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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.
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.
Book
1 online resource.
Fibrosis and regeneration are intimately connected. While fibrotic processes create nonfunctional tissue through the deposition of connective tissue, regenerative processes have the potential to generate new functional tissue. In this manner, efforts to better understand and potentially inhibit fibrosis should be complemented by regenerative approaches. In the first half of this dissertation, mechanisms and targeted inhibition of fibrosis are investigated primarily in the context of mesenchymal lineages. Regenerative approaches focused around cell-based therapies are then explored in the second half of this dissertation.
Collection
Undergraduate Theses, Department of Biology, 2015-2016
Neurons are an astonishing component of our nervous system that intricately control our intended, and unintended, actions. A highly organized microtubule network is essential for multiple cellular functions in neurons including axonal transport, neuronal polarity, and synaptic activity. Disrupted microtubule networks are a hallmark of several neurodegenerative diseases, like Alzheimer’s disease. In order to understand these diseases more comprehensively, the mechanisms behind microtubule organization and regulation must be elucidated. This research aims to provide more insight into microtubule processes. The Yang lab has recently identified nemitin as a novel protein involved in microtubule regulation. Nemitin is enriched in neurons, colocalizes with microtubules and is essential for viability, since knockout of nemitin in mice is lethal embryonically. Using molecular and cellular neuroscience techniques, we further characterize nemitin’s cellular functions and suggest a model for its mechanism of action. Nemitin localizes to the centrosome in Cos-7 cells but no neurons. Using a conditional knockout mouse line, we show that Cre-mediated conditional knockout of nemitin in embryonic neurons suppresses the growth of axons and primary dendrites. To address a potential mechanism, we show using 2D gel electrophoresis that nemitin is phosphorylated in neurons. Finally, we characterize nemitin’s interaction with a putative kinase, STK25, and suggest that phosphorylation may be involved in nemitin’s mechanism of action. Altogether these results suggest that nemitin is necessary for microtubule organization, while also being important for neurite development. This research will strengthen our current knowledge of microtubule regulation in neurons, and could provide novel therapeutic approaches in treating neurodegenerative diseases that exhibit abnormal microtubule networks.
Book
1 online resource.
The transport of sugars is critical for plant growth and development since sugars provide the source of energy. Sugars must be transported from within the cells in which they are synthesized to their destination. Cellular membranes are selectively permeable to sugars, because they contain specific membrane proteins that transport sugars in and out of the cell, either actively against the concentration gradient through the expenditure of energy or passively down the concentration gradient. Here, Arabidopsis nectaries were used as a model system to investigate how sugars, the major components of nectar, are secreted. The molecular mechanisms driving nectar secretion had not been determined. I identified and characterized a novel nectary-specific sugar transporter, SWEET9. I showed that Arabidopsis SWEET9 has all the hallmarks of a transporter responsible for nectar secretion by confirming its expression in nectaries, sucrose transport activity, plasma membrane localization, and its function as a sucrose bi-directional transporter. Importantly, sweet9 knockout mutants lost their ability to secrete nectar, while SWEET9 overexpression led to increased nectar secretion. Based on additional experiments, I propose a model, in which starch-derived hexoses are re-synthesized to produce high concentrations of sucrose in the cytosol of the nectary parenchyma. Sucrose is subsequently secreted into the extracellular space via SWEET9, where it is hydrolyzed by an apoplasmic invertase, potentially creating a large enough osmotic gradient to sustain water efflux into the extracellular space and generate nectar containing a mixture of sucrose, glucose and fructose. Plants carrying mutations in SWEET9 can now be used to study, for example, why Arabidopsis, a predominantly self-fertilizing plant, retains nectar production, or to generate mutants with varying sugar levels in nectar to study plant-pollinator interactions. Our findings led to new questions, regarding the actual sugar gradients, and whether the gradients are sufficient to explain osmotically driven nectar secretion, or whether alternative factors are required. Osmotic gradients play critical roles in many other processes, e.g. the root response to moisture changes in soil, cell expansion and regulation of stomatal aperture. To develop tools that allow us to monitor osmotic gradients, I took advantage of mechanosensitive channels (MS channels) to develop fluorescence-based "membrane tension sensors" that may be able to detect changes in membrane tension that are caused by either osmolality changes or mechanostimulation in intact cells with high spatial and temporal resolution. In response to hypoosmotic stress, MS channels change their conformation to trigger channel opening to adjust turgor. By fusing MS channels to a donor and an acceptor fluorophore, I created Förster Resonance Energy Transfer (FRET) sensors that report conformational rearrangements occurring during changes in membrane tension. The optical readout of these sensors is a change in ratio of acceptor over donor fluorophore intensities. When expressed in yeast, the Oztrac sensors report a shift from hypo- to hyperosmotic conditions, which decrease turgor pressure, and reduce membrane tension. When expressed in Arabidopsis, the sensor showed a ratio change in response to mechanically stimulation in individual root cells as well as to compression forces when roots encountered a barrier, or when roots were exposed to mechanical forces during root growth or mechanical stress treatments. Future goals include the implementation of FRET sugar sensors in nectaries to measure the intra- and extracellular sugar concentration of nectary cells, and the analysis of membrane tension during nectar secretion. The membrane tension sensors will require extensive characterization and optimization, but are expected to have wide applications for studying mechanical forces during plant growth and development. Membrane tension sensors can also be applied to create a temporally resolved membrane tension maps during cellular growth and development and to study cytoskeleton/cell wall-membrane interactions.
Collection
Undergraduate Theses, Department of Biology, 2015-2016
Population growth places great strain on our natural resources, bringing to the forefront the pressing problems such as limited food supplies. One way to address this problem is by improving plant photosynthetic efficiency, resulting in increased plant productivity and higher crop yields. To study photosynthesis and carbon concentration, we use the model organism Chlamydomonas reinhardtii, a single-celled green alga with an inducible carbon concentrating mechanism (CCM) that allows it to perform efficient photosynthesis: our long-term goal is to introduce the algal CCM into higher land plants, which would reduce photorespiration and enhance photosynthesis. It is necessary, therefore, to identify the key components of the CCM and to understand their molecular interactions in order to transfer this mechanism into crop plants. To this end, we have developed a pipeline for large-scale characterization of the algal CCM components and map how these components function together. With fluorescent and affinity tagging coupled to immunoprecipitation (IP) and mass spectrometry to explore these mechanisms, we took the localization data for 40 putative photosynthesis and CCM components to present 381 confident interactions between the proteins involved. The developed pipeline and the foundational knowledge reported here provide valuable tools for continuing work towards bioengineering of the CCM.
Book
1 online resource.
The idea that species can change in response to their fitness effects was famously proposed by Charles Darwin more than a century ago and merged with our burgeoning understanding of genetics as the "modern synthesis" in the early 20th century. The relationship between genotype and fitness has been studied extensively in the decades since, with the goal of not only understanding evolutionary history, but to predict future evolution. Evolutionary prediction is critical for solving problems of biomedical interest such as drug resistance evolution in pests and pathogens or the evolution of cancers. Major questions in the field include understanding the distribution of selective effects of new mutations, the number and types of loci that are targets of adaptive mutations, and the dependency of the fitness effect of a mutation on its genetic background or the environment. In the first chapter of this thesis, I present a theoretical treatment of the predictability of evolution, by studying predictability via historical reconstruction as well as more traditional methods looking at the similarity of independently evolving populations starting from the same initial condition. I relax the assumption made in this prior work that adaptation always proceed through the successive fixation of adaptive mutations, and characterize the changes in evolutionary predictability that result. I find that the two approaches to studying predictability are not identical, and can even be anti-correlated. Remarkably, I find that stable polymorphisms can make the historical reconstruction method unreliable even with perfect sampling, which would never occur under the successive fixation assumption. In order to predict evolution in biological systems, we need a comprehensive genome-wide survey of all possible adaptive mutations in a system and their fitness effects, not just the ones that occurred during a single bout of evolution. In the next chapter, I present a novel and highly extensible approach to overcome the current technical limitations in conducting such a survey in experimentally evolving yeast populations based on recently developed the DNA barcoding technology. In the final chapter, I use the methods developed in the second chapter to study how the genotype-fitness map changes under different growth conditions by remeasuring the fitness of the same clones under many alternative conditions. These conditions were chosen to specifically vary different parts of the yeast growth cycle, such as the exponential growth phase or stationary phase, giving us additional insight into the physiological basis of adaptation in this system. In sum, the work presented in this thesis studies the predictability of evolution using theoretical models and develops the experimental tools necessary to empirically study evolutionary predictability by comprehensively characterizing the genotype-fitness map in experimentally evolving yeast.
Collection
Undergraduate Theses, Department of Biology, 2015-2016
Understanding the mechanisms of cardiac cell lineage commitment is important for improving the understanding of cardiogenesis and the pathophysiological basis of congenital heart disease. Cardiogenesis is regulated by complex interactions between many transcription factors; however, not much is understood about the specific transcription factors that govern cardiac progenitor cell differentiation into cardiomyocytes. One ubiquitous transcription factor, YY1, has been shown to activate and repress genes in other developing tissues, but has not been studied in the differentiation of cardiomyocytes. We have found that YY1 is vastly down-regulated just prior to cardiomyocyte differentiation and think that this down-regulation may play an important part in the differentiation of cardiac progenitor cells into cardiomyocytes. To understand the role of this down-regulation, we created and differentiated mouse embryonic stem cell (mESC) lines that overexpress YY1 during the time that YY1 would normally be down-regulated in the developing embryo. Cells that overexpressed YY1 increased the expression of both cardiac transcription factors and cardiac sarcomeric proteins. We therefore believe that YY1 plays an important role in the process cardiac lineage commitment and these experiments give some insight into one of the many facets that control the differentiation of cardiac progenitor cells into cardiomyocyte.
Book
1 online resource.
Macrophages are immune cells that inhabit almost every tissue of the body and work to maintain organismal homeostasis. They recognize a broad range of deviations from normal (e.g., metabolic imbalance, presence of pathogens, tissue damage) and tailor their responses to return the body to normal. Macrophages are remarkably plastic in their signaling state, which allows them to respond to homeostatic disruptions in a rapid and appropriate manner. Due to this rapid adaptation to new environments, the ability to perturb and observe macrophage signaling in a temporally defined manner is key. Chemical tools allow such rapid manipulation and observation and, in this thesis, I will describe our use of chemical tools such as small molecule inhibitors and optical probes to dissect dynamic macrophage signaling. One mechanism through which macrophages respond to danger is by initiating the formation of protein complexes called inflammasomes. These complexes facilitate activation of the pro-inflammatory cytokines IL-1β and IL-18 and rapid pyroptotic cell death. This causes inflammation, the goal of which is to identify and combat the source(s) of danger. Inflammasome signaling is beneficial for fighting pathogenic bacteria, fungi, and viruses, but aberrant or excessive inflammasome signaling can damage tissues. Chapters 2 and 4 describe the application of small molecule inhibitors to dissect the cellular signals that lead to formation of a specific inflammasome, the NLRP3 inflammasome, which is implicated in the response to bacterial pathogens and is also overactive in metabolic and genetic autoinflammatory disease. Our work in Chapter 2 describes the identification of a small molecule activator of the NLRP3 inflammasome. By characterizing the mechanism of action of this small molecule, we found that disruption of glycolytic metabolism drives NLRP3 inflammasome formation and pyroptotic cell death in macrophages. We find that this mechanism of NLRP3 inflammasome activation is used by host macrophages to sense and react to the intracellular bacterial pathogen Salmonella typhimurium. In Chapter 4, we discuss the development of a live-cell imaging-based screening method that enables comprehensive identification of small molecules that modulate NLRP3 inflammasome formation and pyroptotic cell death. The goals of this project are to further knowledge of cellular signals leading to NLRP3 activation and to identify therapeutic leads for combatting aberrant NLRP3-driven inflammation. Just as macrophages have evolved many ways of recognizing pathogens, pathogens have also evolved many methods of evading detection and can even successfully colonize and replicate within macrophages. For example, Salmonella typhimurium lives in specialized endosomes and lysosomes in host macrophages. In Chapter 3, we describe the development of optical probes that we use to characterize the interplay of endolysosomal Salmonella with host macrophages' endolysosomal enzymes. We focus on a specific family of enzymes, the cysteine cathepsins, because they fulfill anti-bacterial and pro-bacterial functions depending on their localization. We find that upon initial colonization of macrophages, cathepsins can access Salmonella in early endosome-like compartments. In contrast, at later time-points after colonization, Salmonella cause an elevation in endolysosomal pH that impairs cathepsin protease activity and prevents cathepsins from accessing their replicative niche. Taken together, these data convey a complex and changing equilibrium between a bacterial pathogen and the cells that it colonizes.
Book
1 online resource.
Cephalopod chromatophores are small organs that provide the means for much of the elaborate color changing behaviors frequently observed in squid, octopus, and cuttlefish. The vast majority of what is currently known about the anatomy, physiology, and use of chromatophores in squid derives from studies conducted on species of squid from the primarily shallow water dwelling family Loliginidae. This thesis examines the natural chromogenic behaviors and the anatomy and physiology of the chromatophore layer in the ommastrephid squid, Dosidicus gigas, and compares the findings to the chromatophores of the loliginid squid Doryteuthis opalescens. Video of free-swimming Dosidicus from the animal-borne video package Crittercam revealed two novel dynamic displays that differ greatly from the primarily static displays previously documented in loliginid squids. The "flashing" display in Dosidicus is characterized by a rapid oscillation of body coloration from red to white, and is likely a form of intraspecific communication. "Flickering" on the other hand consists of oscillating waves of chromatophore activity that propagate across the skin and is likely a form of dynamic camouflage. These differences in chromatophore use compared to loliginid squids are reflected in the anatomy and physiology of the chromatophore layer in Dosidicus. As in loliginid squids, L-glutamate (L-Glu) and serotonin (5-HT) are both endogenous neurotransmitters to the chromatophore system with excitatory and inhibitory effects respectively. However, the chromatophores of Dosidicus are much more sparsely innervated compared to what has been documented in loliginid squids. Furthermore, spontaneous waves of chromatophore activity are frequently observed in excised skin samples of recently dead Dosidicus. Similar waves can only be elicited in D. opalescens after 5-7 days of chronic denervation of the chromatophore layer, a process that results in almost total degradation of both glutamatergic and serotonergic axons in the chromatophore layer in D. opalescens. In both species, spontaneous wave were resistant to the inhibitory effects of the potent neurotoxin tetrodotoxin (TTX) at concentrations that abolished electrically stimulated chromatophore activity, indicating a separate control mechanism. The waves were sensitive to the inhibitory effects of 5-HT. These results indicate one role of 5-HT in the chromatophore system of both the ommastrephid squid Dosidicus and in loliginid squids may be to regulate spontaneous waves of chromatophore activity without interfering with the excitatory innervation of the chromatophores to different degrees depending on chromatophore use in each species.