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Undergraduate Theses, Department of Biology, 2016-2017
Anthropogenic climate change is causing coral reefs around the world to rapidly deteriorate and disappear, in large part due to thermal stress from increased ocean temperatures. Recent evidence suggests that certain coral species may be better at adapting and acclimatizing to thermal changes than others. Corals form mutualistic relationships with diverse clades of dinoflagellate algal symbionts, in the genus Symbiodinium (spp.). Symbiotic plasticity within this relationship allows the corals to adapt to changes in their environment – there have been many well-documented cases of symbiont clade switching within coral colonies in response to thermal heat stress. The majority of coral reef research, especially as it pertains to symbiont switching, is short-term and therefore, doesn’t provide much insight as to the large-scale global changes that are likely to occur in the wake of climate change. By evaluating symbiont switching in coral colonies around the island of Ofu in American Samoa this study aimed to (i) determine the frequency with which symbiont clades shift within coral colonies, (ii) better understand the influence of climatic factors in promoting the switching of symbiont clades specifically with respect to mass bleaching events, and (iii) provide some of the first temporally significant evidence for symbiont switching in situ. By using DNA extraction and amplification techniques, samples taken from the same coral colonies over the span of several years were assessed for hosted symbiont type and analyzed with respect to species, location, and season; there were also genetically identical replicates that were transplanted to different locations. It was found that corals exposed to greater fluctuations in daily temperature are more likely to host heat resistant symbionts. Additionally, some coral colonies appear to be able to switch their symbiont composition over short time periods (~ 2 months) effectively undergoing rapid adaptation to environmental changes. These findings align with what is a growing consensus that two simultaneous factors, heat resistant symbionts and the ability to rapidly acclimatize to changes in the environment, are necessary for corals to resist heat stress. Understanding the mechanisms that confer differences in resistance responses between corals to environmental stress may allow us to better protect coral reef ecosystems as climate change intensifies.
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.
Collection
Undergraduate Theses, Department of Biology, 2016-2017
Heart diseases remain the leading cause of death in the United States as well as worldwide. Due to the limited proliferative capacity of adult mammalian cardiomyocytes, i.e. heart muscle cells, generating cardiomyocytes de novo remains a topic of significant interest in the cardiovascular field. The de novo creation of cardiomyocytes is seen as a potential means of replenishing cardiomyocytes lost upon cardiac injury and diseases. Previous studies have shown that bioactive lipids such as sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) regulate cellular processes including proliferation, differentiation, and migration in epithelial cells and human embryonic kidney cells. However, their roles in human cardiomyocyte function are poorly understood. I adopted differentiation of human induced pluripotent stem cells (hiPSCs) as a model and examined the ability of biolipids to stimulate cardiomyocyte (CM) differentiation. Using the recently established hiPSC-derived CM (hiPSC-CM) differentiation platform, I demonstrated that treatment with S1P/LPA during the early stage of differentiation can enhance cardiomyocyte formation, generating higher yield of cardiomyocytes from hiPSC differentiation. I showed that combined S1P and LPA treatment facilitated epithelial-to-mesenchymal transition during hiPSC differentiation and enhanced hiPSC-CM formation by increasing nuclear accumulation of β-catenin, a Wnt pathway mediator crucial to the transcriptional activation of genes necessary for cardiomyocyte differentiation. These findings will help improve hiPSC-CM generation for cardiac disease modeling, precision medicine, and regenerative therapies.
Collection
Undergraduate Theses, Department of Biology, 2016-2017
Development in animals is not merely an intrinsically programmed event. Response to environmental conditions plays a large role in an organism’s development. The nematode species C. elegans develop from egg to adulthood through a life cycle known as reproductive development, but in environments of stress, such as starvation, overcrowding, or high temperatures typically found in nature, the worms will enter a dormant larval stage called dauer diapause. Recent work has shown that during the dauer diapause, dendrite outgrowth of the mechanosensory neuron PVD in C. elegans is suspended. Yet, the molecular mechanisms governing the arrest of the development of this neuron remain unknown. To identify the factors involved in pausing PVD dendrite outgrowth during dauer, we tested whether overexpression of four genes that are required for dendrite outgrowth of the PVD neuron during reproductive development (dma-1, mec-3, mbl-1, and rig-1) could bypass the dauer-induced dendrite growth suspension. Additionally, we conducted a forward genetic screen to isolate mutants that fail to suspend PVD development during dauer. The two methods, though, were unsuccessful in identifying the factors governing this phenomenon. Over-expression of the four genes did not cause complete development of PVD’s dendritic arbor, suggesting these genes, individually, do not control the suspension of PVD’s dendritic outgrowth. Also, a mutant of interest with complete development of dendritic outgrowth was not found in the forward genetic screen. However, a mutant with a peculiar phenotype was identified; the PVD neuron of the worm looks like wild-type in reproductive development, but in dauer, the PVD neuron develops abnormally. The mutant is still being studied to understand which gene is affected, but it seems there are multiple genetic events governing the phenotype.
Collection
Undergraduate Theses, Department of Biology, 2016-2017
Eukaryotic gene expression is tightly controlled on multiple levels. Proteins that bind both DNA and RNA are involved in multilevel gene expression by regulating both transcriptional and posttranscriptional processes. Nuclear Factor 90 (NF90) is a DNA- and RNA-binding protein encoded by the Interleukin enhancer-binding factor 3 (Ilf3) gene first cloned based on inducible binding to the Nuclear Factor of Activated T cell (NF-AT) target DNA sequence in the IL-2 promoter. NF90 has been widely studied as a double-stranded RNA-binding protein that participates in stabilization of transcript, splicing, and export of mRNA. The role of NF90 in regulating gene expression as a DNA-binding transcription factor has not been systematically studied. In Jurkat T cells, chromatin immunoprecipitation (ChIP) demonstrated inducible binding of NF90 and NF45 at the IL2 and Pdcd1 promoters in vivo upon T cell activation. In K562 cells, ChIP followed by deep sequencing (ChIP-seq) was used to characterize genome-wide binding patterns of NF90. Proteomic analysis elucidated stimulation-dependent post-translational modifications in Jurkat T cells. NF90 showed a sequence-specific DNA binding activity to a highly-conserved purine-rich motif in its genome-wide binding sites. NF90 colocalizes with histone marks H3K9ac at active promoters and H3K27ac at enhancers, closely associates with core histones in the nucleus, and exhibits a novel histone acetyl transferase activity. Taken together, these results characterize NF90 as a transcriptional activator with histone acetyl transferase activity.
Collection
Undergraduate Theses, Department of Biology, 2016-2017
Asymmetric cell division is an important process that can link stem cell development to the cell cycle. Arabidopsis stomatal guard cell development, a model for plant stem cell lineages, requires asymmetric divisions and has been well characterized in terms of the distinct developmental stages and the transcription factors involved with each transition. To date, the effects or linkages of these developmental transitions on the cell cycle have not been defined in this system. Further, the existence and effects on the cell cycle of the putative plant DREAM complex, whose homologues regulate the cell cycle in animals, has not yet been demonstrated. In this thesis, I implemented two different methods of visualizing cell cycle stages in Arabidopsis leaves: a method that monitors nucleotide incorporation during DNA synthesis (EdU staining) and the CDT1A reporter line that marks the S and G2 stages of the cell cycle for live cell visualization. The latter method was used to begin to characterize how cell cycle timing is affected by differentiation or by members of the DREAM complex. Here, I present evidence that the critical transcription factors for stomatal development appear beginning in G1 and persist, likely with activity, through G2, as well as evidence that the DREAM complex is active from G1 through G2. To facilitate quantitative measurements of cell behaviors, I developed tutorials for MorphoGraphX, a software program that can segment and measure cell size, shape, and growth over time and that will be essential for continued studies of cell cycle and fate.
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.
Collection
Undergraduate Theses, Department of Biology, 2016-2017
Diabetes is a devastating disease that affects over 400 million people worldwide. Most of those affected with diabetes have some degree of insulin resistance. Peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulatory receptor often targeted by drugs such as Rosiglitazone to overcome insulin resistance, and plays a major role in cardiovascular health and metabolism regulation. This research aims to elucidate the mechanism underlying PPARγ function by knocking out the gene using CRISRP/Cas9 as well as by assessing the effects of Rosiglitazone treatment on cardiomyocytes. In this study, a human induced pluripotent stem cell (hiPSC) platform was utilized for the first time to successfully induce a PPARγ knock-out (KO) and allow for further study of the KO’s effect on cellular metabolic function in human cardiac cells. A CRISPR-based approach was employed to knockout PPARγ in an iPSC line obtained from healthy individuals. Successful KO was verified via TOPO screening and Sanger sequencing. These cells are currently being differentiated for further analysis. In parallel, cardiomyocytes treated with varying doses of Rosiglitazone were assayed for changes in gene expression and cell viability. These cells were characterized by measuring mRNA expression of PPARγ and related genes implicated in cardiovascular and metabolic regulation. qRT-PCR of these genes, including PPARγ showed a significant increase in mRNA expression in treated cardiomyocytes compared to controls. Importantly, these genes have been suggested to be regulated by PPARγ and to participate in control of cellular metabolic function. Therefore, analysis of their differential expression following Rosiglitazone treatment may provide insight into PPARγ-dependent regulation of cardiovascular health. These results contribute to the molecular and genetic understanding of PPARγ and Rosiglitazone treatment in development of cardiovascular diseases, as they show effects on key metabolic genes and cell function.
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.
Collection
Undergraduate Theses, Department of Biology, 2016-2017
Medulloblastomas are a malignant (WHO grade IV) brain tumor that comprise the majority of pediatric brain tumors. They are classified into four molecular subgroups each highlighted by amplification of a certain oncogenic pathway. Group 3 medulloblastomas are characterized by MYC amplification and have the worst prognoses of the subgroups, with a dismal overall survival (OS) rate of less than 50% and debilitating toxic side effects for those that survive current modes of therapy. A potential novel therapeutic target for medulloblastomas is casein kinase 2 (CK2), a tetrameric enzyme implicated in MYC regulation. Here, I investigated the role of CK2 activity in the expression of c-myc and one of its phosphorylated forms at serine-62 (p-c-myc). I demonstrated that while CK2 activity and expression did not seem to have a significant effect upon the overall expression of c-myc and p-c-myc, CK2 inhibition exhibited a significant decrease in nuclear c-myc and p-c-myc, suggesting that CK2 can regulate c-myc activity by controlling its localization to the nucleus. This is an important discovery, as c-myc is a transcription factor and requires localization to the nucleus for proper function. The data generated here suggest a crucial relationship between two established oncogenic proteins and help identify CK2 as a promising therapeutic target to more effectively treat a particularly harmful and as-of-yet uncured pediatric brain cancer.
Collection
Undergraduate Theses, Department of Biology, 2016-2017
Huntington’s Disease (HD) arises through pathogenic aggregation of Huntingtin (Htt) protein, causing the formation of fibrillar beta-sheet amyloid aggregates, which can create a toxic environment by disturbing cellular transport, altering mitochondrial function, and undermining transcription regulation [1-4]. Pathogenic aggregation of Htt is mediated by the first 17 residues of Htt exon 1 (N17), which act as an “on” switch for this aggregation [2,5,6]. While a significant amount of research has focused on Htt aggregates, less work has focused on soluble Htt oligomers. This is partly due to the fact that these oligomers are heterogeneous and transient in nature. However, recent research has shown that these oligomers may be the cytotoxic species present in HD [7]. Interestingly, these oligomers also appear to form in an N17-dependent manner [Shen et al., in preparation]. However, the mechanism by which N17 promotes formation of these toxic oligomers, and therefore pathogenesis, remains unclear. The following research utilizes a model protein with slower aggregation kinetics, mutagenesis experiments, and various biochemical assays to elucidate the properties and formation of these oligomers. We find that our protein (HttQ17) serves as an informative model system for pathogenic Htt, based on aggregation and oligomerization in each system. Surprisingly, HttQ17 oligomers do not seed further HttQ51 aggregation, implying that these species may be interacting with each other in unexpected ways. Experiments with the HttQ17 system further confirm the importance of N17 in the formation of Htt oligomers as Htt lacking this N-terminal sequence (HttQ17△N17) is unable to oligomerize to the same extent as HttQ17. Furthermore, altering the polarity of residues within this motif alters formation of oligomers and aggregates.
Collection
Undergraduate Theses, Department of Biology, 2016-2017
An active field of research within bioengineering is the control of gene expression using small molecules, given the broad impact such a tool would have. A reversible, tunable method for gene expression regulation that does not require frequent intervention or maintenance is optimal for this strategy. Current systems, such as CRISPR/Cas9, are capable of transcriptional activation and repression, but are activated by small molecules not currently in clinical use. Here, we describe a synthetic architecture for a set of transcription factors that allows an endogenous gene to be either repressed or activated through the choice of drug. This work was adapted from the small molecule activated shutoff (SMASh) tag, a self-cleaving protein tag derived from the Hepatitis C virus NS3/NS4a protease. Two sets of mutations were introduced to the NS3 protease to create two protein tags, each inhibited by a different drug. We demonstrate chemical control over the transcription of the secreted protein vascular endothelial growth factor (VEGF) in human cell lines. VEGF is commonly overexpressed in cancers and therapeutically desirable in some applications, so control over its secretion is clinically relevant. This new tool offers the opportunity to study downstream effects of gene suppression or constitutive activation in research contexts. In addition, its dose-dependent and bidirectional nature represents the first step toward a new tool in the gene therapy arsenal to tightly regulate protein levels within either implanted therapeutic cells or afflicted cells over a broad dynamic range.
Collection
Undergraduate Theses, Department of Biology, 2016-2017
Brain cancer is a devastating disease that affects both adults and children, yet effective therapies remain elusive. Glioblastoma (GBM) makes up 15.1% of primary brain tumors, and represent one of the most common types of brain cancers. One key hallmark of brain tumors is the significantly increased tissue stiffness as the cancer progresses, likely due to changes in cell fates and extracellular matrix (ECM) compositions of tumor tissues. Elucidating the role of ECM cues such as matrix stiffness in modulating brain cancer progression would be critical in improving the therapeutic outcomes of this devastating disease. Previously, the Fan Yang laboratory has engineered a novel gradient platform to study tissue zonal organization in a 3D environment. In this project, we focused on employing such platform for high-throughput screening of matrix stiffness effects on brain cancer cells, and we chose patient-derived GBM 270 as a model cell type. Using a syringe pump and gradient maker system, we were able to create and characterize a PEG-based gradient hydrogel with brain-mimicking biochemical cues and tunable stiffness. We found that patient derived glioblastoma cells were able to survive and proliferate in this gradient hydrogel platform over 21 days. We then evaluated the effects of matrix stiffness in modulating patient-derived GBM cell proliferation and invasion in 3D using such platform. We observed significant differences in cell morphology between different zones, specifically in cell spreading indicated by their actin filament organization. We found zonal- dependent upregulation of genes responsible for the ECM degradation, which is integral to cell spreading and cancer metastasis. We finally examined the drug resistance of GBM 270 cells encapsulated within different zones in gradient hydrogels, and found that they exhibited differential resistance to Temozolomide (TMZ), indicating the significant effect of matrix stiffness on cell phenotype changes. This project has demonstrated that 3D gradient platform can be broadly adapted with tunable biochemical and mechanical matrix cues for elucidating complex niche interactions in the progression of brain cancer or other cancer types in 3D using reduced materials, cells and time.
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.
Collection
Undergraduate Theses, Department of Biology, 2016-2017
Recent human genome-wide association studies have implicated microglia, the immune cells of the central nervous system, in contributing to the progression of Alzheimer’s disease. Oligomeric amyloid-beta (Aβ42), the precursor to Aβ plaque formation, elicits pro-inflammatory responses in microglia. Recently, using two separate AD-immune models, our lab has identified the kynurenine pathway (KP) as being highly regulated in microglia and in peripheral monocytes. The KP metabolizes tryptophan (TRP) through the KP, forming quinolinic acid (QUIN), a precursor of de novo nicotinamide adenine dinucleotide (NAD+). Recent data from the lab also shows that microglia suppress the KP enzyme quinolinate phosphoribosyl transferase (Qprt) when stimulated with Aβ42, resulting in accumulation of QUIN and decrease production of NAD+. Considering that NAD+ also functions as the main electron carrier in oxidative phosphorylation (OXPHOS), we sought to examine whether the immune KP regulates the metabolic state of microglia and macrophages. Using an immune model of AD by stimulating human monocyte-derived macrophages (MDMs) with Aβ42, we characterized the KP using transcriptomics and quantitative western analysis. We also conducted seahorse and NAD+ assays to assess cellular respiration and NAD+ levels, respectively. Finally, we ran metabolomics on mouse macrophages as a preliminary experiment to document metabolic changes that occur due to genomic and pharmacological inhibition of the KP. Our findings show that in the presence of Aβ42, MDMs exhibit a decrease in OXPHOS and down-regulate Qprt. Pharmacological inhibition of various KP enzymes replicates the decrease in OXPHOS and also induces the MDMs to adopt a pro-inflammatory phenotype. Last, untargeted metabolomics reveals enrichment of metabolic processes that are implicated in inflammation. Our study strongly suggests that modulation of de novo NAD+ through the KP can influence the metabolic activity and the inflammatory state of the cell.
Collection
Undergraduate Theses, Department of Biology, 2016-2017
Neural progenitor cell (NPC) proliferation and differentiation is influenced by factors like local cell-to-cell interactions, soluble mitogens and extracellular matrix (ECM) molecules. These factors have been shown to maintain NPC pools and restrict the production of new neurons to neurogenic zones in the adult brain. Previous research has not yet addressed whether the interaction between ECM molecules localized to the subgranular zone, and the NPCs via the αvβ3/β5 integrin receptor could enable the maintenance of the stem cell pool and the production of neurons. Thus, we investigated the gating effects of various (Arg-Gly-Asp) RGD-motif ECM molecules through the αvβ3/β5 integrin receptor on growth factor mediated NPC proliferation. Using hippocampal mouse-derived NPCs that express or lack the integrin αvβ3/β5, we investigated the difference in endogenous proliferation rates, and then tested the gating effect of various ECM substrates via the integrin αvβ3/β5 on Shh, VEGF, and IGF-1 mediated NPC proliferation. Here, we show that NPCs with the αvβ3/β5 integrin have increased NPC proliferation compared to those that lack the integrin αvβ3/β5 when plated on the ECM substrates vitronectin, fibronectin, and collagen. Next, we show that IGF-1 yields a greater increase in proliferation in NPCs that express the integrin αvβ3/β5 compared to those that lack the integrin αvβ3/β5 when plated on vitronectin, fibronectin, laminin, and MFG-E8. This work improves our understanding of the interaction between NPCs and the local microenvironment of the adult brain as we seek to develop the tools to understand and artificially manipulate NPC proliferation and differentiation.