may be much closer to the new environment, and adaptation may occur much faster. While we are beginning to understand the processes of local adaptation in marine environments, little is known about how genetic diversity distributes across environmental variability at very small spatial scales. In Chapter 1, I examined the spatial scale of genetic variability in the California mussel, Mytilus californianus, at the spatial scale of one to two meters. By using transcriptomic techniques, we identified a suite of candidate single nucleotide polymorphisms (SNPs) that appeared to have different frequencies of alleles between mussels living in sun-exposed and shaded beds within the same region of the intertidal zone. Further validation of these candidate loci revealed many false positive outlier identifications, though genetic differentiation remained within a critical thermal tolerance gene, HSP70kDa 12B, at these spatial scales. In Chapter 2, I extend the concept of small-scaled spatial genetic variability to spatiotemporal genetic variability in the Atlantic cod, Gadus morhua, within the Gulf Maine. Using deep DNA sequencing of a small number of individuals, we showed that three previously known 'islands of divergence' in cod that have been examined across ocean basins and between ecotypes of cod are present and segregating between mating cohorts within the same bay. Using the newly assembled genome, we go further and describe the genic content of those islands of divergence and argue that they may be supergenes, as one of these regions is highly enriched for genes coding for chromatin structuring proteins and chromatin alteration genes. Lastly, in Chapter 3 I expand the concept of genetic variability to include community variability at small spatial scales, by investigating the community structure of coralline algae encrusted cobbles sampled within the same kelp forest canopy in Pacific Grove, CA. By using both morphological taxonomic techniques and DNA metabarcoding on the same samples, we were able to compare both of these methods directly. We found that, while all samples were dominated by malacostracans and polychaetes, subtle differences remained between samples taken at different depths within the same 'community.' Together, these studies show that the spatial scale of sampling can make significant differences in the findings of both intra-species genetic diversity and community composition, and environmental variability at these small scales should be taken into account when sampling at these scales.
Book
1 online resource.
Here you will learn more about IgE and molecular basis of allergic disease. The overarching goal of this work has been to better understand the characteristics of human B cell antibody repertoires, the role they play in the pathology of allergic disease and their contribution in modifying allergic symptoms, and leading to the acquisition of tolerance during allergen specific immunotherapy. In Chapter 2, (previously published as Levin and King, et. al, (2016) in JACI), we examine how IgE repertoire persistence and evolution may hold promise as markers for events accompanying specific immunotherapy of allergic disease. In this chapter, we have combined the use of use phage display technologies to determine IgE specificities, along with high throughput sequencing of blood and tissue B cell repertoires. In this way we were able to identify and track the fates of allergen-specific clones over the course of immunotherapy. We discovered that members of the same allergen-specific B cell clones could be found in both nasal mucosa and the blood, and found evidence of large clonal expansions, persistence, and isotype switching of members of allergen specific IgE containing clonal lineages as immunotherapy progressed. Chapter 3 follows up on this study presented in Chapter 2. In this chapter, we ask what happens in the B cell repertoires of the patients over a longer term of treatment. We look at the fates of allergen specific clones 3 years after the start of immunotherapy, when patients are expected to have reached the optimal improvement of symptoms and are preparing to go off treatment. In our data from this timepoint, we observed a trend toward detection of fewer IgE expressing cells as members of the known allergen specific clones, and more detection of non-IgE expressing clone members, including IgG4, supporting the idea that specific immunotherapy can give rise to IgG4-expressing members of allergen-specific B-cell clones in allergic patients, in a time frame associated with the therapeutic effect of the treatment. In Chapter 4 we discuss more generally the characteristics and differences in B cell repertoires between the blood and nasal biopsy. We found that B cell repertoires in nasal mucosal tissue differed from those observed in the peripheral blood in many ways, which we describe in this chapter. We detected, for instance, distinct antigen experienced IgD and IgM cell populations in tissue, not previously described, and saw a higher proportion of IgG3 usage in nasal tissue, with less IgG2 present. Specific immunotherapy did not appear to elicit global changes in the B cell repertoires in either the nasal tissue or blood. Chapter 5 (previously published as Hoh, et. al, (2016) in JACI), on patients with food allergies and compliments many of our findings from patients with sensitivities to aeroallergens. Investigating allergen-specific B-cells in peanut allergic patients, we found that these cells usually tended to express mutated antibody genes, were typically of switched isotypes, and were often able to bind to both linear and conformational epitopes. Even well-characterized linear epitopes of allergen proteins could be recognized by multiple independent B-cell clones within a single patient. We saw that oral immunotherapy was associated with increased frequencies of allergen-binding B-cells in the blood, and, in one example, progressive somatic mutation of IgG4, but not IgE, members of an allergen-specific clone. Peanut allergen-specific B-cell clones in allergic patient blood typically expressed mutated isotype-switched antibodies, including IgE, and target common epitopes. Oral immunotherapy increased frequencies of specific clones and may also preferentially induce IgG4 somatic mutation. Chapter 6, (previously published as Looney, et. al, (2016) in JACI) discusses methods for generally evaluating somatic mutation patterns in antibody heavy chain genes of B cell clones containing IgE-expressing to search for evidence of direct (from IgM or IgD) versus indirect (from an intermediate switched isotype) isotype switching, a topic that has been a matter of much debate within the field. In this study, we found that both allergic and healthy individuals showed evidence to support the model for indirect switching, particularly from IgG1, as the predominant pathway to IgE expression in humans. Analysis of antibody mutation patterns in allergic and healthy human subjects indicated that most IgE is derived from B cells that previously expressed IgG and had encountered antigen, rather than from naïve IgM+ B cells. Prior to the work presented in this dissertation, studies of B cell repertoires in allergic patients had largely relied on few sequences, or not delved into differences between isotypes. By the use and combination of new technologies and methods, applied to larger cohorts of patients, and across multiple timepoints and/or tissues we have been able to add both depth and breadth to the prior knowledge of human IgE lineages, their origins, and the ways they are being modified over the course of allergen specific immunotherapy. There is still much to learn, but we hope that this work will provide a basis from which to evaluate allergen-specific human antibody repertoires in healthy and diseased individuals, and lay a foundation for future studies aimed at increasing and applying our knowledge for continued improvement in the diagnosis, treatment, and ultimately, the prevention of allergic disease.
Book
1 online resource.
The human-dominated epoch of the Anthropocene is putting new pressures on species, threatening the persistence of many. One such pressure is anthropogenic climate change. In many species, climate change is causing parts of their range, generally lower latitudes and lower elevations, to no longer be habitable. As a result, we are seeing a general movement of species to higher latitudes and higher elevations. However, as montane species move to higher elevations, they will encounter greater physiological stress due to high-elevation hypoxia -- the decrease of available oxygen at high elevations due to a reduction in barometric pressure. The limitations that hypoxic stress may put on species' ability to alter their distributions have never been explored. One montane organism particularly vulnerable to climate change and already experiencing elevational range shifts at up to ten times the global average are pikas, genus Ochotona. There are at least 28 pika species, each occupying a unique elevational range between 0 -- 6,100 m with at least 14 species inhabiting elevations above 4,000 m. Little is known about how pikas can tolerate the hypoxic stress of their high-elevation habitat. By learning more about the mechanisms underlying hypoxia tolerance in pikas, I aim to gain understanding of what role hypoxic stress may play in their future range adjustments in response to projected climate change. Through the utilization of museum tissue samples, extensive field work in the Indian Himalayas, and the only captive colony of pikas in the world, I have investigated hypoxia tolerance in pikas from three different angles: (1) molecular evolution in mitochondrial candidate genes; (2) variation in gene expression along an elevational gradient within a population between 3,600 and 5,000 m; and (3) changes in gene expression within an individual when exposed to varying levels of hypoxia for a 5-day period. Our results indicate that while different pika species have genetic adaptations seemingly specializing them to the general elevational range of the species, on a local and even individual scale, changes in gene expression may offer a mechanism by which pikas can rapidly acclimate to hypoxic conditions. Our findings suggest that pika species may be genetically adapted to the elevational range they occupy, potentially limiting range movement; however, changes in gene expression may enable rapid range shifts within a species' elevational envelope.
Book
1 online resource.
This thesis explores the importance of host nutrient transporter systems in plant pathogen infections focusing on the one hand on UmamiT amino acid and SWEET sugar transport proteins, and on the other hand on bZIP transcription factors as potential regulators of transporter genes using the plant model Arabidopsis thaliana and three pathogens. The pathogens investigated are the hemibiotrophic bacterium Pseudomonas syringae pv. tomato DC3000 as well as the hemibiotrophic fungus Colletotrichum higginsianum and the necrotrophic fungus Botrytis cinerea.
Book
1 online resource.
Reconstructing human evolutionary history is of central importance to many fields, from medical genetics and biological anthropology to archaeology and environmental studies. Human population histories, such as where and when people move, and how populations grow or adapt to their environments, affect human genetic and phenotypic variation, and disease risk. The past decade has reinforced the importance of recent human population history for the distributions of variation; over the past fifteen thousand years, populations around the world have undergone dramatic demographic changes related to environmental and cultural adaptation. This thesis develops multiple new methods to leverage genetic and archaeological evidence to reconstruct past human demography during the past ~15,000. Chapters 2-4 derive a set of mechanistic models describing the impact of sex-biased admixture on distributions of genetic ancestry in an admixed population. Chapters 3 and 4 infer the sex-specific admixture history of major human migrations during the last ten thousand years. Finally, Chapter 5 leverages models from ecological statistics and demographic archaeology to test the relationship between human population growth, local environments, and agricultural development. Together, these chapters interrogate the impact of environmental and cultural change on human demography during the last fifteen thousand years using new mathematical and statistical methods.
Book
1 online resource.
Cell migration requires the large-scale coordination of force generation. This coordination can occur on a mechanical level by physical coupling of interconnected cytoskeletal components, and on a biochemical level by feedback interactions among the signaling molecules that direct actin polymerization. The study of large-scale coordination in cell motility has been hampered by the fact that the cell types best suited for experimental exploration by mechanical perturbations are usually not ideal for experimental exploration by molecular perturbations, and vice versa. Fish epidermal keratocytes have proved to be particularly useful for experimentation and modeling of the mechanics of large-scale coordination because of their simple and stereotyped shapes, their large uniform actin-rich protrusive lamellipodia, and their extremely rapid and persistent movement. However, molecular manipulations in these primary cells, typically cultured from adult members of fish species that have not been genetically well-characterized, have been limited. I have developed a new method for keratocyte culture from zebrafish embryos, enabling me to take full advantage of the molecular experimental methods including morpholino-based gene knockdown that are available for zebrafish, which also has a fully sequenced genome. Using this new molecularly tractable experimental system, I have identified a novel role for myosin light chain kinase in regulating overall cell polarization independently of previously known polarity regulators such as the small GTPase Rho and membrane tension. My investigations of the biology of embryonic zebrafish keratocytes have also shed new light on other aspects of large-scale cell movement coordination. For example, I have found that some zebrafish embryonic keratocytes exhibit an interesting traveling wave behavior, where an actin-rich protrusion appears to propagate laterally around the perimeter of the cell. While superficially similar behavior has been previously observed in keratocytes isolated from adults from other fish species when they are cultured on highly adhesive substrates, I have been able to demonstrate that the mechanism of wave propagation differs in the two cases. These observations have the potential to illuminate the biochemical feedback interactions that are most crucial for the rapid actin polymerization found in keratocytes and other fast-moving cell types. Finally, I have used the embryonic zebrafish keratocyte system to study the mechanical origin of the regular wrinkles that can form perpendicular to the leading edge in fish keratocyte lamellipodia, which were first described more than 90 years ago. I have found that characteristics of these wrinkles can be well explained by a physical model assuming that the mechanical coupling within the lamellipodial cytoskeleton involved in actin-based cell motility is characterized by largely elastic behavior, suggesting that forces exerted on one side of the cell are rapidly transmitted over the entire length of the lamellipod. This result stands in contrast to previous expectations based on observations in other cell types suggesting that the actin cytoskeleton behaves as a viscous fluid over the relatively slow time scales associated with whole-cell motility.
Book
1 online resource.
Coastal hypoxia represents an important environmental change that has the potential to impact a wide range of species and communities in shallow subtidal habitats. While eutrophication-driven hypoxia has long been of concern in sheltered water bodies such as bays and estuaries, recently hypoxic events caused by advection of deep oceanic oxygen minimum zone waters into shallow shelf areas, have been observed. While a large component of this process is naturally occurring. Strong evidence exists which suggests that these events are also increasing due to climate change, through global deoxygenation and increases in coastal advective processes. While advective processes are commonly thought of as spatially uniform at the regional scale, this dissertation presents evidence throughout which, in fact, suggests that coastal advective processes (especially nearshore internal waves) are highly complex spatially, mostly due to their interactions with shallow topographic features such as rocky reefs. This observation is novel in two important ways. First, this small-scale spatial interaction between internal waves and rocky reefs represents novel complex habitat layer to the fluid environment, which has not been previously observed, nor considered for nearshore subtidal ecological studies. Secondly, when nearshore internal waves contain hypoxic water, the resulting small-scale spatial variability in the dissolved oxygen landscape may span super-saturated and severely hypoxic oxygen concentrations on the scale of meters within the reef. Together, the results from this dissertation suggest that small scale spatial variability in the fluid environment arising from internal wave/reef interactions, is a crucial, but poorly understood, component of nearshore habitats, which can complicate and extend hypoxic events. The results here also suggest variability at this scale may feasibly impact local organisms, especially through alterations to behavior. These dynamics are complicated, and much more work should be conducted in the future to better understand the whole impacts to shallow subtidal communities.
Book
1 online resource.
Water is necessary for all known forms of life. Due to their sessile lifestyle, land plants employ an array of physiological and developmental responses to enhance uptake of water from the environment and limit its loss in times of water deficit. These responses occur at several spatial scales of organization, including at the level of single cells, multicellular tissues, organs, and organ systems. Despite the importance of these acclimatory processes to plant survival, the mechanisms of water perception upstream of acclimation responses are poorly understood. To gain a better understanding of these processes, I performed experiments to examine the genetic and physiological basis for hydropatterning, a recently discovered developmental response to water availability. During hydropatterning, lateral root branches are induced in regions of the main root directly contacting sources of available water, and are inhibited in regions exposed to low water availability. I used hydropatterning as a model to understand water perception in plants using two experimental strategies. In my first approach, I asked whether developmental competence to respond to water was limited, and if so, what biological processes were required to establish competence. Through a combination of physiological and mathematical-modeling experiments, I revealed growth to be a requirement for competence, and demonstrated that growth-associated changes in tissue biophysical properties were predictive of future lateral root patterning decisions. In my second approach, I sought to identify genetic loci required for hydropatterning. I uncovered phenotypic variation in hydropatterning through a mutant screen and a survey of natural maize accessions, and identified a 1-Mb interval of the maize genome associated with this phenotype. I conclude with a discussion of how my findings will inform future studies on water sensing, and what key experimental and technological advances will be necessary to move this field forward.
Book
1 online resource.
As life evolves and generation upon generation accumulates mutations and experiences selection, the resulting genetic changes provide a record of the evolutionary dynamics. Population genetic variation, consisting of the allele frequencies of variants in a population, provides a detailed record of the contemporary dynamics of evolution. Of particular relevance here, population genetic variation can track "evolution in action" in systems where selection is occurring on spatial and rapid temporal scales. In addition, the distribution of allele frequencies (site frequency spectrum, or "SFS") can illuminate the strength of selection acting on groups of sites in the genome. While the concept of using population genetic data to study these processes is decades old, the ability to study these processes genome-wide is relatively new. Population genomic studies allow us to quantify the extent that selective processes are affecting populations, and how that differs between species. In this dissertation I utilize population genomic data to characterize patterns of selection and demography across spatial and temporal transects and to detect patterns of selection on synonymous variation. I use the model system, Drosophila melanogaster, which, in addition to having a conveniently short generation time and a small and well-annotated genome, has been studied since the 1920's as model system for understanding ecology, evolution and genetics. D. melanogaster is also one of the most well sequenced organisms. I utilize pre-existing population genomic data as well as produce a substantial amount of D. melanogaster and D. simulans genome sequence data to conduct three separate studies: 1) the comparative genomics of latitudinal genetic variation, 2) seasonal genetic variation across populations, and 3) the causes and consequences of selection on synonymous variation. In the first study, I focus on the genomic patterns of variation with latitude. Examples of clinal variation in phenotypes and genotypes across latitudinal transects have served as important models for understanding how spatially varying selection and demographic forces shape variation within species. I examine the selective and demographic contributions to latitudinal variation through the largest comparative genomic study to date of D. simulans and D. melanogaster, with genomic sequence data from 382 individual fruit flies, collected across a spatial transect of 19 degrees latitude and at multiple timepoints over two years. Consistent with phenotypic studies, I find less clinal variation in D. simulans than D. melanogaster, particularly for the autosomes. Moreover, I find that clinally varying loci in D. simulans are less stable over multiple years than comparable clines in D. melanogaster. D. simulans shows a significantly weaker pattern of isolation by distance than D. melanogaster and I find evidence for a strong contribution of annual re-migration to D. simulans population genetic structure. While population bottlenecks and migration can plausibly explain the differences in amount and stability of clinal variation between the two species, I also observe a significant enrichment of shared clinal genes, suggesting that the selective forces associated with climate are acting on the same genes and phenotypes in D. simulans and D. melanogaster. In the second study I focus on temporal variation. One large source of temporal variation is seasonal fluctuation in the environment. Seasonal environmental heterogeneity can act as a fluctuating selective pressure, which can result in the maintenance of genetic variation if there is a fitness trade-off across seasons. I use pooled population genomic sequence data for 26 populations, sampled seasonally (spring and fall), and sampled across years, to assess the extent of seasonal genetic fluctuation and the consistency of seasonal variation across geographic regions. I find that there is an excess of genetic variants that behave in the same seasonal way across geographic regions. However, this enrichment is weak, suggesting that the identity of seasonal variants shifts temporally and spatially. As seasonal changes in the environment mirror some of the changes seen along a latitudinal cline, we also test for parallelism between seasonal and clinal variants. I find a strong enrichment of sites that change in allele frequency in the same manner from the south to the north as from the fall to the spring. The global consistency, from across Europe and North America, in seasonal and latitudinal variation strongly suggests that these patterns result from selection rather than demography. Finally, I take a departure from temporal and spatial heterogeneity to look at genomic heterogeneity in selective pressures. Specifically, I assess the cause and extent of selection on synonymous sites. Strikingly, I find evidence for strong purifying selection on synonymous sites associated with biased codon usage. Although biased codon usage is a well-documented phenomenon, the extent of selection on biased codon usage was previously not well understood. Using genome sequence data from two D. melanogaster populations, I performed an SFS-based maximum likelihood estimation of purifying selection on fourfold degenerate synonymous sites using short introns as a neutral control. In addition to finding strong purifying selection on synonymous sites due to codon bias, I also find a significant positive relationship between the change in codon usage bias (ancestral to derived) and polymorphism. This is suggestive of purifying selection on derived unpreferred alleles and positive selection on derived preferred alleles. Synonymous sites in alternatively spliced genes, RNA binding protein bound regions and splice junctions are also under detectable amounts of strong purifying selection; however, codon bias explains the greatest proportion of sites under selection. My finding of strong selection on codon bias directly conflicts with previous models of codon bias that predict uniformly weak selection and indicates that the functional effect of biased codon usage has been underestimated.
Book
1 online resource.
Revolutionary methods developed in the last decade have yielded new insights into many biological systems. Despite these technological advances, many complicated structures such as the brain have continued to defy our understanding. In part, this veil exists because of the necessary trade-offs made by most methods, sacrificing resolution of some dimensions (e.g. functional, temporal) in order to more precisely measure others (e.g. structural, molecular). Recent methods for unbiased, whole-sample analysis with high resolution could eliminate some of these trade-offs and yield a more complete understanding of systems-level interactions in complex structures. In this thesis, I apply whole-system methodologies to characterize a behavioral neural network spanning multiple brain regions and a developmental process in an entire organ, the pancreas. In the first section of this work, I use a model organism, the larval zebrafish, to study the whole-brain response in passive coping. I develop a behavioral challenge protocol that induces passive coping in the larval zebrafish and perform brain-wide calcium imaging of neural activity during the behavioral transition. Recordings of neural activity reveal a slow but striking ramping of activity confined to the lateral habenula, as well as a gradual transition to a reduced level of activity in both the raphe nuclei and the dorsal thalamus. Additionally, I use optogenetic stimulation of the lateral habenula combined with brain-wide imaging to show that activation is sufficient to reduce mobility as well as reduce activity in the raphe. These results provide unbiased evidence of a critical role for the lateral habenula in regulating both immediate and prolonged effects of stress on action selection, whereby either synaptic or membrane properties of lateral habenula neurons encode both prior and on-going experiences. In the second section of this work, I adapt CLARITY, a tissue-clearing technique, to be easily compatible for clearing a variety of heterogeneous and soft tissues and for integration into a standard clinical workflow. After developing a biphasic hydrogel methodology and an automated analysis platform for high-throughput quantitative volumetric analysis of biological features, I validate and apply this approach in the examination of a variety of organs and diseased tissues with a specific focus on the dynamics of pancreatic innervation and islet development in laboratory mouse and human clinical samples. Together, these two sections demonstrate unbiased, whole-sample techniques for: (1) probing the brain-wide neural response in disease-relevant behaviors in a model organism; and (2) characterizing molecular-level phenotypes and development processes in a variety of intact systems.
Book
1 online resource.
Microbes that associate with plants form taxonomically and functionally diverse communities. These microbial communities are shaped by many different aspects of plant identity, such as phylogeny, taxonomy and functional group. However, plant-associated microbial communities are also shaped by abiotic environment and a plant's response to abiotic environment. This dissertation explores how plant habitat specialization, species, and genotype shape soil and root microbial communities across hydrologic and abiotic stress gradients in field settings. In chapter 2, ectomycorrhizal communities associated with seven willow species across a hydrologic gradient respond primarily to soil moisture, organic matter and pH; communities were not strongly differentiated across willow species. In chapter 3, I utilized a field experiment to test how water availability, willow habitat specialization and willow species influence soil microbial communities underneath a willow plant. Microbial communities were most dissimilar in contrasting water availability treatments. Both plant habitat specialization and species identity influenced microbial community composition, but the degree and direction depended on the abiotic environment and microbial group. In chapter 4, I tested the influence of willow genotype and wind exposure, which alters soil moisture and nutrients, on root associated fungal and bacterial community structure. Microbial community composition differed by wind exposure and seven of the ten willow genotypes hosted different microbial communities, but again direction depended on the wind exposure and genotype. In conclusion, these three studies show that the turnover in plant microbiome communities will be dependent on plant traits, species, and genotype as environmental conditions change.
Book
1 online resource.
Factors related to the temporal scale of fungal community assembly remain poorly defined. In this dissertation I explore two temporal determinants of fungal community assembly, ecosystem age and species arrival order. First, I explore the effect of ecosystem age using an observational study of the fungi associated with the roots of an ericaceous plant, Vaccinium calycinum, across a 4.1 myr soil chronosequence in Hawaii. I show that soil development promotes greater diversity in ericaceous root-associated fungal communities and that soil-age related nutrient limitation facilitates colonization of ericaceous roots by a greater diversity of non-mycorrhizal fungi in both young and old soils. Second, I use a laboratory microcosm study of root-associated fungi to show that fungal species pools from older ecosystems include species that are more likely to coexist within the roots of a single seedling. Finally, I use a community of wood-decomposing fungi in a microcosm experiment to show that the interactive effects of top-down (grazing) and bottom-up (nutrient availability) forces determine the importance of immigration history (i.e., priority effects) for community composition and function. Taken together these results demonstrate that temporal processes occurring at both short and long time scales can be important determinants of fungal community assembly.