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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.
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.
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.
Collection
Undergraduate Theses, Department of Biology, 2015-2016
Noncoding RNAs can orchestrate eukaryotic gene expression programs through diverse yet only partially understood mechanisms. One noncoding RNA in particular, 7SK, is known to repress mRNA transcription by blocking RNA polymerase II activity at gene promoters. Recent studies suggest that 7SK may be physically associated with chromatin, the target of RNA polymerase II, but the functional significance of this association has not been explored. Here we discover novel roles for 7SK at distinct genomic loci and find that it coordinates the functions of multiple protein complexes. 7SK binds both promoters and enhancers across the genome. At enhancers, mass spectrometry and co-immunoprecipitation reveal a direct, previously unknown interaction between 7SK and a major eukaryotic chromatin remodeling complex called BAF. Depletion of 7SK disrupts the BAF complex, causing aberrant transcription as well as increased DNA damage signaling at thousands of enhancers. These results suggest that 7SK has a multifaceted role in controlling gene regulation. By scaffolding at least two separate nuclear protein complexes at distinct genomic elements, 7SK provides a conceptual framework for how noncoding RNAs may operate as versatile regulators of information flow through the nucleus.
Collection
Undergraduate Theses, Department of Biology, 2015-2016
The Wnt pathway is an instructive signaling system in development, tissue homeostasis, and cancer. Although many core components of the canonical Wnt signaling cascade have been identified, additional layers of regulation likely remain undiscovered. To identify genes and regulatory elements involved in Wnt signaling, we performed unbiased forward genetic screens in a human haploid cell line using insertional mutagenesis with a retrovirus. The work in this thesis is divided into two experimental sections based on the analysis of these haploid genetic screens: 1) design and implementation of an improved and generally applicable computational pipeline to map the genomic insertion sites of the retroviral mutagen used in these screens and 2) the experimental analysis of a class of unexpected mutants we discovered using this pipeline. Analysis of insertional genetic screens in cultured cells has traditionally been “gene-centric”, focused on mapping the insertions in genomic regions with annotated, protein-coding transcripts. We hypothesized that recurrent insertions in non-protein coding regions or unexpected patterns of insertions in coding regions of the genome may identify regulatory elements or cryptic transcripts that regulate Wnt signaling. Therefore, we developed a bioinformatics pipeline designed to be “gene blind” by identifying recurrent insertions in one-thousand base-pair bins across the human genome, chosen consecutively across each chromosome without regard to gene boundaries. Using this pipeline, we found several genomic regions, including unannotated areas near the genes LRP6, TCF7L2, TFAP4 and APC, that are strong candidates to play important regulatory roles in Wnt responsiveness. These genomic regions may represent regulatory elements such as enhancers, promoters, or cis-acting non-coding RNAs (ncRNAs). A human tumor suppressor gene that is a central negative regulator of the Wnt pathway, the adenomatous polyposis coli (APC) gene is one of the most commonly mutated genes in colorectal cancer. Therefore, we designed a screen to search for genes that positively regulate the high-level oncogenic signaling observed when the APC gene is inactivated using CRISPR/Cas9 engineering. Our bioinformatics pipeline identified a perplexing pattern of retroviral insertions in the APC gene that were enriched for in cells selected for reduced Wnt signaling in the absence of APC. This was paradoxical since APC had already been engineered to contain a frameshift mutation in these cells. We found that these insertions somehow increase the level of APC protein, leading to the suppression of signaling in cells still containing a putatively inactivating lesion in the APC gene. In summary, the combined use of haploid screens and a custom bioinformatics pipeline has the potential to probe the genomic regulome of a signaling pathway that plays a central role in development and cancer. Future detailed understanding of these genetic elements and their mechanism of function may reveal new strategies that could be used to target human cancers resulting from deregulation of the Wnt pathway.
Collection
Undergraduate Theses, Department of Biology, 2015-2016
Autism spectrum disorders (ASDs) are fundamentally social and behavioral disorders with a range of comorbidities and social and financial impacts. Recent studies have estimated ASDs to have a heritability of around 50%, while indicating that there is also a significant environmental component. It is clear that neither genes nor environment in isolation can explain the etiology of autism. Large-scale genomics studies have identified a set of genes that have been shown to have a high association with ASDs. In addition, recent studies have identified certain environmental factors associated with an increased risk for developing ASDs, with pre- and perinatal hypoxia as one of the more salient factors. However, the interaction between genes and environment through the lens of hypoxia has yet to be evaluated. This study aimed to find and characterize the intersection between genes associated with autism and the genes associated with the cellular response to hypoxia. Every gene in a database of autism-associated genes was interrogated, through a thorough literature search and comparison with a set of microarray data, for evidence of its regulation by hypoxia. This process created a set of genes associated with both autism and the hypoxia response. A statistical test for overrepresentation indicated that hypoxia-regulated genes were overrepresented in the ASD database; the proportion of ASD genes also responsive to hypoxia was roughly twice what would be expected by chance. Functional and network analyses then showed that specific biological functions were overrepresented in the ASD/Hypoxia gene set, indicating that the number of hypoxia-regulated genes among all ASD genes, were indeed greater than would be expected by chance, and fell into specific networks and pathways. This lays the groundwork for functional characterization of variations in these genes in a population affected with autism as well as a neurotypical population. With a better understanding of how variations in certain genes can affect an individual’s response to a potential hypoxic event, researchers and medical professionals alike can open up better therapeutic avenues for children and adults affected by ASDs.
Collection
Undergraduate Theses, Department of Biology, 2015-2016
The microtubule cytoskeleton is spatially reorganized from the centrosomes to new subcellular sites during cell differentiation in many cell types, yet the importance of possessing distinct microtubule organizing centers (MTOCs) and the mechanisms governing MTOC reassignment remain poorly understood. Centrosomal and non-centrosomal microtubules are nucleated by a conserved nucleating complex, the γ-tubulin small complex (γ-TuSC), which is comprised of GIP-1/GCP3, GIP-2/GCP2, and TBG-1/γ-tubulin. Tissues of the nematode C. elegans display dualistic MTOC localization, in which γ-TuSC proteins are capable of localizing to both MTOCs, but do so at distinct times in development. This makes C. elegans an ideal system for studying the process of MTOC reassignment. We have proposed a model of MTOC reassignment whereby regulation of γ-TuSC protein localization dictates the sites of functional MTOCs. The gip-1 locus is predicted to encode multiple isoforms, presenting the possibility that differential expression of GIP-1 isoforms may play a role in this process. We therefore set out to investigate the role of GIP-1 in the developing C. elegans intestine. In this tissue, MTOC proteins are translocated from the centrosomes to the future apical cell membrane following their final round of divisions, resulting in a relatively simple dichotomy of MTOC function. Because a complete loss of functional MTOCs leads to an early arrest in embryonic development, we adapted an endogenous protein degradation pathway to deplete GIP-1 exclusively in the developing intestine around the time of MTOC reassignment. We found that GIP-1 is required for normal apical localization of GIP-2 and TBG-1, but not for apical localization of microtubules or the polarity protein PAR-3. Embryos depleted of intestinal GIP-1 have fewer and abnormally enlarged intestinal nuclei as compared to control embryos. Using a structure–function approach to identify GIP-1 domains required for MTOC localization and function, we have identified two domains (GIP-1[N], GIP-1a[G+C]) that localize to centrosomes but not apical membranes. Together, these results demonstrate that GIP-1 is vital for normal mitosis in the early embryonic intestine, and highlight the presence of redundant microtubule nucleating and anchoring mechanisms in this tissue. Furthermore, we provide the first evidence that GIP-1 may localize to centrosomal and apical membrane MTOCs through distinct protein domains. Future efforts focused on understanding the consequences of GIP-1 depletion for microtubule dynamics will greatly inform our understanding of the role of GIP-1 in regulating the microtubule cytoskeleton. With aberrant microtubule organization linked to some epithelial cancers and invasive cell behavior, understanding this basic cellular process may inform future efforts to inhibit cancer proliferation and metastasis.
Collection
Undergraduate Theses, Department of Biology, 2015-2016
Chronic wounds afflict over 6.5 million individuals and cost over $25 billion annually in the United States alone. These nonhealing wounds are caused in large part by impairments in neovascularization, the formation of new blood vessels. Stem cell therapies have emerged as a potentially viable approach to treat chronic wounds. In particular, adipose-derived mesenchymal stem cells (ASCs) are promising due to their ease of harvest and production of pro-regenerative molecules, including those that promote neovascularization. However, a challenge with stem cell therapies is maintaining cell survival in the harsh wound environment. Therefore, stem cell delivery to the wound must be optimized to make cell-based therapies for wound healing translatable. Previously, the Gurtner laboratory found that delivering ASCs by seeding these cells in a hydrogel composed of collagen and the polysaccharide pullulan and topically applying the ASC-seeded hydrogel to a cutaneous wound accelerated wound closure. This project focused on further elucidating how ASC-seeded hydrogels have this therapeutic effect. Specifically, we tested the hypothesis that ASC-seeded hydrogels increase the recruitment and heighten the functionality of endogenous progenitor cells that promote neovascularization. We used techniques including a model in vivo to study cell recruitment to cutaneous wounds, fluorescence activated cell sorting (FACS) of recruited cells, and in vitro measurement of recruited cell proliferation, migration, and expression of provascular genes. We found that application of ASC-seeded hydrogels to wounds in vivo, compared to injected ASCs or a saline control, increased the recruitment of circulating bone marrow-derived mesenchymal progenitor cells (BM-MPCs, p < 0.05), a population we previously found plays a role in neovascularization. In vitro, we found an increase in expression of genes related to angiogenesis, as well as BM-MPC proliferation, migration, and tubule formation, all of which play a role in neovascularization (p < 0.05 for all assays). These data support our hypothesis, demonstrating that treating cutaneous wounds with ASCs seeded in hydrogel increases recruitment of progenitor cells that contribute to neovascularization and improves recruited cell provascular functionality. Overall, this study demonstrates that our pullulan-collagen hydrogel can optimize delivery of ASCs to cutaneous wounds, underscoring the potential of ASC-seeded hydrogels to be used clinically in cell-based therapies for chronic wound healing.
Book
1 online resource.
Tandem mass spectrometry (MS/MS) enables the high-throughput identification and characterization of complex protein mixtures, and depends critically on bioinformatics tools to interpret mass spectra as peptide sequences. There exist two general techniques for the interpretation of mass spectra: de novo sequencing and database search. In de novo sequencing, a mass spectrum is directly interpreted as a protein sequence. In database search, a mass spectrum is identified from its best match in an existing sequence or spectrum database. Though more unbiased and less restrictive than database search algorithms, de novo sequencing algorithms are less popular due to their relatively lower accuracy and lack of automated statistical validation tools. However, database search algorithms suffer greatly in both speed and sensitivity as database search spaces increase through the addition of protein sequences and post-translational modifications. To able to apply MS/MS to more diverse systems, I developed the de novo sequencing algorithm Label Assisted De novo Sequencing (LADS). LADS utilizes chemical strategies to bolster introduce signatures into mass spectra which improve sequencing accuracy, and employs a support vector machine-based model to discriminate true from false identifications. I also developed a method by which to empirically estimate false discovery rates (FDRs) from any de novo sequencing algorithm. In the last stage of my PhD, I developed TagGraph, an unrestricted database search tool able to match peptides to mass spectra from sequence databases without assuming any protease specificity or requiring a user-specified set of modifications. I demonstrate the utility of TagGraph on the recently published human proteome dataset, matching over four million spectra to modified peptides, and identifying new functional roles and disease associations for protein hydroxylation. Both TagGraph and the de novo FDR calibration technology described herein have the potential to greatly extend the scope and depth of tandem MS analyses.
Book
1 online resource.
Many marine invertebrates have complex life cycles in which embryogenesis gives rise to a planktotrophic larva with its own body plan, distinct from the adult. In the phylum Hemichordata, both direct and indirect developing species occur. While data from the direct developing enteropneust Saccoglossus kowalevskii have provided insights into the evolution of the hemichordate adult body plan, comparatively little is known about the development and evolution of the tornaria larva of indirect developing species. In this dissertation, I establish the indirect developing hemichordate Schizocardium californicum as a new system for molecular developmental studies, and explore the evolution of the hemichordate larval body plan by investigating the molecular mechanisms that regulate development of its tornaria larva. In Chapter 1, I describe the information necessary to use Schizocardium californicum as a model system for developmental studies. I describe methods for adult collecting, induction of spawning and larval rearing in Schizocardium californicum, and provide a detailed description of embryonic development, larval development and metamorphosis, using light microscopy, immunolabeling and confocal microscopy. In Chapter 2, I ask whether the differences between larval and adult body plans in hemichordates are associated with fundamental changes in axial patterning. I characterize the expression patterns of a large number of transcription factors with conserved roles in deuterostome anteroposterior patterning throughout development of Schizocardium californicum. Using expression of these transcription factors to identify homologous body regions between larva and adult, I show that most of the ectoderm of the tornaria larva has an anterior identity, and lacks a body region equivalent to the adult trunk. Addition of a Hox-patterned trunk to this anterior larval territory prior to metamorphosis ensures the transition between larval and adult body plans. This work identifies a large-scale molecular correlate of a difference between larval and adult body plans, and demonstrates that modulating the timing of Hox expression may have played an important role in the evolutionary transition between direct and indirect development in hemichordates. In Chapter 3, I explore a potential cause of the delay in trunk development in Schizocardium californicum by characterizing the expression patterns of components of the Wnt signaling pathway. These results show that unlike Saccoglossus kowalevskii, early activation of Wnt signaling is insufficient to activate expression of trunk patterning genes. Together, these studies establish Schizocardium californicum as a new system for molecular developmental studies, with the potential to rapidly expand our knowledge of larval evolution in hemichordates, and identify large-scale differences in the molecular architecture of hemichordate larval and adult body plans. These results provide insights into the evolution of the body plan of the tornaria larva, and improve our ability to compare developmental data from hemichordates with other phyla.
Book
1 online resource.
Lysine methylation is a signaling mechanism conserved from yeast to humans that is critical for many basic cellular processes. Moreover, these processes have been linked to many human diseases like developmental disorders and cancer. While many proteins related to lysine methylation signaling have been identified and characterized, many components of methyl-lysine signaling are poorly understood and require further mechanistic insight. In this work, we further to our understanding of methyl-lysine signaling by addressing technical and biological queries related to methyltransferase activity and identification and characterization of methyl-lysine binding proteins. In Chapter 2, we perform a systematic characterization of lysine methyltransferases on histone H3, a concentrated center for nuclear signaling, and put forth data highlighting the importance of nucleosomes when characterizing histone-modifying enzymes. In Chapter 3, we identify the PZP domain of AF10 to bind unmodified H3K27 and this binding event is regulated by modification of that residue. In cells, the binding interaction between H3 and AF10 regulates DOT1L and the deposition of H3K79 methylation, which manifests in leukemia cells sensitive to H3K79, suggesting a possible target for pharmaceutical intervention. H3K79 methylation is an important modification thought to be important for transcriptional elongation, however the mechanism linking these processes remains elusive. In Chapter 4, we suggest that the FACT complex may provide this link that can read H3K79 methylation signals. FACT is a histone chaperone complex important for relaxation chromatin ahead of RNA polymerase II to facilitate transcription elongation. Modest efficiency in remodeling H3K79me2 histones coupled with compensation of H3K79 depletion with increased FACT complex provide the strongest experimental links between H3K79 methylation and transcription elongation. Together, we contribute to the field of lysine methylation signaling by characterizing the biochemical and biological properties of writer and reader proteins with special consideration to how recombinant chromatin reagents may influence the results.
Collection
Undergraduate Theses, Department of Biology, 2015-2016
Tumor protein p53 is one of the best known tumor suppressors, controlling various regulatory pathways including apoptosis and cell cycle arrest. Among p53’s main functions is its activity as a transcriptional activator of downstream target genes. Binding of p53 to target gene regulatory regions promotes gene expression and subsequent protein formation. We discovered that Pard6g, the gamma isoform of an apical-basal polarity regulator, was transcriptionally activated by p53, suggesting a potential role of apical-basal polarity maintenance in p53-dependent tumor suppression. Cells lacking Pard6g demonstrate increased anchorage-dependent growth, while not exhibiting significantly different anchorage-dependent proliferation, suggesting that Pard6g functions as a tumor suppressor through mechanisms involving three-dimensional cell morphology or polarity.
Book
1 online resource.
Rho GTPases are central regulators of cell polarity whose activity must be tightly regulated. In S. cerevisiae, the essential small type Rho GTPase Cdc42 regulates cell polarity during vegetative growth and coordinates cell signaling with morphogenesis in the mating response. Three Rho GTPase activating proteins (GAPs), Bem3, Rga1, Rga2, contribute to the specific spatial and temporal regulation of Cdc42, serving both overlapping and unique regulatory functions. Phosphorylation of these GAPs are known to regulate their function as during the G1 phase of the cell cycle, the cyclin-dependent kinases phosphorylate and inhibit Rga2 at S/TP sites to promote Cdc42 activation during bud emergence. Regulation of these phosphosites by phosphatases and other kinases remains to be examined. A recent systematic screen identified Rga2 as a substrate for the Ca2+-activated phosphatase, calcineurin. Our studies reveal that calcineurin dephosphorylates Rga2 during pheromone signaling to activate its function in limiting Cdc42 signaling. Calcineurin regulation of Rga2 is important to dampen pheromone-induced gene expression and promote cell survival during prolonged exposure to mating factor. Together with the known functions of calcineurin in promoting endocytosis of the pheromone receptor and inhibiting transcription factor activity, calcineurin signaling provides critical negative feedback during the pheromone response that is essential for cell survival.
Collection
Undergraduate Theses, Department of Biology, 2015-2016
Background: S. meliloti is a nitrogen-fixing bacteria that infects the roots of its legume host plants to form nodules, where it converts atmospheric nitrogen into ammonia that plants can use in a symbiotic relationship. Much of the mechanism of this symbiotic relationship is still unknown, but genes required for proper cell envelope function have been implicated, and more of such genes likely exist. At the same time, recent research has uncovered possible genes that may be involved in cell growth and cell envelope synthesis in the closely related A. tumefaciens, which may also play a role in S. meliloti cell envelope function and symbiosis. Methods: Tn5 mutagenesis followed by screening on chlorophenol red-β-D-galactopyranoside (CPRG) was conducted on S. meliloti strain MB782. Mutants with possible cell envelope defects were selected and the location of Tn5 insertions identified by arbitrary PCR and sequencing. Mutant sensitivities to detergents were tested by spot assays with SDS or deoxycholate. SMc00038 and SMc00039 were identified to be possible candidates required for cell growth and cell envelope synthesis based on recent research of A. tumefaciens, and were deleted from the chromosome of S. meliloti CL150 to create the strain D217. Effects of deletion on symbiosis, growth rates, cell shape, and deoxycholate and NaCl sensitivity were studied. Results: 3 genes previously reported to affect both cell envelope function and symbiosis were amongst the list of genes identified using CPRG screening, and 48% of mutants identified showed increased sensitivity to detergents. D217 demonstrated slower growth and abnormal cell branching, but no effects on symbiosis, deoxycholate, or NaCl sensitivity were observed. Conclusion: Tn5 mutagenesis followed by screening on CPRG is a promising method of identifying genes required for cell envelope integrity and symbiosis. SMc00038 and SMc0039 may play a role in proper cell growth.
Collection
Undergraduate Theses, Department of Biology, 2015-2016
The majority of cancer survivors with a history of chemotherapy treatment endure long-term deficits in neurological performance. This chemotherapy induced cognitive and motor decline is known colloquially as chemobrain. The symptoms associated with chemobrain may be attributed to damage to myelin, the insulating sheath surrounding neurons allowing for rapid saltatory conductions, or myelin-forming cells, such as oligodendrocyte precursor cells (OPCs) thus altering brain function. To confirm the extent of OPC population depletion in children receiving chemotherapy, we examined post-mortem brain samples from the frontal lobes of children treated with multi-agent traditional chemotherapy. We found that OPCs are depleted specifically in subcortical white matter. In contrast, grey matter OPCs are preserved in comparison to age-matched control subjects. Methotrexate (MTX), an antimetabolite chemotherapeutic, is a commonly used agent in pediatric cancer therapy and is particularly associated with white matter injury and cognitive dysfunction. We have developed a mouse model of juvenile methotrexate chemotherapy exposure in which mice are treated with MTX or PBS (one dose each week from P21-35 for a total of three doses). One month following the last dose of MTX, mice exhibit behavioral deficits in motor speed and attention, as well as depletion of deep cortical grey matter and subcortical white matter OPCs. As in human subjects, superficial grey matter OPC density was preserved. Concomitant with deep cortical and subcortical OPC depletion, we observed an increase in immature oligodendrocytes, suggesting increased but incomplete differentiation of OPCs. Experiments allografting healthy, GFP-labeled OPCs into the environment of the previously chemotherapy-treated brain suggest a persistent change to the gliogeneic microenvironment underlies the accelerated OPC differentiation. OPCs isolated from whole brain exhibit an IC50 less than the measured MTX concentration achieved in the brain with this paradigm, suggesting that a direct cytotoxic effect on OPCs may also plays a role in depletion of the subcortical OPC population. Also, MTX treated mice exhibit increases in the number of activated microglia within the corpus callosum in MTX treated mice. Additionally, we found an increase in TGFβ signaling in MTX-exposed OPCs as indicated by an increase in phosphorylated Smad2/3 (pSmad2/3) and a subsequent increase in TGFβ gene expression in the activated microglia. Collectively, these findings suggest MTX chronically activates microglia and elevates TGFβ signaling to inhibit the ability of OPCs to proliferate and accelerate OPC differentiation respectively.
Collection
Undergraduate Theses, Department of Biology, 2015-2016
The ochre sea star (Pisaster ochraceus) is a keystone predator that can control the structure and maintain the diversity of rocky intertidal communities. In 2013, a densovirus instigated a large sea star die-off that caused populations of Pisaster across the West Coast of North America to collapse. In the wake of their absence, the rocky intertidal zone faces potential change in community structures. For example, prey populations, specifically mussels, could now expand without predation by Pisaster to restrict them. Field surveys and experiments examined the possible impacts of the die-offs of the ochre sea star on the intertidal zone. Specifically, impacts were measured on the California Mussel (Mytilus californianus), and two predatory whelks Ocinebrina circumtexta and Nucella analoga compressa. Eighteen 0.5 m2 plots within mussel beds at two sites, within the Lovers Point State Marine Reserve, in Monterey Bay, California, were photographed on seven dates between June 2014 and July 2015 to measure changes in mussel percent cover and tidal heights of the mussel beds. To examine whether whelks have the potential to replace P. ochraceus in controlling the M. californianus population, I conducted counts of the dominant species, Ocinebrina sp. & Nucella sp., in the plots on five dates and I performed a lab experiment to measure the mortality rates of M. californianus with and without the predatory whelk Nucella sp. Counts of P. ochraceus were also conducted at the two sites, on each monitoring date, and compared to J. Pearse’s (2010) historical counts done for the same areas since 1950. Percent mussel cover showed a small but significant increase over the 13-month monitoring. Plots that had a greater cover to begin with showed a slightly greater increase. However, some mussel patches with low cover but with recently recruited M. californianus grew faster because smaller mussels grow faster than larger ones. The lower limit of the mussel beds has shifted up to 32 cm lower in some plots, possibly due to recruitment and higher survival occurring at lower levels in the absence of sea stars. Whelk counts revealed a decrease in densities over the 15-month monitoring. The abundance of P. ochraceus is now the lowest it has been since 1950: sea star population size has seen a 90-95% decline over the past 25-50 years. Most of this mortality is not due to the 2013 outbreak of the sea star die-off. Finally, the presence of Nucella increases the mortality of M. californianus in laboratory feeding experiments, but there is little evidence for size selection. However, the potential for the whelks to fill in the niche left vacant by Pisaster population collapse seems limited based on my laboratory estimates of mortality from whelk predation and field estimates of whelk densities. Overall, the predicted mussel expansion did occur, though patchily. However, processes other than predation could drive or limit mussel expansion. Continued monitoring and field experiments are needed to examine possible changes in intertidal communities and elucidate their drivers.