David Levin - US grants

Long Term Objectives: Since magnetic resonance (MR) imaging, x-ray computerized tomography (CT), and positron emission tomography (PET) provide complementary information about brain anatomy and function, a given patient often undergoes more than one of these procedures. The physician is then presented with a large number (e.g. 100) of cross-sectional images which may have different geometrical orientations and which portray different aspects of brain anatomy and function. He must integrate this huge amount of fragmented information into a coherent mental picture of the brain. The long term objective of this proposal is to develop software for displaying this information in an explicitly integrated fashion. Specifically, spatial integration will be achieved by using computer graphics to create 3-D renditions of brain anatomy or function from each modality. Multimodality integration will be accomplished by fusing these separate 3-D views into a single, comprehensive model of the brain. Interactive software will be developed for exploring and manipulating this 3-D model, so that anatomical and functional information can be examined at any point and from any viewing angle. This software is expected to be useful for medical diagnosis, surgical planning, radiation therapy planning, and medical education. Specific Aims: Preliminary work in the applicant's laboratory has demonstrated the feasibility of achieving the following aims: 1. Techniques will be developed for using MR images to create 3-D renditions of the surface of the brain, surface of the skin, selected internal structures (e.g. lesions), and blood vessels. The accuracy of each type of rendition will be measured experimentally. 2. PET data will be used to create 3-D views of metabolic activity in the brain's cortex. An existing technique for retrospective image registration will be used to fuse the data from MR and PET into a single integrated 3-D model of brain anatomy and function. Clinical tests of the accuracy of the combined display will be performed. 3. Software "switches" will be developed so that the user can view any combination of anatomical features from MR and functional data from PET. Other tools will enable the operator to "roam through" the 3-D model and inspect cross-sectional images at any selected point. Software for surgery simulation will be written so that the user can "rehearse" surgical procedures on the 3-D model.

DESCRIPTION (Adapted from Applicant's Abstract): Members of the family of serine/threonine-specific protein kinase known collectively as protein kinase C (PKC) are thought to play a pivotal role in the regulation of metazoan cell proliferation through their activation by growth factors and other agonists. The long-range goal of this project is to use a combined molecular genetic and biochemical approach to understand the role of PKC in the growth control of the budding yeast, S. cerevisiae. The PKC1 gene of S. cerevisiae encoded a homolog of mammalian isozyme of PKC.Loss of PKC1 function results in a cell cycle-specific osmotic stability defect. Specifically, mutants in PKC1 undergo cell lysis shortly after bud emergence. Thin section electron microscopy of pkc1-arrested cells reveals small holes in the cell walls located at the bud tips - the site of new growth in budding yeast. This suggests a defect in the ability of pkc1 mutants to remodel their cell walls to accommodate new growth. Electron micrographs also reveal a thinning of the beta-glucan layer of the cell wall in the region surrounding the bud tip. The level of beta- glucan synthase and beta-glucanase activities in yeast cell extracts lacking PKC1 function will be examined to determine if either activity is regulated by PKC1. A class of dominant extragenic suppressors of a pkc1 deletion results from activation of novel protein kinase that may act downstream of Pkc1p. Deletion of the suppressor gene, designated BCK1, results in a cell lysis defect similar to that of pkc1 mutants. The sequence changes identified in suppressor alleles of BCK1 suggest that, under normal conditions, Bck1 may be activated in response to phosphorylation by Pkc1p. To test this hypothesis, protein kinase assays for Pkc1 and for Bck1p will be developed initially. Then, the ability of Pkc1p to phosphorylate Bck1 will be tested, and the effect of such phosphorylation on the protein kinase activity associated with Bck1p will be examined. Finally, if these initial experiments indicate that Bck1p is regulated by Pkc1p, dominant suppressor forms of Bck1p will be tested for Pkc1p-independent activity. A distinguishing feature of PKC isozymes is their requirement of several and 3) diacylgylcerol (DAG). PKC isozymes possess a catalytic domain, which is responsible for their protein kinase activity and a regulatory domain to which activating cofactors bind. Exogenous phorbolester tumor promoter can substitute for DAG in the activation of PKC. it is this feature of phorbol esters that is believed to be responsible for their tumorigenic properties. Mutant alleles of the PKC1 gene will be generated by oligonucleotide-directed mutagenesis for use in structure/function analysis of the regulatory domain of Pkc1p. The biological effects of these mutations (as measured in yeast cells bearing chromosomal deletion of PKC1) should provide insight into the specific amino acid residues involved in cofactor activation of Pkc1p. Enzymological studies of mutant Pkc1 forms will allow direct examination of the cofactor requirements of these enzymes. Existing pkc1 mutants will be exploited for the purpose of isolating additional pathway components. Expression of presumptive cofactor-independent forms of Pkc1p results in growth inhibition of yeast cells. Genes that are involved in the regulation of Pkc1p, or that encode proteins that modulate the effects of pkc1 activity, such as specific protein phosphatases, will be isolated through their ability to suppress this growth defect. Other known cell lysis mutants will also be examined for genetic interactions with PKC1 and BCK1.

The last 15 years have seen dramatic advances in the technology for acquisition of brain images. Magnetic resonance imaging (MRI), positron emission tomography (PET), and x-ray computed tomography (CI) produce scores of cross-sectional images which play a crucial role in diagnosis and therapy. Three-Dimensional models of the patient's brain, created from these data, can be used to Perform computer simulations of neurosurgical procedures or radiation treatments. However, these methods of therapy planning have suffered a missing link namely, the absence of a convenient quantitative method of transferring these highly precise treatment plans from images to the patient's body.The PIs have recently developed an interactive localizer which bridges this gap by registering images with the patient's anatomy in a non-invasive, retrospective manner. The further development of this type of frameless stereotaxy will make it possible to perform fully quantitative image-guided neurosurgery and radiation therapy. Such methods may be less time consuming, less expensive, more convenient, and entail less post-procedure morbidity than conventional qualitative techniques. Specific Aims 1. Construction of a mobile localizer which is suitable for use in inpatient hospital rooms, clinics, operating rooms, or radiation treatment suites. 2. Development of applications software for neurosurgical planning and radiotherapy planning. 3. Testing of the neurosurgical localizer on phantoms, volunteers, and brain tumor patients. 4. Testing of the radiotherapy localizer on phantoms, volunteers, and brain tumor patients.

DESCRIPTION: Members of the family of protein kinases known collectively as protein kinase C (PKC) are thought to play a pivotal role in the regulation of a host of cellular functions in metazoans. The PKC1 gene of S. cerevisiae encodes a homolog of mammalian isozymes of PKC that is essential for cell growth. Loss of PKC1 function results in a cell lysis defect that is due to a deficiency in cell wall construction. PKC1 regulates a phosphorylation cascade that culminates in the stimulation of the MPK1 MAP kinase. This phosphorylation cascade is known as the "cell integrity pathway", because mutants that are deficient in protein kinase signaling display cell lysis defects that are exacerbated by growth at high temperature and by pheromone-induced morphogenesis. The long-range goal of this project is to use a combined molecular/genetic and biochemical approach to delineating the entire cell integrity pathway from the plasma membrane-associated receptor molecules to the ultimate nuclear and cytoplasmic targets of signaling. It is anticipated that this signaling pathway will serve as a paradigm for understanding the role of PKC signaling in growth control and stress responses in animal cells. It is also expected that identification of the components of the cell integrity pathway that are involved in cell wall construction will provide potential targets for the development of antifungal drugs that are specific to fungal species. The specific aims of this project are to: 1) identify additional genes whose expression is controlled by the cell integrity pathway. Genes that have been implicated in cell wall construction will be examined for induced expression in response to cell integrity pathway signaling. Additionally, a "promoter trap" screen has been devised to identify novel genes whose regulation is controlled by this pathway. From these studies, it is anticipated that PKC1/MPK1-dependent upstream activating sequences (UASs) will be identified in the promoter regions of regulated genes. 2) To identify and characterize PKC1/MPK1-regulated transcription factors. We will screen genomic and cDNA libraries for sequences that, when overexpressed, increase the basal level of MPK-1 dependent transcription. Potential MPK-1 regulated transcription factors will be characterized biochemically with respect to their ability to interact with UASs identified in Aim #1. 3) To determine the role of RHO1 in PKC1 signaling. The small G-protein encoded by RHO1 is required for MPK1 activation, probably by interacting with and stimulating PKC1. This notion will be tested in vitro using purified PKC1 and RHO1. The GTPase-activating protein anticipated to interact with RHO1 will also be identified using glucan synthase activity as an assay for RHO1 function. 4) To identify additional upstream components of the cell integrity pathway. Novel signaling components will be identified through synthetic lethal and suppressor screens using a conditional allele of the SIT4 gene, which encodes a Pl-4 kinase that has been implicated in PKC1 signaling.

There is substantial evidence that the central nervous system of young children is marked by "plasticity" or the ability to make compensatory changes in its functional architecture. For example, children may redevelop the ability to speak or to walk after damage to the speech or motor areas. Adults are much less likely to respond in this way. Similar phenomena have been studies in animal experiments where it has been possible to crate detailed cortical maps showing which parts of the remaining brain tissue assume the functions of damaged areas. In the last few years, magnetic resonance imaging (MRI) techniques have been developed to detect signals in functionally active parts of the brain. With appropriate computer graphic and image processing techniques, functional MRI (FMRI) signals can be combined with conventional MR images to create integrated 3-D models of cortical structure and function of individual subjects. However, these computational methods need to be automated so that it is practical to apply them to the huge volumes of data produced by fast MRI acquisitions. Our long-term goal is to develop practical computational tools and MRI technology for the creation of 3-D maps of cortical structure and function. We will use this technology to study the functional reorganization of brains of young adults who sustained anatomical brain damage at an early age. Theoretically, such experiments would increase our understanding of the spatial, temporal, and functional limits of human brain plasticity. Practically, this type of information could help neurosurgeons plan the resection of brain lesions so that important functional areas are left intact. The project will be divided into three parts: 1. Image Segmentation Methods. Software will be developed for automatic identification of the brain surface in MR head images. By greatly reducing the labor required to perform this type of image segmentation, this software will promote the routine use of MRI-derived 3-D brain models for many purposes, including neurosurgical planning and multimodality display. 2. Intraoperative Validation of FMRI. It is important to characterize the accuracy of FMRI as a brain-mapping tool. To do this, FMRI will be sued to predict the location of the sensorimotor cortex in patients scheduled to undergo resection of a small brain tumor. Stereotactic surgical methods will be used to compare the FMRI prediction with the location of the sensorimotor cortex determined by electrocorticography. 3. Brain Mapping of Hemiplegic Subjects. FMRI data and image processing will be used to create cortical maps of bilateral sensorimotor areas in normal volunteers and in age-matched hemiplegic subjects with neonatal brain damage. The spatial distribution of functional activity in these tow groups will be compared with the aid of these cortical maps and also by using a common reference frame, derived from a stereotactic brain atlas. Since the FMRI data from all subjects will be mapped into an atlas-defined reference frame, these data can be readily utilized by other investigators.

DESCRIPTION (Adapted from Applicant's Abstract): There is a growing medical need for safe and effective antifungal agents that stems from the rapidly increasing population of immune-compromised patients. Currently available therapeutic agents act on targets that are also found in mammalian cells. Because human cells do not possess the machinery needed to construct cell walls, the process of wall construction in fungal pathogens provides an attractive target for novel therapeutics. The long-term objective of this project is to understand how yeast cells maintain the structural integrity of their cell walls during growth and morphogenesis. The principal mechanism by which yeast cells detect and respond to wall stress is a signaling pathway mediated by a family of cell surface sensors, a small GTPase (Rho1), protein kinase C (Pkc1), and a MAP kinase cascade, although additional pathways also contribute to the structural integrity of the wall. The study of yeast cell wall integrity is likely to reveal suitable molecular targets for the development of antifungal agents that display selective toxicity against fungal cells. The specific aims of this project are: 1) To understand the function of the Hcs77 and Mid2 cell surface sensors for cell wall integrity signaling. Genetic and two-hybrid analyses will be used to test a model for the interaction of these sensors with the Rom1 and Rom2 guanosine nucleotide exchange factors for Rho1. If the model provides to be incorrect, three unbiased genetic and biochemical approaches will be taken to identify the signaling components with which these sensors interact. Finally, a model for these sensors as probes of the cell wall will be tested by examining the effect on signaling of length mutations in the extracellular domain of Hcs77. 2) To identify signaling components that function on a second branch of the Pkc-activated signaling pathway. Pkc1 has been proposed to regulate a bifurcated pathway with the MAP kinase cascade on one branch. A genetic screen for defect additivity with a component of the MAP kinase cascade will be used to identify signaling components that function on a second branch of this pathway. 3) To identify signals that activate the Ypk1 protein kinase, and the output of its signaling. Ypk1 and its redundant homologue, Ypk2, serve a function in the maintenance of cell wall integrity, and have been connected recently to phosphoinositide metabolism at the plasma membrane. Ypk1 protein kinase activity will be used as an assay to identify conditions that activate signaling through this molecule. The effect of Ypk1 activity on plasma membrane phosphoinositide levels will be examined in ypk1/2 mutants and under conditions of Ypk1 activation. 4) To understand the function of the Ypk1/Stt4 complex. Ypk1 resides in complex with Stt4-PI-4-kinase. Moreover, ypk1/2 mutants are suppressed by over expression of the Mss4 pI-5-kinase. These results implicate Ypk1/2 in the regulation of phosphoinositides at the plasma membrane. The level at which this regulation takes place will be explored.

[unreadable] DESCRIPTION (provided by applicant): The Ras family of small guanine 5'-triphosphatases (GTPases) comprise a group of molecular switches that link receptors on the cell surface to signaling pathways that regulate cell proliferation and differentiation. Ras proteins are highly conserved among eukaryotic species. Mutant forms of Ras that are locked in the guanine 5'-triphosphate (GTP)-bound state, and thus continuously drive cell proliferation by interacting with their target proteins, are found in 30 percent of human tumors. This application describes a novel protein in budding yeast that appears to serve two separate functions, despite the fact that it is only 68 amino acids in length. The gene encoding this protein, designated RIN1 (for Ras inhibitor 1) behaves genetically as an inhibitor of Ras, and the Rin1 protein associates in vivo with GTP-bound Ras in a manner that suggests it competes for the same binding site as Ras target proteins. Because Rin1 resides in the membrane of the ER, rather than on the cell surface with the majority of Ras, it is hypothesized that one function of Rin1 is to maintain Ras in an inactive state during its posttranslational modification in the ER. The long-term objective of this project is to develop a form of Rin1 that effectively inhibits GTP-bound Ras on the cell surface for the purpose of treating Ras-involved malignancies. The second function of Rin1 is in the processing or transport of glycosyl-phosphatidylinositol (GPI)-anchored proteins from the ER. It is hypothesized that this function is carried out through association with a second member of the Ras family that is involved in protein secretion. The specific aims of this project are: 1) To understand the membrane topology of Rin1, and to identify the Ras-interaction region. This is the first step toward developing a form of Rin1 that is optimized for Ras inhibition at the cell surface. 2) To understand the function of Rin1 in GPI-protein anchoring or secretion. This will involve identification of the second target of Rin1. It is also likely that this aim will contribute to the new area of protein sorting in the ER. 3) To determine if yeast Ras can signal from the ER, and to create a more effective Rin1-based inhibitor of Ras. This aim is focused on assessing the function of Ras expressed in the ER, and the function of the Ras-interacting domain of Rin1 expressed at the cell surface. These experiments will help to test the applicant's model of Rin1 function as an inhibitor of Ras signaling in the ER. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): There is a growing need for safe and effective antifungal agents that stems from the rapidly increasing population of immunecompromised patients. Because human cells do not possess the machinery needed to construct cell walls, the process of wall construction in fungal pathogens provides an attractive target for novel therapeutics. The long-term objective of this project is to understand how yeast cells maintain the structural integrity of their cell walls during growth and in the face of osmotic stress. These studies are likely to reveal suitable molecular targets for the development of antifungal agents that display selective toxicity against fungal cells. The principal mechanism by which yeast cells detect and respond to cell wall stress is through the Cell Wall Integrity (CWI) signaling pathway, which transmits stress signals generated at the cell surface to a GTPase switch that activates a MAP kinase cascade. However, there is an additional pathway that contributes to the structural integrity of the cell wall in response to hypo-osmotic shock. This pathway culminates in the reduction of turgor pressure by release of intracellular glycerol through the Fps1 glycerol channel. The specific aims of this project are 1) To determine the function of the Mpk1 cell wall stress MAP kinase when bound to the Paf1-RNA polymerase transcription elongation complex (PafC). Recent discoveries have revealed that Mpk1 activates a subset of its transcriptional program through a non-catalytic mechanism that involves its association with the promoters and coding regions of its transcriptional targets. New data suggests that Mpk1 moves from the promoter to the PafC. Experiments are described to dissect the function of Mpk1 within the context of this complex. 2) To establish the nature of the relationship between CWI signaling and DNA damage checkpoint signaling. Mpk1 is activated in reponse to DNA damaging agents, but its cell wall transcriptional program is not activated under these conditions. It is hypothesized that novel serine phosphorylations on Mpk1 provoked by DNA damage checkpoint kinases redirect it from cell wall targets to other functions relevant to the DNA damage response. Experiments are proposed to test this hypothesis and to identify the role of Mpk1 in this response. 3) To establish the mechanism by which a pair of novel regulators of the Fps1 glycerol channel, Rgc1 and Rgc2, activate Fps1 and to identify regulatory phosphorylation sites on these proteins in response to various stress signals. Preliminary data suggest that Rgc1/2 serve as regulatory nodes for multiple protein kinases. Experiments are proposed to identify pathway components both upstream and downstream of Rgc1/2. 4) To determine if the Pkc1 protein kinase of the CWI pathway contributes to the G2/M transition through the RSC chromatin remodeling complex. A recently-discovered physical interaction between Pkc1 and the Rsc1 subunit of the RSC complex suggests a mechanism by which Pkc1 contributes to the G2/M transition. Experiments are proposed to test this possibility.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The MAP kinase Mpk1 is a key regulatory protein controlling activation of transcription (via Rlm1) of cell wall genes. Our lab has previously shown Mpk1 has both a kinase dependent and a kinase independent mechanism of transcriptional activation. Here we try to not only identify novel binding partners of Mpk1 kinase domain, but we also wish to identify surfaces of interaction of the protein by screening an Mpk1 bait that contains surface mutations on 2 major MAPK binding areas-the substrate binding domain and the docking domain.

DESCRIPTION (provided by applicant): Cell survival depends on the ability to respond to stress signals from the extracellular environment. Diverse stress signals induce the expression of specific genes that function in the physiologic response to the stress. In the absence of stress, expression of many of these genes is maintained at a minimal level. We have found in the yeast S. cerevisiae, a model eukaryotic system, that the basal expression of many stress-induced genes is minimized by a novel mechanism - premature transcriptional termination, or transcriptional attenuation. Genes induced by cell wall stress require the MAP kinase Mpk1 to carry out two separate steps in the transcription process, neither of which requires its protein kinase activity. The first is to recruit a transcription factor to promoters of target genes. The second involves blocking attenuation, which occurs within the promoter-proximal region of target genes under non-inducing conditions. Attenuation is mediated by the Sen1 termination complex and is blocked by the translocation of Mpk1 to the elongating RNA polymerase (Pol II). Under inducing conditions, gene expression depends upon the relief of attenuation. For Mpk1-induced genes, this happens through the association of Mpk1 with the elongation factor Paf1, which blocks the recruitment of the Sen1 complex to Pol II. This interaction is conserved in the human ortholog of Mpk1, ERK5, suggesting that regulated transcriptional attenuation operates in humans. Based on our preliminary findings, we propose that a wide variety of stress-induced genes are silenced by transcriptional attenuation under non-inducing conditions and that a constellation of transcription factors are likely to relieve attenuation under inducing conditions through interactions with the Paf1C (a complex containing Paf1). The long-term objective of this project is to provide a novel approach to blocking the expression of specific genes, or groups of genes, by inhibiting relief of transcriptional attenuation. We propose to elucidate the mechanisms that regulate transcriptional attenuation and the degree to which various stresses use similar or different attenuation-relief factors to regulate a variety of target genes. One immediate goal will be to determine if other MAP kinases that respond to different signals also function as attenuation-relief factors. Another project will identify non-MAP kinase attenuation- relief factors that allow the induction of a variety of stress-induced genes we have found to be under attenuation control. A third goal will be to understand the role of the Paf1C in the recruitment of the Sen1 termination complex to Pol II. Overall, these studies will yield a mechanistic understanding of regulated transcriptional attenuation and reveal the ubiquity of the process in yeast, which will inform subsequent studies on human cells.

Even as security has long been a tenet of good programming practice, developers continue to produce insecure software resulting in a litany of data breaches and other compromises. This project aims to improve education on secure software development and add evidence to understanding methods, tools, techniques, and other factors that best contribute to writing secure code. The project centers on a novel multiphase programming competition that combines ideas from two traditionally disparate kinds of contests: those for building code and those for finding bugs in others' code. In phase one, contestants are tasked with building secure code. In phase two, contestants perform vulnerability analyses to attempt to break the code submitted by the other contestants in the first phase. The original builders finally aim to fix exploits discovered in phase two to recover lost points. Educators, practitioners, and policymakers broadly view secure code as important, and yet there is little consensus as to how best to teach and encourage secure-programming practices. By developing a competition, this project creates a setting that is more engaging to students, improving learning outcomes, and moreover enables greater insight into both practice and pedagogy through the analysis of data on how the participants approach secure programming, what techniques they use, and what methodologies succeed or fail for different programming tasks. The educational impact is significant, as the competition scales to hundreds of participants over two years, improving the design of the contest based on each offering. The artifacts and data produced by this project are made freely available to assist secure-programming endeavors across educational institutions. Finally, the students involved in the design, implementation, and execution of the contest are trained in advanced research and pedagogical methods.

Over the past few years, cloud computing services like Amazon's EC2 have commoditized computing resources. Despite this success, cloud computing primarily targets users whose virtual machines (VMs) are rarely idle, as cloud users are typically billed for the amount of time the VM is awake, not how much work it does. Thus, services that are long-lived but mostly idle a significant fraction of the time are prohibitively expensive for many potential users of cloud computing. In this project, the PIs are developing a finer-grained model of cloud computation based around lightweight instances. Lightweight instances are more akin to processes than virtual machines; they allow clients to only pay for their actual usage of cloud resources, and to not have the complexity and overhead of running an entire operating system. The lightweight instance approach has the potential to bring the benefits of cloud computing to a variety of users for whom it is a poor fit today. The PIs are exploring a new, finer-granularity model for cloud computing, wherein the cloud makes it appear that all client instances are running all the time, but may actually swap them out while idle to permit statistical multiplexing. Compared to today's VM-based model and container-based model, the proposed process-based model provides a much higher level of abstraction, and lets clients run long-lived, mostly-idle services much more cheaply than is possible today. If successful, the research would open up a wide variety of new applications and architectures: processes could cheaply be run in the cloud on behalf of end users, providing them with the ability to run long-lived services cheaply.

DESCRIPTION (provided by applicant): There is a growing need for safe and effective antifungal agents that stems from the rapidly increasing population of immunecompromised patients. Because human cells do not possess the machinery needed to construct cell walls, the process of wall construction in fungal pathogens provides an attractive target for novel therapeutics. The long-term objective of this project is to understand how yeast cells maintain the structural integrity of their cell walls during growth and in the face of osmotic stress. These studies are likely to reveal suitable molecular targets for the development of antifungal agents that display selective toxicity against fungal cells. The principal mechanism by which yeast cells detect and respond to cell wall stress is through the Cell Wall Integrity (CWI) signaling pathway, which transmits stress signals generated at the cell surface to a GTPase switch that activates a MAP kinase cascade. However, there is an additional pathway that contributes to the structural integrity of the cell wall in response to hypo-osmotic shock. This pathway culminates in the reduction of turgor pressure by release of intracellular glycerol through the Fps1 glycerol channel. The specific aims of this project are 1) To determine the function of the Mpk1 cell wall stress MAP kinase when bound to the Paf1-RNA polymerase transcription elongation complex (PafC). Recent discoveries have revealed that Mpk1 activates a subset of its transcriptional program through a non-catalytic mechanism that involves its association with the promoters and coding regions of its transcriptional targets. New data suggests that Mpk1 moves from the promoter to the PafC. Experiments are described to dissect the function of Mpk1 within the context of this complex. 2) To establish the nature of the relationship between CWI signaling and DNA damage checkpoint signaling. Mpk1 is activated in reponse to DNA damaging agents, but its cell wall transcriptional program is not activated under these conditions. It is hypothesized that novel serine phosphorylations on Mpk1 provoked by DNA damage checkpoint kinases redirect it from cell wall targets to other functions relevant to the DNA damage response. Experiments are proposed to test this hypothesis and to identify the role of Mpk1 in this response. 3) To establish the mechanism by which a pair of novel regulators of the Fps1 glycerol channel, Rgc1 and Rgc2, activate Fps1 and to identify regulatory phosphorylation sites on these proteins in response to various stress signals. Preliminary data suggest that Rgc1/2 serve as regulatory nodes for multiple protein kinases. Experiments are proposed to identify pathway components both upstream and downstream of Rgc1/2. 4) To determine if the Pkc1 protein kinase of the CWI pathway contributes to the G2/M transition through the RSC chromatin remodeling complex. A recently-discovered physical interaction between Pkc1 and the Rsc1 subunit of the RSC complex suggests a mechanism by which Pkc1 contributes to the G2/M transition. Experiments are proposed to test this possibility.

The Public Key Infrastructure (PKI), along with the Secure Sockets Layer (SSL) and Transport Layer Security (TLS) protocols, are responsible for securing Internet transactions such as banking, email, and e-commerce; they provide users with the ability to verify with whom they are communicating online, and enable encryption of those communications. While the use of the PKI is mostly automated, there is a surprising amount of human intervention in management tasks that are crucial to its proper operation. As a result, there have been numerous instances where mismanagement of the PKI has harmed the security of end users. This project is developing techniques to better understand and improve the management of the PKI, helping to better secure the Internet.

This project has four research foci, each examining the management challenges faced by different players in the PKI: Content Distribution Network (CDN) administrators, Certificate Authorities (CAs), end-users, and non-Web protocols. First, the project is conducting measurements to better understand the frequency of sharing private keys between sites and their CDNs, and to improve the security of this practice. Second, the project is developing new incentives for CAs to ensure information about their revoked certificates reach end users. Third, the project is aiming to better understand how the PKI will evolve as the Internet of Things (IoT) grows and the PKI is forced to quickly scale up. Fourth, the project will expand existing measurement approaches to understand the difficulties of PKI management in non-Web protocols (e.g., IMAPS), which have traditionally been less-well maintained.

Residential networks lack empirical information about service outages and reliability. The absence of clear information, coupled with the relative inexperience of ordinary users combines to hide the sources of connectivity and performance problems. The goal of this proposal is to provide external measurement of outages in residential Internet connections, empirically determining the reliability of different providers and technologies, and recording outage information suitable for diagnosis. Although some projects instrument home networks from within the home, this work looks from the outside, searching for rare events across many more networks and countries for comparisons. This work addresses two key challenges in measuring network reliability from the outside. The first important problem is to determine whether address changes are actually the cause for a network device becoming unreachable to external measurement. The second key problem is in separating individual failures from other failures. For example, with an approaching storm, many users may disable their equipment to avoid lightning damage, but with a passing storm, an outage may affect many users at once. Engineering the measurement infrastructure to adapt to these events and identify simultaneous outages will permit classifying outages.

Reliable networks are particularly important as users transition away from dedicated landlines toward voice-over-IP, while still needing the ability to make emergency calls, particularly during events that challenge networks such as earthquakes and severe weather. Predicting reliability permits informed decision-making about migrating to new network technologies--concretely, whether removing conventional wired lines is safe.
Datasets from this research will be public and we will maintain a website that presents Internet outage data in a manner that is accessible by the general public.

? DESCRIPTION (provided by applicant): Stress-activated MAPK signaling pathways respond to a wide variety of stress conditions, including DNA damage, oxidative stress, heat shock, endoplasmic reticulum stress, hyper- and hypo-osmotic stress, shear stress, and a growing number of chemical toxins. Despite their organization with sensors at the cell surface connected to signaling components that typically culminate in the control of transcription factors and other proteins that impact cell physiology, there is growing evidence that many stressors activate these pathways through intracellular inputs rather than by signals that emanate from the cell surface. We have developed evidence in baker's yeast that a multitude of stress signals stimulate two stress-activated MAPK pathways, the Cell Wall Integrity (CWI) pathway and the High Osmolarity Glycerol (HOG) pathway, through intracellular inputs at various points along these pathways. This proposal is focused on identifying and characterizing the stress-specific components that feed into the MAPK cascades with a special focus on DNA damage-induced activation of the CWI pathway. Aim 1 extends our recent discovery that protein kinase C (Pkc1), which is the top protein kinase of the CWI MAPK cascade is a target of DNA damage checkpoint signaling. Pkc1 plays important roles in the response to and survival of DNA damage. Thus, we propose to take a combined phospho-proteomic, biochemical, and molecular genetic approach to understanding the impact of checkpoint signaling on the regulation of Pkc1 and the role of this pathway in the maintenance of genomic stability. Aim 2 seeks to identity stress-specific components involved in the intracellular activation of the CWI pathway MAPK cascade. These will focus on DNA damage, which activates the CWI MAPK (Mpk1) without the need for regulation of its upstream pathway components. This aim will also examine cell wall stress inputs, which surprisingly enter the pathway at a point within the MAPK cascade. We will use a combination of biochemical and mass spectrometric approaches, as well as genetic screens. Aim 3 is to identify and characterize intracellular stress inputs to the HOG MAPK cascade. The toxic metalloid arsenite and citric acid both activate the MAPK (Hog1) through an input that does not require regulation of upstream pathway components. Curcumin, a promising colon cancer and Alzheimer's therapeutic, signals to Hog1 through an input within the MAPK cascade. Completion of these aims will provide novel insights into the mechanisms by which stress signals activate MAPK pathways and will delineate a novel branch of the DNA damage response pathway.

Far too many talented students are not pursuing graduate school, or a career in higher education and research. We believe this is because students do not understand what to expect in graduate school, or what such a career entails. To address this problem, University of Maryland, in collaboration with faculty at Cornell University and the Max Planck Society in Germany are starting a series of research summer schools. The goal of the school is to introduce world-class research to top quality students, and engage them, one-on-one and in small groups, with leading researchers in the field.

This proposal seeks funding for approximately 25 students from the United States to attend the first iteration of the school, which will be held on the premises of the Max Planck Institute for Software Systems, in Saarbruecken Germany, from August 8-13th, 2017. Students have applied online, and are evaluated based on grades, a statement and recommendation letters. Our central thesis is that short, hands-on experience with leaders from a wide range of interesting areas of Computer Science will effectively entice many undergraduates to commit to longer-term Research Experience for Undergraduates (REUs), graduate school, and ultimately careers in research. To evaluate this, we will track participants' progress and trajectory, both during and after the research school, using questionnaires, one-on-one advising sessions, and post-school research projects.

The research school is designed around the belief that exposing students, especially women and minorities, to the breadth and diversity of Computer Science research, especially early in their careers, will make them more likely to pursue REUs and, eventually, careers in research. Being exposed to high quality research and being able to work with leaders in their fields will make the option of a research career "real", and have a transformative impact for the positive in the attendee's academic careers.

This project designs and implements programmable system elements to be run within the anonymity networks, such as Tor (The Onion Routing network). The central idea is that users can inject new code into the network that is then run within a protected execution environment. The motivation is to enable the creation of new and significantly enhanced anonymity services, such as content distribution networks, of use in today's and future anonymity networks. The project carries potential to demonstrate that an anonymity communication network, such as today's Tor network, could deliver a much larger range of services that could greatly enhance the potential range of offerings over secure, anonymous networks, motivating further developments.

The proposed research has four main and original thrusts: (1) Programmable Tor middleboxes,
(2) Censorship-resistant hidden services, (3) Hidden-services-based content distribution network (CDN), (4) Decentralized anonymous credentials. Much thought and development will also go into the applications that can be built upon the proposed functionalities, and as a result, if successful, the end possibilities are expected to be transformative. The training obtained by students during the investigation spanning across cryptography, secure computation, and anonymized communication networks are expected to result in a cohort with a distinctive, important and useful set of skills.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

A major hurdle to having more women researchers and professors in computer science is that many undergraduate women are simply not exposed to what computer science research entails or provided opportunities to explore as a career path. This project will design and run the Tech+Research workshop, an all-female Computer Science research hackathon with the goal of highlighting the diversity and opportunities in the field of computing research. The weekend event will be hosted in collaboration with Technica (https://gotechnica.org), the largest all-female hackathon. Computing faculty from institutions across the state of Maryland will mentor groups of attendees on projects in their prospective research areas. Along with providing hands-on research experience in a dynamic hackathon setting, Tech+Research will include sessions introducing attendees to the basics of computing research and highlight the exciting opportunities that come with pursuing a graduate degree in computer science.

This hackathon-centered approach to introducing students to research has the potential to be a low-overhead step towards a more equitable, inclusive field of computer science. To measure its impact, this project will perform surveys and track the students' progress in their pursuit of Research Experiences for Undergraduates (REU) funding and graduate school. Finally, this project will share its findings and materials to help foster similar workshops at other institutions.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Project Summary Arsenic is the most prevalent toxin in the environment. This natural metalloid enters the biosphere from geochemical sources and, to a lesser degree, from anthropogenic sources. Human exposure to arsenic is mainly through food, water and air, and contamination of groundwater poses a worldwide health problem. Inorganic aqueous arsenic exists mainly as oxyanions of trivalent arsenite [As(III)] and pentavalent arsenate [As(V)]. As(V) is much less toxic than As(III), which is thiol reactive and binds covalently to cysteine residues in proteins. Chronic exposure to inorganic arsenic is associated with cardiovascular disease and hypertension, diabetes mellitus, neurological disorders, and various forms of cancer. It has been proposed that both direct modification of biomolecules by As(III) and reactive oxygen species (ROS) generated by arsenicals are responsible for its toxicity and carcinogenicity. Despite these health effects, As(III) is used as a highly effective treatment for certain types of cancers. Therefore, it is important to understand the cellular responses mobilized by arsenic-induced stress. Both As(V) and As(III) exposure stimulate the yeast stress-activated MAPK (SAPK) Hog1, whose activity is critically important for the cellular response to arsenic. We are interested in two general questions. First, how do diverse stressors activate a small number of SAPKs? We have found that many stressors activate yeast SAPKs by intracellular routes that interface with SAPK pathways in atypical ways, rather than signaling from the cell surface, which may influence the behavior of the SAPK. Second, how does the cell mobilize coherent, stress-specific outputs from an activated SAPK? This proposal centers on the cellular responses to arsenic exposure. We have developed evidence that both As(III) and its methylated metabolite, MAs(III), are important signaling molecules that allow cells to mobilize protective, stress-specific responses through modification of specific cysteine residues in target proteins. We refer to this as an arsenic stress signaling code. Aim1 extends our recent findings that cells respond differently to As(V) and As(III) exposure. We propose to understand the mechanistic bases of distinct regulatory events driven by these stressors. We will identify key targets of arsenic modification for the regulation of the glycerol channel Fps1 [the major port of entry for As(III)], and test the role of newly discovered arsenic modifications of proteins involved in the regulation of the oxidative stress response and replication initiation. Aim 2 is to understand how Hog1 activated by As(III) drives stress-specific outputs. This aim extends our recent finding that Hog1 itself is modified by arsenic and that this modification is important for its role in the response to As(III). Using mass spectral approaches, we will determine the Hog1 phosphorylome in response to As(III) and As(V) and establish whether Hog1 target specificity is altered by arsenylation. Aim 3 is to delineate the novel pathway by which As(V) activates Hog1 and to determine its significance for As(V) entry to cells. Completion of these aims will establish a novel paradigm centered on the regulatory nature of protein arsenylation.