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Book
online resource (xii, 132 pages) : illustrations (some color)
Global analysis of post-translational modifications (PTMs) by mass spectrometry facilitates interrogation of complex signaling networks. We have utilized this technology and developed analytical tools to combine peptide level data into high confidence PTM site identifications and to assess the quality of quantification measurements. We have applied this workflow in order to study three systems: 1) mitochondrial acetylation and phosphorylation during cardiac ischemia, 2) phosphorylation regulation Toxoplasma gondii egress from host cells, and 3) early phosphorylation events during hedgehog signaling. 1. Acetylation is a highly abundant modification in the mitochondrion, but we lack a general understanding of both its regulation and functional relevance, especially with regard to the acetyltransfer mechanism. To explore the nature of mitochondrial acetylation, we compared several features of mitochondrial phosphorylation and acetylation sites, showcasing stark differences between these two PTMs. We found that mitochondrial acetylation is biased towards ordered secondary protein structures, and that acetylation and succinylation modify the same residues in an unbiased, unenriched sample. We suggest that these results are consistent with low levels of background, non-functional acetylation. In order to understand acetylation regulation, we must know the mechanism of acetyl transfer. Our findings indicate that either a putative mitochondrial acetyltransferase or family of acyltransferases exist that operate non-specifically and recognize substrates in a matter entirely distinct from kinases, or else that a substantial fraction of observed mitochondrial lysine acylation results from non-enzymatic modification by acyl-CoAs. 2. We report the first phosphoproteomes of T. gondii and P. falciparum. These datasets demonstrated for the first time tyrosine phosphorylation in P. falciparum, and unusual phosphorylation-site motifs for P. falciparum. We observed extensive phosphorylation beyond the intercellular parasites' boundaries. In a follow-up study in T. gondii, we focused on the role of calcium-dependent protein kinase 3 (TgCDPK3), which regulates parasite egress from the host cell in the presence of a calciumionophore. We measured relative phosphorylation site abundance in wild type and TgCDPK3 mutant and knock-out parasites by quantitative mass-spectrometry using stable isotope-labeling by amino acids in cell culture (SILAC). Our results demonstrate that TgCDPK3 regulates several functions in addition to egress. We also observed novel phosphorylation events on proteins predicted to play a role in egress, and which implicate TgCDPK3 in regulating other calcium-dependent signaling pathways, including TgCDPK1. 3. Finally, we have initiated a study of the early phosphorylation signaling events in response to hedgehog signaling. Here, we report preliminary results that implicate a kinase, casein kinase 2 (CK2), in hedgehog signaling. Work is currently being undertaken in the Scott Lab to further characterize the role of CK2 in hedgehog signaling while we analyze biological replicates to expand proteome coverage and validate our initial hits.
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Book
online resource (x, 116 pages) : illustrations (some color)
Epithelial cells sometimes display polarity along the axis orthogonal to the apicobasal axis -- a phenomenon referred to as planar cell polarity (PCP). This polarization biases organization of the cytoskeleton, and is required for a range of fundamental processes including, but not limited to, oriented cell division, polarized function of cilia, and the control of collective cell migration events during development. Consequently, deregulation or dysfunction of PCP genes has been linked to disorders including deafness as a result of misorientation of inner ear cells, neural tube closure defects because of a failure to execute convergent & extension, and tumor invasiveness. Despite having identified some of the key proteins required to achieve PCP, much is still unknown about their molecular functions. Evolutionarily conserved proteins, known as the core PCP proteins, are distributed asymmetrically within wing epithelial cells and form complexes within the proximal or distal cortical domains. Here, we demonstrate that these diametrically opposed complexes participate in a bi-directional negative feedback loop that simultaneously produces their asymmetric distribution within cells and reinforces the orientation of polarity to neighbors by transmitting polarity information intercellularly. We found that one of the core PCP proteins, Prickle (Pk), is important for negative feedback as it is required to mediate the exclusion of distal and proximal complexes from the plasma membrane during acquisition of asymmetry. Moreover, we found that the level of Pk is regulated by the Cullin1 (Cul1)/SkpA/Supernumerary limbs (Slimb) E3 ubiquitin ligase complex. Our observations lead us to believe that loss of, or excess, Pk perturbs PCP signaling through the disruption of feedback. Ultimately, these results highlight an underlying molecular mechanism for the generation of planar polarity. Finally, the approaches presented in this monograph can further help elucidate the molecular functions of the other core PCP proteins.
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Book
online resource (xvi, 99 pages) : illustrations (some color)
Cardiomyopathies are diseases of the myocardium resulting in heart failure, arrhythmia, and sudden death. Cardiomyopathies represent a major cause of morbidity and mortality in all age groups and recent application of human genomic technologies has revealed thousands of gene mutations that cause inherited and sporadic cardiomyopathies. Many mutations in the beta-cardiac myosin heavy chain (beta-MHC) gene, which encodes the motor that powers ventricular contraction, have been identified to cause inherited cardiomyopathies, including dilated cardiomyopathy (DCM). However, the molecular mechanisms by which these mutations alter the force generation and kinetic properties of the myosin molecule have not been elucidated. As a result, there are no available therapies targeted toward treating the underlying cause of these diseases to date. We have successfully adapted a system to produce recombinant human cardiac myosin motor domain, known as subfragment-1 (S1), using a mammalian myoblast cell line. This technique has allowed us to obtain significant quantities of highly purified recombinant human beta-cardiac myosin S1 for in-depth functional analyses of wild type and DCM-causing mutants. Characterization of biomechanical properties of DCM causing human beta-cardiac S1 demonstrated that DCM mutations results in an overall hypo-contractile state, although the fundamental mechanistic changes that lead to decreased power output vary. In the future, more complex systems can be analyzed by performing assays with regulated thin filaments instead of actin and double-headed myosin motors to gain further insights into the effects of beta-MHC mutations on cardiac myosin function. Furthermore, using emerging induced pluripotent stem cell (iPSC) technology, insights into the effects of these mutations of myosin biomechanical function in vitro can be correlated with their effects on cardiomyocyte function at the cellular level. Further understanding of these mechanisms can guide the search for specific therapeutic targets and lead to the development of small molecules to modulate the effects of the DCM-causing mutations and potentially prevent or reverse this clinically devastating disease.
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Book
online resource (xvi, 206 pages) : illustrations (some color)
Organismic evolution involves both selective and neutral forces, although their relative contributions are often unknown. This thesis proposes novel statistical methods for analyzing genetic data from a variety of organisms, including yeast, Mycobacterium tuberculosis, and humans. The chapters of this thesis provide complimentary perspectives on the relative roles of selection and neutrality, from the molecular to the population level, and present various statistical tools for genetic data analysis. Chapter 2 proposes a maximum-likelihood based method with which to classify and identify interactions, or epistasis, between pairs of genes. Chapter 3 details a study of genetic data from Mycobacterium tuberculosis isolated from human Aboriginal Canadian communities; our analyses suggest that the bacterium spread to these communities via the Canadian fur trade in the 18th and 19th centuries. Chapter 4 discusses the detection of signatures of natural selection in the genomes of 12 diverse African human populations, and proposes novel considerations for identifying biological functions under selection and for comparing signals of selection between populations. Finally, Chapter 5 details the inference of the genetic basis and evolutionary history of light skin pigmentation and short stature in the genetically diverse [not equal to sign] Khomani Bushmen of the Kalahari Desert of South Africa, believed to be one of the world's most ancient human populations. These chapters emphasize that a more complete understanding of the evolutionary history of humans and other organisms requires not only the consideration of neutral and selective processes, but also both phenotypic and genetic information. The statistical methods and approaches presented in the following chapters have the potential to improve inferences of natural selection and demography from genetic data, as well as provide insight into the relative roles of both.
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Book
online resource (xiii, 90 pages) : illustrations (some color)
Using U.S. Department of Veterans Affairs (VA) records, this dissertation explores the staffing mix in substance use disorder treatment programs (SUDTPs) and the effectiveness of the current clinical practice guideline in reducing suicide risk associated with opioid therapy. The VA treats a large number of Veterans diagnosed with substance use disorders (SUDs) in four types of specialized SUDTPs at an annual cost of more than $350 million. The staffing mixes were not significantly different from 2001 to 2003, the years under review. However, given differences in treatment focus and provided services, the four types of SUDTPs may require different staffing mixes. We demonstrate how cost-effective staffing mixes for each type of VA SUDTP can be defined empirically. We derive prediction functions for benefits and costs based on patients' treatment outcomes at VA SUDTPs nationally from 2001 to 2003, and use them to formulate optimization problems to determine recommended staffing mixes that maximize net benefits per patient for four types of SUDTPs while conforming to limits of current practice. Compared to the actual staffing mixes in 2001-2003, the recommended staffing mixes would lower treatment costs while improving patient outcomes. We also introduce an approach to building a decision support system for staffing each type of SUDTP reflecting the dynamics of effectiveness and costs associated with the staffing changes. Compared to the actual staffing mixes, the staffing mixes suggested by the staffing decision support system are expected to treat significantly more Veteran patients under the existing staffing budget while improving patient outcomes. We also examine associations between adherence to guideline recommendations and the risk of suicide attempt based on Veterans Health Administration (VHA) records. Opioid therapy is often provided to reduce chronic pain in at-risk patients. Unfortunately, available opioid medications have been also used as a means for attempting suicide. In 2010, the VHA and the Department of Defense released a jointly developed clinical practice guideline (CPG) to reduce the risk of adverse events such as suicide attempt in patients prescribed opioids, primarily based on expert consensus. We conduct multivariate mixed-effects logistic regression analyses to define the associations between facility-level CPG adherence rates and risk of suicide attempt while controlling for the associations between individual-level delivery of CPG-based services and the risk of suicide attempt. Major findings include the following: suicide attempts were negatively associated with facility-level adherence rates for both consistent urine drug screenings and follow-up appointments, and were positively associated with facility-level risky sedative co-prescription rates.
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Book
online resource (x, 107 pages) : illustrations (some color)
Mobilization of the T cell response against cancer has the potential to achieve long-lasting cures. However, it is not known how to optimally harness antigen presenting cells to achieve an effective anti-tumor T cell response. In this study, we show that anti-CD47 antibody-mediated phagocytosis of cancer by macrophages can initiate an anti-tumor T cell immune response. Using the ovalbumin model antigen system, anti-CD47 antibody-mediated phagocytosis of cancer cells by macrophages resulted in increased priming of OT-I T cells (CD8+), but did not prime OT-II T cells (CD4+). The CD4+ T cell response was characterized by a reduction in Foxp3+ regulatory T cells. Macrophages following anti-CD47-mediated phagocytosis primed CD8+ T cells to exhibit cytotoxic effector function in vivo, protecting animals from tumor challenge. We conclude that anti-CD47 antibody treatment not only enables macrophage phagocytosis of cancer, but can also initiate an anti-tumor cytotoxic T cell immune response. Anti-CD47-mediated phagocytosis of cancer cells by macrophages leads to priming of a predominant CD8+ T cell response without priming of CD4+ T cells, demonstrating differential priming of T cell responses by macrophages. Priming of effector CD8+ T cells is difficult to achieve with existing vaccines for cancer and infectious diseases. Anti-CD47-based vaccination strategies serve as a promising new strategy for overcoming this challenge for treating or preventing human disease.
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Book
online resource (xxi, 169 pages) : illustrations (some color)
The fruit fly, Drosophila melanogaster, is a key model species for biological research. Trained humans can manipulate, inspect and dissect individual flies, but these operations are often rate-limiting bottlenecks for screening and experimentation. Here I present a high-speed, economical robot for handling non-anesthetized adult flies. Using machine vision the robot tracks a fly's thorax and gently grabs it ~400 ms after targeting. The robot can then translate and rotate the picked fly, inspect its phenotype, dissect or release it, and thereby rapidly prepare multiple flies sequentially for a wide range of experimental formats. In one illustration, the robot restrained flies and dissected the cuticle to permit two-photon imaging of neural dynamics. In another, the robot sorted flies by sex. The robot's tireless capacity for accurate, repeatable manipulations will enable experiments and biotechnology applications that would otherwise be totally infeasible, especially those requiring high-throughput capture, testing and assessment of individual fly attributes.
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Book
online resource (xi, 109 pages) : illustrations (some color)
Axl, Sky, and Mer receptor tyrosine kinases (RTKs) are increasingly implicated in a host of cellular responses including cell survival, proliferation, migration, phagocytosis, chemoresistance, and epithelial-mesenchymal transition (EMT). Furthermore, the Gas6/AXL system is implicated in several types of human cancer as well as inflammatory, autoimmune, vascular and kidney diseases. Since the discovery of Gas6/AXL in 1988 in chronic myelogenous leukemia, many more cancers have been found to express AXL. In chapter 1, I will describe my contribution to the field that includes identifying the role of AXL in metastatic ovarian cancer and validating the therapeutic target in ovarian cancer. In the second chapter, I have investigated the role of AXL in chemoresistance in ovarian cancer. Within the past decade, AXL crosstalk has emerged as a dominant pathway for ligand-independent activation. With the recent identification of AXL as an important factor in mediating tyrosine kinase inhibitor resistance, understanding this cross-talk is important. There has been a recent report of c-MET and AXL cross-talk in breast cancer. c-MET has become a potential target for cancer therapy with numerous clinical trials using c-MET inhibitors as monotherapy or in combination with other targeted agents or chemotherapy. I have further explored this cross-talk in ovarian cancer. In my final chapter, I have identified AXL expression in ovarian cancer tumors formed in a conditional genetically modified invasive ovarian cancer mouse model. In the future, AXL therapy should be further investigated in patients with metastatic ovarian cancer.
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Book
online resource (xvi, 195 pages) : illustrations (some color)
The skin is a classical example of a tissue maintained by stem cells, but the identity of the stem cells that maintain the different epidermal compartments and the signaling mechanisms that control their activity remain unclear. Using lineage tracing and quantitative clonal analyses, we show that the Wnt-target gene Axin2 marks epidermal stem cells that compete neutrally and require Wnt/[beta]-catenin signaling to proliferate. By RNA in situ hybridization, we show that the Axin2-expressing stem cells produce their own self-renewal signals in the form of Wnt proteins. These cells also express secreted Wnt inhibitors, including Dkks, which accumulate at high levels around more differentiated cells. We propose a new model for skin maintenance, in which epidermal stem cells produce short-range Wnt signals to maintain their own identity and function, while simultaneously secreting longer-range inhibitors that suppress Wnt signaling to promote differentiation of the stem cell progeny.
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Book
online resource (xvii, 183 pages) : illustrations (some color)
A majority of biological microscopy investigations involve the focusing of visible light with conventional lenses. Fluorescence microscopy is one of the most widely used tools in biology but its resolution has historically suffered from the diffraction limit to about 200 nm laterally and 800 nm axially. In the past decade, this resolution problem has been overcome by the rapidly emerging field of super-resolution microscopy. The first demonstrated super-resolution technique, STimulated Emission Depletion (STED) Microscopy, is the topic of this Dissertation. This Dissertation has two primary areas of focus: the design optimization of a STED microscope, covered in Chapters 2-4, and its application to super-resolution imaging in cells and tissues, covered in Chapters 4-6. Chapter 2 describes the STED apparatus and experimental methods used. This chapter covers the guiding principles behind the design of a STED microscope, which forms a basis for understanding the logic underlying the homebuilt STED microscope which was constructed for this research. This STED microscope has a typical resolution of approximately 60 nm (full-width-at-half-maximum) or 25 nm (sigma) and has the sensitivity to image single fluorophores. In Chapter 3, a framework for evaluating and optimizing STED performance in the presence of several key tradeoffs is presented. Chapter 4 describes both developments in STED Microscopy required to utilize far-red-emitting dyes and the challenges associated with performing super-resolution imaging in intact Drosophila tissue. In Chapter 5, the optimization of labeling density revealed the 9-fold symmetry of a centriole protein structure, an important organelle in cell development. In Chapter 6, Huntingtin protein aggregates are resolved beyond the diffraction limit in a cell model of the neurodegenerative Huntington's disease.
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Book
online resource (xxviii, 140 pages) : illustrations (some color)
The ability of organic chemists to design and synthesize functional molecules has revolutionized the ways in which we can probe biological systems. From catalysts capable of releasing bioactive molecules from bioinactive precursors to oligomers that enable or enhance the uptake of molecules across cell membrane and cell wall barriers, the work described herein emphasizes the power of designing for function. These new tools allow for control over the location of many biologically relevant molecules, including drugs, probes, imaging agents, metabolic modulators, and pesticides, with ramifications for a variety of biochemical, agricultural, and medicinal applications. Chapter 1 reviews the development of selective catalysts designed for use in biological systems. The catalysts have primarily been used for bioconjugations, expanding the classical repertoire of bioconjugation techniques to allow for selective chemistries at many more functional groups than was traditionally possible. Emerging strategies for imaging in biological systems using transition metal catalysis are also reviewed, as are transition metal-based catalytic therapeutics. The synthesis and evaluation of a novel bioorthogonal ruthenium catalyst designed for release of biologically active molecules from inactive precursors is the focus of Chapter 2. Although the challenges associated with using transition metals in biological systems are apparent, creative design ultimately led to the development of a useful bioorthogonal catalyst / substrate pair. This system allows for real-time visualization of transition metal catalysis to generate a biologically active compound that releases photons when it encounters its intracellular enzyme target. Chapters 3 and 4 detail the synthesis and application of molecular transporter scaffolds. Chapter 3 introduces a novel organocatalytic ring-opening oligomerization of guanidinylated cyclic carbonates to access molecular transporter scaffolds in 1 step. This strategy allows for rapid access to molecular transporters of varying lengths. In addition, the synthesis allows for concomitant probe or drug attachment and the carbonate-based backbones of the resulting transporters are biodegradable on a timescale allowing for cellular uptake and intracellular degradation. The ability of molecular transporters to cross not only cell membranes, but also cell walls, is discussed in Chapter 4. These studies focused on the delivery of small molecules and proteins across the algal cell wall and cell membrane barriers using D-octaarginine-based molecular transporters. With this method, it was shown that fluorescein-octaarginine conjugates were able to cross these barriers in a variety of algal species from the class Chlorophyceae, although several species showed no uptake or only cell surface staining. It was also shown in the algal model organism Chlamydomonas reinhardtii that octaarginine-protein conjugates could cross the cell wall and cell membrane barriers to deliver a functional protein to the intracellular algal space.
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Book
online resource (xxii, 210 pages) : illustrations (some color)
The work presented herein is concerned with the development of biophysical methodology designed to address pertinent questions regarding the behavior and structure of select pathogenic agents. Two distinct studies are documented: a shock tube analysis of endospore-laden bio-aerosols and a correlated AFM/NanoSIMS study of the structure of vaccinia virus. An experimental method was formulated to analyze the biological and morphological response of endospores to gas dynamic shock waves. A novel laser diagnostic system was implemented to provide time resolved data concerning the structural decomposition of endospores in shock-heated flows. In addition, an ex situ methodology combining viability analysis, flow cytometry and scanning electron microscopy was employed to both assess the biological response of the endospore to the shock event, as well as to provide complementary data regarding the structural state of the treated endospore. This methodology was implemented to model the shock wave induced response of a variety of Bacillus endospores. The results are subsequently synthesized into a theoretical framework to be employed in modeling the interaction of endospore-laden bio-aerosols with blast waves. An analytical method combining atomic force microscopy (AFM) and nanometer-scale secondary ion mass spectrometry (NanoSIMS) was developed to examine the spatial localization and depth distribution of specific biological elements in viral systems. This methodology was implemented to analyze the distribution of 13C labeled fatty acids as well as 15N labeled thymidine in individual nanometer sized vaccinia viral particles. Based upon the 13C and 15N signals, three-dimensional depth-resolved data regarding the architecture and localization of the virion lipid membrane and the nucleoprotein complex was generated. In addition, this methodology was employed to provide direct correlation of architectural and chemical data for isolated sub-viral structures. The technique and results presented herein represent a novel tool for the structural and chemical study of both intact viral particles as well as specifically targeted sub-viral elements.
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Book
online resource (xviii, 204 pages) : illustrations (some color)
Our bones are complex and beautiful structures that highlight that Nature is a masterful materials scientist. These composite structures of minerals, proteins, and cells are capable of maintaining a remarkable, ever-changing balance based on an individual's biomechanical needs. Growing, running, jumping, sitting, sleeping -- all of our actions and inactions are chronicled and inform the processes of new bone formation and old bone resorption. The hierarchical microstructure, building from calcium phosphate nanocrystals embedded in collagen fibers, underscores the importance of mineral and organic components that synergistically contribute to the toughness of bone needed daily. Unfortunately, due to trauma or disease, at times our bones fail and are unable to heal themselves. It is for these instances that the field of Regenerative Medicine works to develop therapies built on expertise from materials science, engineering, and medical fields. Using protein engineering and bone biology as the starting foundation, my thesis work has focused on the development of two protein-engineered biomaterials for the improvement of regenerative medicine therapies focused on osseointegration of implants and bone regeneration. Engineered protein biomaterials harness the extensive toolkit provided to us by Nature, which includes the machinery to synthesize protein materials and myriad functional pieces to mix and match in our novel designs. With these tools I've helped develop an engineered elastin-like protein to be a photocrosslinkable, cell-adhesive, thin-film coating to improve the osseointegration of implants used to stabilize fractures. The material demonstrates increased speed and extent of cell attachment to coated surfaces, serving as proof of principle for use of this material in stimulating integration of coated implants through improved implant-cell interactions. Focusing my attention on non-healing skeletal defects, I worked with MITCH, our Mixing-Induced, Two-Component Hydrogel, to develop it for stem cell delivery and bone regeneration applications. MITCH employs molecular recognition of a peptide domain binding pair for gentle, on-demand, 3D cell encapsulation at constant physiological conditions. Further using this binding strategy to emulate the intimate interface between organic and mineral phases in native bone by crosslinking mineral nanoparticles into the hydrogel network via specific molecular interactions, I created a material capable of delivering adipose-derived stem cells and stimulating fast bone regeneration in critical-size calvarial defects. Regenerative medicine brings together the renewing power of stem cells and the rational design of biomimetic niches to help the body heal when it is incapable of doing so without assistance. Taken together, this body of work validates the strategy of designing protein-engineered biomaterials by taking cues from Nature to further the development of regenerative medicine therapies, improving their success and widespread adoption.
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Book
online resource (ix, 189 pages) : illustrations (some color)
Nervous system function, from sensory perception to motor control, requires precisely patterned networks of synaptic connections. Each synapse is the fundamental unit of connection and proper establishment of this complex structure is essential for cognitive processes. Therefore, elucidating the molecular mechanisms and characterizing the proteins important for the development and structural organization of a synapse is fundamental to our understanding of the brain. Using C. elegans, I have explored the process of presynaptic development for my thesis work. I demonstrate that specification and initiation of synapse formation by clustering of SYG-1/Neph and SYG-2/Nephrin, two immunoglobulin superfamily transmembrane proteins, is dependent on their cell adhesion strength and interaction topology during development. Once this extracellular instructive cue is established at nascent synapses, an Arp2/3 dependent actin cytoskeleton is built locally that is dependent on the WVE-1/WAVE regulatory complex (WRC). This is mediated by interaction between a WRC interacting receptor sequence (WIRS) motif on the intracellular tail of SYG-1, which recruits the WRC. Next, an adaptor protein NAB-1/Neurabin binds to F-actin and recruits active zone proteins, SYD-1 and SYD-2/Liprin-[alpha] by forming a tripartite complex. SYD-2/Liprin-[alpha] is a key scaffold protein that interacts with multiple active zone components including UNC-10/Rim, ERC/ELKS-1, and GIT to form the synaptic active zone. SYD-2/Liprin-[alpha]'s pro-synaptogenic function can be regulated by interaction between its N-terminal coiled-coil domains and C-terminal sterile alpha motif (SAM) domains. Taken together, this insight into how synapses assemble in a simpler organism may lead to better understanding of development and function of the mammalian brain and its dysfunction in disease.
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online resource (xi, 134 pages) : illustrations (some color)
In response to hyperosmotic shock, cells of the budding yeast Saccharomyces cerevisiae lose up to 50% of their volume. This drastic decrease in cell size results in excess plasma membrane, which forms large infoldings that must be quickly removed. How these plasma membrane invaginations are resolved to restore the cell surface is not well understood. We show that hyperosmotic shock activates calcineurin, the Ca2+/calmodulin-dependent protein phosphatase, leading to restoration of normal membrane morphology. During hyperosmotic stress, actin patches (sites of endocytosis) become depolarized; we find that under these conditions calcineurin accumulates at sites of polarized growth and promotes actin patch repolarization. Hyperosmotic stress causes calcineurin to bind to the yeast synaptojanin Inp53/Sjl3 and dephosphorylate its proline rich tail. Dephosphorylation by calcineurin activates Inp53, which in turn dephosphorylates PI(4,5)P2 at the plasma membrane and promotes repolarization of the actin cytoskeleton. Consistently, cells lacking the partially redundant synaptojanins Inp51/Sjl1 and Inp52/Sjl2 require calcineurin for growth even in the absence of hyperosmotic stress, and display abnormal plasma membrane morphology when calcineurin activation of Inp53 is blocked. Finally, we find that calcineurin binding to Inp53 stimulates its association with the yeast amphiphysin Rvs167, suggesting model where calcineurin stimulates Inp53 and Rvs167 mediated membrane scission, promoting recovery from excess membrane stress and allowing resumption of polarized growth. In neurons, periods of intense synaptic vesicle release also result in excess membrane, and calcineurin stimulates association of synaptojanin with amphiphysin to promote synaptic vesicle endocytosis. Our findings in yeast suggest that stimulation of endocytic complex formation by Ca2+/calcineurin is a fundamental and conserved feature of the eukaryotic response to excess plasma membrane.
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online resource (x, 143 pages) : illustrations (some color)
Recurrent gene fusions and chromosomal translocations have long been recognized for their roles in oncogenesis. This dissertation employs genomic approaches to discover and characterize novel gene fusions in several cancer types. First we developed a "breakpoint analysis" pipeline for gene fusion discovery and applied this method to a collection of nearly 1,000 human cancer samples profiled on DNA microarrays. This approach led to the discovery and characterization of twelve new gene fusions in diverse cancer types including angiosarcoma, pancreatic cancer, and colon cancer. Separately, we performed RNA Sequencing on a series of 36 breast cancer specimens and used a suite of computational tools developed in-house to discover ~350 candidate gene rearrangements. Notably, we discovered recurrent fusions of the sterile 20 (STE20)-like kinase TAOK1, and functional studies suggest that these fusions encode potent oncoproteins that drive breast carcinogenesis. Many of the alterations discovered in this dissertation represent the first gene fusions reported to date in the corresponding cancer type, and many represent potentially druggable targets with therapeutic implications for patients.
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online resource (xiii, 199 pages) : illustrations (some color)
Catalytic promiscuity, the property of enzymes possessing low levels of activity toward non-cognate reactions, can be exploited as a functional tool to investigate conserved and non-conserved mechanism of enzyme specificity and catalysis between members in the same superfamily. Members of the main branch of the Alkaline Phosphatase (AP) superfamily have a structurally conserved core and active site bimetallo site. Other active site features that confer specificity for a given reaction differ between the families. Families within this superfamily catalyze a wide range of reactions, and enzymes within different families show catalytic promiscuity toward reactions catalyzed by other families within this branch. Structural comparisons and phylogenetic analysis suggests that all of the families in the superfamily arose from a common ancestor whose active site consisted of the bimetallo site alone, absent of peripheral, specificity determining features. Experimental data with a member of the nucleotide pyrophosphatase/phosphodiesterase (NPP) family suggests that a "minimal" mutant version of this enzyme that lacks peripheral, specificity determining features, has equal activity toward the two major reactions catalyzed by the AP superfamily, phosphate monoester and phosphate diester hydrolysis reactions. Together, the structural comparisons, phylogenic analysis, and experimental results lead to the hypothesis that the common ancestor of the AP superfamily is a "generalist." By using a variety of techniques, we provide support for a mechanism of evolution in which a "generalist" enzyme may give rise to multiple enzymes specific for, related, but individual reactions. Support for this mechanism, first proposed in 1976, has, until, now been limited. We suggest that this "generalist" mechanism is a valid mechanism for the evolution of ancient enzymes, present in the early evolution of life, into diverse superfamilies in which each family possesses specificity for one given reaction.
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online resource (xvi, 192 pages) : illustrations (some color)
Accurate maintenance and transmission of DNA from one cell to another is crucial for the propagation of a species, as accumulation of random mutations can result in loss of critical cellular functions. During DNA replication, DNA lesions encountered by the replication machinery cannot be repaired, as unwinding of the parental DNA separates the damaged DNA from the undamaged template which would normally be used for repair. These replication-blocking lesions can be seriously detrimental to the cell, as stalled replication forks can collapse into double-stranded DNA breaks and lead to genomic rearrangements. To prevent the accumulation of stalled and collapsed forks, the cell uses DNA damage tolerance (DDT) pathways to bypass the DNA lesion and complete replication. There are two known DDT pathways -- translesion synthesis, which uses specialized translesion synthesis polymerases to replicate directly over a DNA lesion, and template switching, which promotes invasion into the sister chromatid and extrusion of the newly synthesized strand for use as a template. These processes rely on a series of E3 ubiquitin ligases in mammalian cells, including Rad18, SHPRH, and HLTF. As a result, we wished to examine the role of ubiquitin modification, and ubiquitin-related SUMO modification, on the control of DDT. Together, the data presented here provide important new insight into how cells control the response to DNA damage and, importantly, how this response is repressed in the absence of damage. As misregulation of Rad18 and SHPRH, as well as several other DDT components, has been implicated in cancer development and progression, knowing more about this regulation may help us understand how cells avoid the generation of mutations, and ultimately the development of disease.
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online resource (viii, 88 pages) : illustrations (some color)
Choosing the right physician has important consequences for patient satisfaction and health outcomes. How do people decide which physician to choose? Although research has demonstrated that how people actually feel (their "actual affect") influences their health care preferences, we predicted that how people ideally want to feel (their "ideal affect") would play an even more important role. Consistent with this prediction, the more college students (Study 1) and community adults (Study 2) wanted to feel high arousal positive states on average ([ideal HAP]; e.g., excited), the more likely they were to choose a HAP-focused (vs. low arousal positive [e.g., calm] or LAP-focused) physician. Experimentally increasing the value of HAP also increased participants' choice of a HAP (vs. LAP) physician (Study 3). Wanting to feel low arousal positive states (ideal LAP) did not predict physician choice until participants were given a neutral (non-emotional) option: under these conditions, ideal LAP predicted choice of the LAP physician, and ideal HAP predicted choice of the HAP physician (Study 4). Across studies 2-4, the association between ideal affect and choice was mediated by perceived physician trustworthiness. When community adults were assigned to either a HAP or LAP virtual physician (Study 5), ideal HAP predicted greater self-reported adherence to the HAP physician's recommendations, and ideal LAP predicted greater self-reported adherence to the LAP physician's recommendations. Across all five studies, actual affect did not predict preferences for physicians. These findings suggest that people choose and listen to physicians who express the affective states that they ideally want to feel, in part because they trust those physicians more. Together, these studies demonstrate the importance of ideal affect in health-related decision making.
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online resource (ix, 210 pages) : illustrations (some color)
During embryonic development, dynamic remodeling of the chromatin landscape is required for the transcriptional machinery to access the genomic DNA in a spatiotemporally controlled manner. This intricate level of regulation allows precise gene regulation, necessary for complex morphogenetic processes. The Brg1/Brm-associated factor (BAF) complex is a multisubunit, ATP-dependent chromatin remodeling complex. Among the subunits, Brg1 is an ATPase that is critical for the function of the complex. Here, we study the role of Brg1 in foregut and cardiac outflow tract development. In the foregut, Brg1 functions with HDAC proteins in the ventral epithelium to regulate Nkx2-1 expression, essential for foregut septation into trachea and esophagus. In the absence of Brg1, embryos develop a single-tube foregut, reminiscent of human patients with tracheoesophageal fistula. In the developing heart, Brg1 functions in the secondary heart field-derived myocardium to control Semaphorin 3c (Sema3c) expression, essential for the outflow tract to septate into pulmonary artery and aorta. Deletion of Brg1 in the secondary heart field results in decreased neural crest cell colonization of the outflow tract, leading to a phenotype similar to the persistent truncus arteriosus pathology. Brg1 cooperates with HDAC proteins and Chd7 in the outflow tract myocardium to control Sema3c expression. These studies demonstrate novel functions and molecular mechanisms of Brg1 in foregut and cardiovascular development.
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