%{search_type} search results

1,533 catalog results

RSS feed for this result
View results as:
Number of results to display per page
Collection
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.
Book
1 online resource.
G protein coupled receptor proteins (GPCRs) couple extracellular soluble ligand binding to intracellular signaling pathway activation. GPCRs are excellent drug targets due to their positioning at the plasma membrane upstream of signaling cascades, and due to their ability to respond to ligand binding with a range of signaling outputs. Crystallography has revealed the structure of drugs bound to the inactive and active state GPCRs at high resolution; each crystal structure represents a single, low-energy GPCR conformation. Crystal structures have also defined the canonical activation mechanism for GPCRs as an opening up of the intracellular surface; for Family A GPCRs, the intracellular portion of transmembrane helix 6 (TM6) undergoes the largest conformational change upon ligand binding. Here I present the design and development of two novel fusion proteins for GPCR crystallography. Crystal structures of the M3 muscarinic receptor fused to both novel crystallization aids improved the resolution of the overall structure and extended the view of residues comprising TM6. In addition to the states seen by crystallography, recent spectroscopy studies revealed that GPCRs sample a wide variety of conformations. This raises the question of when and whether high-energy (non-crystallographic) conformations are relevant for GPCR function. In this work, I both employ and validate a high-pressure electron paramagnetic resonance (EPR) spectroscopy technique to understand the basal and ligand activation of an archetypal GPCR, the Beta-2 adrenergic receptor (β2AR). The studies demonstrate a pre-existing equilibrium between inactive and active receptor states, providing a structure-based explanation for the observed phenomenon of basal activity. Clinically, inverse agonists are used to inhibit basal activity of GPCRs, and these high-pressure studies also reveal a structural mechanism for inverse agonists: they inhibit the outward movement of TM6. Studies of β2AR endogenous and synthetic agonists under ambient and pressurized conditions reveal a distinct conformational profile for each agonist. These conformational differences may be responsible for different ligand efficacies towards two signaling pathways downstream of GPCRs: arrestin-mediated versus G-protein-mediated signaling. Overall, these studies provide further support for a general mechanism for GPCR activation, conformational selection. The ability to fine-tune drug efficacy is the next frontier in GPCR drug discovery. To this end, I contributed to a collaborative drug discovery project to find novel β2AR allosteric ligands by docking to a hypothetical allosteric binding site, at a distinct location from where endogenous ligands bind. We built upon the principles of conformational selection, searching for negative modulators by docking to the inactive crystal structure, and finding positive modulators from active-state structure docking. Some of the new allosteric ligands discovered have signal bias properties, targeting arrestin over G-protein coupling. Though the underlying mechanisms of ligand bias are difficult to assess by most physiologic and biophysical methods, EPR spectroscopy revealed subtle differences in the conformational ensemble, which may correspond to arrestin versus G protein-specific drug effects.
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.
Root-associated fungi (RAF) shape the interface between plants and soil. This ubiquitous group of organisms influences ecosystem processes such as nutrient cycling, and shapes plant community diversity and composition. Until recently, methodological difficulties have prohibited extensive study of RAF community composition. Thus, questions as to how tropical RAF communities vary between hosts, and across space and environmental conditions remain unanswered. In this dissertation, I present three studies that employ DNA metabarcoding to characterize the community composition of RAF in Neotropical forests. In Chapter 1 we employed a transect-based systematic survey of RAF to quantify relative contributions of host phylogeny and spatial distance to structuring RAF communities. In Chapter 2 we used a targeted sampling scheme that controlled for host phylogeny and location to compare diversity and composition of RAF associating with rare vs. common tree species. In Chapter 3 we investigated plant-RAF interaction patterns among plants in the diverse tribe Psychotrieae (Rubiaceae), and compared the relative influence of edaphic conditions, host phylogeny, and spatial distance in structuring RAF communities. Results show that RAF communities were structured by host phylogeny, such that RAF community similarity decreased with phylogenetic distance between hosts. This pattern persisted across the range of phylogenetic distances included in these studies, from closely related congeners to members of highly divergent host clades. Analyses were repeated with fungal communities pooled at deeper taxonomic levels, and by trophic mode. Patterns of host phylogenetic structure show that closely related host plants shared more phylogenetically similar root fungal microbiomes than distantly related host plants. Thus, core components of the RAF microbiome are conserved across the host plant phylogeny. Edaphic conditions also influenced RAF distributions, but geographic distance was generally a stronger predictor of RAF composition, consistent with the possibility that dispersal limitation structures RAF communities at fine spatial scales. Results v differed between fungi from different trophic modes; putative symbiotrophs, which were dominated by arbuscular mycorrhizal fungi, showed relatively weak host preference, while pathotrophs showed stronger host preference. Janzen-Connell processes involving distance-dependent mortality due to species-specific natural enemies likely maintain forest diversity by inhibiting conspecific regeneration beneath parent trees. Other studies have shown that RAF communities drive these processes, but have treated fungal communities as a black box in doing so. The present dissertation illuminates this black box. Results from each chapter consistently show that Neotropical RAF meet the prerequisites of host preference and dispersal limitation necessary to drive forest diversity maintenance processes under the Janzen-Connell hypothesis. Furthermore, common plant species supported less diverse RAF communities than rare host plants, especially for mutualistic symbiotrophs. This correlation between host abundance and RAF diversity is consistent with the idea that differences in Janzen-Connell processes between rare and common plants may be driven by differences in their RAF communities. The implications of uncovering emerging patterns in RAF community composition for plant ecology are discussed throughout, highlighting that RAF community ecology represents an important frontier in understanding Earth's biodiversity.
Book
1 online resource.
G6PD deficiency, an enzymopathy affecting 7% of the world population, is caused by over 160 different amino acid variants in glucose-6-phosphate dehydrogenase (G6PD). This essential enzyme plays a critical role in maintaining redox homeostasis, and while variants that reduce activity are protective against malaria, complete loss of G6PD activity is lethal. G6PD deficiency is a major risk factor for hyperbilirubinemia and kernicterus, and may contribute to many health conditions including neurodegenerative diseases, heart disease, diabetes, and aging. The clinical presentation of G6PD deficiency is diverse, likely due to the broad distribution of variants across the protein and the potential for multidimensional biochemical effects -- previously characterized G6PD variants have been shown to affect catalytic activity, thermostability, and protein folding. However, the relationship between the structural, biochemical, and phenotypic effects of a G6PD variant remains largely unexplored. Recent developments in the fields of bioinformatics and genome sequencing have allowed us to combine existing phenotypic, biochemical, and genomic information about G6PD to develop new hypotheses about its evolution, structure, and function. In this study, we use existing databases of characterized and uncharacterized G6PD variants to interpret the importance of various structural regions of G6PD. Using biochemical analyses, we identify a trade-off between protein stability and catalytic activity as a major determinant of a G6PD variant's clinical phenotype. Additionally, we examine the evolution of G6PD using a recently developed sequence coevolution analysis method. We identify three coevolving sectors of amino acids that are enriched in different classes of G6PD variants; these three sectors also correspond to functionally characterized structural regions. Based on two sectors that span multiple structural regions, we develop novel hypotheses about conformational and allosteric regulation of G6PD. This work expands the current understanding of the structural and biochemical underpinnings of G6PD variant pathogenicity, and suggests a promising avenue for correcting G6PD deficiency by targeting essential structural features of G6PD.
Book
1 online resource.
Cells use a variety of mechanisms to mitigate the threat of genetic invasions and to prevent the propagation of parasitic information. A family of prokaryotic adaptive immune systems associated with Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) arrays has been shown to protect cell populations from "selfish DNA" such as plasmids and viruses. CRISPR-mediated immunity begins with an "adaptation" phase in which the host acquires short sequence segments (spacers) from the genome of the infectious agent. The spacers are stored within CRISPR arrays in the host genome and serve as heritable molecular memories of the invader. This information is used by CRISPR-associated (Cas) nucleases in the subsequent "interference" phase to identify and disrupt future infections by the same invader. Most CRISPR-Cas systems can be phylogenetically classified into three major types. Type I and Type II systems acquire spacers from (and subsequently interfere with) DNA sequences, while Type III systems additionally target RNA sequences for degradation. In the absence of any known mechanisms for CRISPR adaptation to RNA, this was believed to be an auxiliary mode of attack against DNA parasites. We discovered that some Type III systems can acquire spacers directly from RNA, suggesting that these CRISPR-Cas systems could learn to fend off RNA parasites as well. This system represents, to our knowledge, the first identified cellular process in which sequence information is actively acquired from RNA and deposited (as DNA) into the genome at the behest of the host cell. Moreover, direct acquisition of RNA spacers into CRISPR arrays represents a novel and uniquely sequence-agnostic mode among the handful of known mechanisms for reverse flow of genetic information from RNA into DNA genomes, including reverse transcription machineries associated with retroviruses, telomeres, retrotransposons, mobile group II introns, and phage diversity generating elements.
Book
1 online resource.
The formation of complex but highly organized neural circuits requires precise recognition and interactions between cells that constitute the nervous system. A growing body of research has discovered numerous neuro-neuronal interactions underlying neural development, while the important roles played by glia are appreciated only more recently. In this thesis, I utilized the olfactory circuit of Drosophila melanogaster as a model to study the roles of both neurons and glia which together establish the unique structure of the olfactory system. During the assembly of the Drosophila olfactory circuit, ~50 olfactory receptor neuron (ORN) classes and ~50 projection neuron (PN) classes form one-to-one synaptic connections in ~50 glomerular compartments in the antennal lobe, each of which represents a discrete olfactory information processing channel. Several cell surface molecules have been reported to mediate neuro-neuronal interactions and determine the specific connectivity of olfactory neurons. However, we are still far from a complete understanding of this process. In a genetic screen to look for additional wiring molecules in this process, I identified Fish-lips (Fili), a leucine-rich repeat transmembrane protein, to be expressed in a subset of olfactory neurons. Loss- and gain-of-function experiments indicate that Fili can instruct PN dendrites to project to proper glomerular targets. Besides olfactory neurons, the antennal lobe is also permeated by several types of glia. Specifically, each glomerular compartment is separated from the adjacent compartments by membranous processes from the ensheathing glia. In a genetic screen designed to reveal molecular mechanisms underlying glia morphogenesis, I identified that Thisbe, a fibroblast growth factor released from olfactory neurons particularly local interneurons (LNs), controls ensheathing glia to wrap each glomerulus. The FGF receptor, Heartless, acts cell-autonomously in ensheathing glia to regulate process elaboration so as to insulate each neuropil compartment. Overexpressing Thisbe in ORNs or PNs causes over-wrapping of glomeruli to which their axons or dendrites target. Failure to establish the FGF-dependent glia structure disrupts precise ORN axon targeting and discrete glomerular formation. In summary, I have identified two molecular mechanisms underpinning the assembly of the olfactory circuit. Fili, combined with previously identified cell surface cues, can define dendrites from different PN classes and instruct them to target correct glomeruli. After the initial innervation of PN dendrites and ORN axons, ensheathing glia respond to a neuronal cue, Thisbe, to pattern the boundaries of the nascent glomerular compartments; neural compartments in turn require such glial barriers to separate themselves from neighboring compartments, so as to ensure the correct organization of the olfactory circuit. These findings highlight the synergism of neurons and glia in shaping the intricate network of the nervous system.
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.
Budding yeast cells link their rate of division to growth via the transcriptional inhibitor Whi5. Cell growth dilutes, and thereby partially inactivates, Whi5, allowing the expression of two cyclins, Cln1 and Cln2. Cln1 and Cln2, in complex with cyclin-dependent kinase (Cdk1), further inactivate Whi5, completing a positive feedback loop that drives cells into S-phase by inducing expression of hundreds of genes. An additional cyclin-Cdk1 complex, Cln3-Cdk1, has been thought to contribute to the initial inactivation of Whi5 through phosphorylation. Using phos-tag SDS-PAGE to assay the phosphorylation state of Whi5 in vivo, we show that, although Cdk1 activity is required at S-phase entry, Whi5 phosphorylation during G1 does not require Cdk1. We identify the specific residues on Whi5 that are phosphorylated during G1 and show that their ablation has a moderate effect on cell cycle progression, as assayed by cell size. We also find that two of these phosphosites match the consensus motif for PKA, suggesting a possible additional Whi5 regulatory mechanism. We show that rapid Whi5 phosphorylation at S-phase entry depends on the phospho-threonine binding activity of the cyclin-Cdk1 subunit Cks1. Finally, we find that blocking Cks1 binding to Whi5 phospho-threonines yields a size phenotype equivalent to blocking all Whi5 phosphorylation by cyclin-Cdk1. This suggests that Cks1 binding to Whi5 phospho-threonines is essential to the function of Whi5 phosphorylation by cyclin-Cdk1.
Book
1 online resource.
The development of novel drug-regulable tools that allow manipulation or control of target proteins has the potential to meet many unmet needs in basic and applied science. In 2008, with the TimeSTAMP technique, a new class of genetically encoded, drug-regulable protein tags was introduced. This original TimeSTAMP tag allowed for drug-dependent epitope labeling of a protein of interest, allowing the researcher to selectively visualize only the protein copies synthesized within a drug-specified time window. One novel feature of this new class of drug-regulable module was its property of self-cleavage: in addition to a distal epitope tag, TimeSTAMP contained the hepatitis C virus (HCV) NS3/4A protease, and was covalently linked to the target protein via a protease substrate. Thus, the tag underwent autocleavage and removed itself by default. A second novel feature of the system was that it relied on a clinically approved class of small molecule drugs (HCV NS3/4A serine protease inhibitors) to enforce preservation of the tag. Since the tag is only retained on new protein copies synthesized in the presence of drug, this allows selective tagging of the newly synthesized pool of the protein of interest. The ability of drug-regulable protease-based tags to selectively interrogate only the newly synthesized pool of a protein of interest makes it an especially attractive technique for probing the role of de novo protein synthesis in biological processes known to rely on carefully choreographed new protein expression -- for instance, synaptic plasticity. While de novo activity-dependent protein synthesis is known to be necessary for synaptic plasticity and memory consolidation, the question of which specific proteins' new synthesis is required for these processes has proven refractory to investigation, due to the lack of tools with which to selectively interrogate the newly synthesized protein pool for a given protein. Likewise, the properties of drug-regulable protease-based tags offer advantages for biotherapeutic applications. The fact that these protease-based modules can be controlled by clinically relevant small molecules makes them attractive for possible gene therapy applications using protein- or cell-based approaches. While interest in gene therapies, particularly those whose activity or persistence is regulable by small molecules as a safety feature, is currently experiencing a resurgence, the lack of clinically approved bioinert drugs with which to regulate them complicates translation to the clinic. This dissertation details efforts to expand the toolbox of drug-regulable genetically encoded protease-based modules, and leverage them towards answering outstanding questions in neuroscience, and also towards developing drug-activatable proteins relevant to gene therapy. By adapting the TimeSTAMP tag such that it contained a degradation-promoting sequence, a new protease-based tag, SMASh, was developed that can mediate drug-enforced shutoff of the further expression of a protein of interest. The SMASh tool was characterized and subsequently deployed to shut off the synaptic protein PSD95, which is known to be relevant to synaptic plasticity and memory. To apply this tool towards the study of endogenous PSD95, PSD95-SMASh knock-in mice were generated in order to test the hypothesis that de novo hippocampal PSD95 synthesis is necessary for normal expression of fear memory in mice and for normal expression of long-term potentiation (LTP) in hippocampal slices. While an effect on fear memory was not observed, new PSD95 shutoff in hippocampal slices attenuated the rate of passive LTP decay after LTP induction. TimeSTAMP knock-in mouse strains for PSD95 and the synaptic protein Arc were also developed as additional tools for interrogating the newly synthesized pools of key synaptic proteins. Finally, a generalizable NS3 protease-based module called StaPL was developed in order to exert drug control over therapeutically relevant proteins. An inserted StaPL module cleaves a protein into nonfunctional pieces, but application of NS3 protease inhibitor preserves linkage, and thus function. The NS3 protease was also mutagenized in order to produce two orthogonal StaPL modules that are able to be controlled independently by two different NS3 protease inhibitors. StaPL regulation was shown to be extensible to controlling zinc finger-based bidirectional transcriptional effectors, dCas9-based transcriptional effectors, and a caspase-9 suicide switch.
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.