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Record Count: 7
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DESCRIPTION (provided by applicant): Living organisms are under constant assault by mutagenic agents, including radiation from various sources and a wide range of chemicals in the environment. Cell survival is dependent upon effective repair of DNA damage caused by these agents. The most lethal form of DNA damage induced by these agents is double-strand breaks (DSBs) and, in the absence of DSB repair, cells die rapidly. However, some DSB repair pathways are dangerous because they can lead to genomic instability, resulting in chromosomal rearrangements. Chromosomal rearrangements have been implicated in oncogene activation and are the hallmark of many cancers. Therefore, characterization of pathways that predispose cells to genetic instability is critical for the understanding of tumorigenesis and may help to elucidate targets for cancer prevention and treatment. The proposed research will investigate the mechanism of break-induced replication (BIR), a poorly understood DSB repair pathway, which can lead to genetic instability. The current BIR model suggests that the junction made between the invading broken strand and the undamaged molecule initiates DNA synthesis on the intact chromosome, thereby acting as an origin of replication. This can result in copying of hundreds of kilobases of DNA from the donor molecule while a large piece of the unrepaired, broken DNA is lost during the next round of replication. BIR has been suggested to play an important role in the repair of collapsed replication forks, and also in several cancer-related phenomena, including telomere maintenance in the absence of telomerase, loss of heterozygosity, and formation of non-reciprocal translocations. The relevance of BIR to mechanisms underlying tumorigenesis has made this model a critical starting point for many branches of research. However, the basic tenet of BIR - bona fide replication to repair breaks in DNA was deduced based on genetic data but has never been demonstrated directly by physical analyses of intermediates and/or the final products of this process. The objective of the proposed research is to use two powerful technologies, dynamic molecular combing coupled with fluorescent in situ hybridization and two- dimensional gel electrophoresis, to test the central hypothesis of BIR; i.e., that DSB repair is achieved by the assembly and progression of a replication fork. The proposed research will determine the structure of BIR intermediates and products in yeast Saccharomyces cerevisiae (a model genetic organism). Also, it will estimate the speed of BIR and its ability to replicate through centromeres and known barrier sites. In addition, the roles of individual proteins involved in BIR will be identified. The proposed approach is unique because, unlike other studies of BIR that rely only on indirect genetic or population physical data, it will enable visualization of BIR in individual DNA molecules using dynamic molecular combing, while two-dimensional gel electrophoresis will help to analyze replication fork intermediates.Narrative. The proposed research is aimed to determine the mechanism of break-induced replication (BIR) by using two powerful technologies: molecular combing and two-dimensional gel electrophoresis. This research will test the basic tenet of the BIR model, namely, the assembly of a bona fide replication fork to repair double-strand breaks in DNA. This knowledge is critical to further the understanding of several cancer-related phenomena, including telomere maintenance in the absence of telomerase, loss of heterozygosity, and formation of non-reciprocal translocations.
Crisp Terms/Key Words: Saccharomyces cerevisiae, molecular genetics, centromere, fungal genetics, mutagen, mutant, genetic recombination, in situ hybridization, DNA replication, DNA repair, environmental radiation, DNA damage, telomere, hazardous substance, fungal protein, two dimensional gel electrophoresis
DESCRIPTION (provided by applicant):
Cr(VI) contamination of soil and groundwater is a significant problem worldwide. In the United States, chromate is the third most common contaminant of hazardous waste sites and the second most common inorganic contaminant found in the environment. In situ and ex situ bioremediation processes that exploit the intrinsic metabolic capabilities of dissimilatory metal ion-reducing bacteria (DMRB) remain potent, potentially cost-effective approaches to the reductive immobilization or detoxification of environmental contaminants. The microbial catalysis of Cr(VI) reduction to sparingly soluble, less bioavailable Cr(III), for example, is a promising remediation strategy for Cr(VI)-contaminated subsurface soil and groundwater environments. The genus Shewanella represents one of the few groups of microorganisms that have received intensive investigation because of their wide ecological distribution, diverse respiratory capacities, and environmental relevance. Despite several advances made in elucidating Shewanella biology as it relates to chromate transformation, fundamental questions about the specific chromate reduction mechanism remain unclear. This information gap includes (i) the identity of dedicated chromate reductase(s), (ii) the cellular localization of chromate transformation (e.g., distal appendages, outer cell surface, periplasm, cytoplasmic membrane, cytosol), and (iii) the environmental parameters under which microbial populations have the greatest specific chromate reduction rates. The problem in predicting and assessing bioremediation performance is compounded by the lack of fundamental knowledge of the molecular basis, regulatory mechanisms, and biochemistry enabling bacterial metal-reducing capabilities. We propose to engineer nanoscale methodologies, comprising of chromate-tagged nanoparticles and intracellularly grown gold nanoislands to function as enhancers for Surface Enhanced resonance Raman scattering probing to generate chemical maps of chromate reduction sites as well as to monitor the reduction dynamics in exquisite molecular and single-organism detail. Objectives of this study are to 1) assess the impact of gold nanoparticle composition, geometry, and functionality on cell viability, growth, and efficacy of microbial chromate reduction using S. oneidensis as a model system; 2) track the localization of chromate transformation at single-cell resolution using functionalized gold nanostructures as well as using intracellularly grown gold nanoislands by Raman chemical imaging; and 3) evaluate the influence of bioremediation-relevant environmental factors on chromate transport, localization, and reduction rates. The development of passive and active nanoprobes in conjunction with confocal Raman chemical imaging will constitute a significant step in enabling a platform for dynamic monitoring of intracellular events and compartmentalization of metal reduction sites at single-cell resolution. The knowledge gained from this novel study will contribute to the development of scientifically grounded strategies for improving bioremediation efficacy.
DESCRIPTION (provided by applicant): Hepatocellular carcinoma (HCC) is mainly a male disease; incidence in both men and women has increased dramatically over the past 20 yrs. Evidence indicates that estrogens protect against HCC while androgens promote it. The infant mouse treated with diethylnitrosamine (DEN) serves as a model to study these hormonal influences. Although the liver expresses estrogen receptor (ERa), the protective effects of estrogen may be mediated through prolactin (PRL); whether liver ERa or PRL receptor (PRLR) is required for the protective effect is not known. It is also unknown if liver androgen receptor (AR) is essential for androgen promotion of HCC in males. The long term goals are to determine the molecular mechanisms accounting for gender differences in HCC and to determine if environmental contaminants impinge on those mechanisms. The aims of this exploratory investigation are: 1) Determine the role of hepatic hormone receptors in modulation of liver carcinogenesis. The working hypothesis is that ERa and AR regulate expression of genes, cell proliferation and apoptosis, thereby inhibiting or promoting, respectively, tumor growth. The hypothesis will be tested using wildtype and receptor knockout mice in tumorigenesis experiments. 2) Determine the molecular mechanisms of hormonal effects in the liver. The working hypothesis is that estrogen protection and androgen enhancement derive from opposing gene regulatory events. We will determine the genes that are reciprocally regulated in the liver by estrogen and androgen. Carcinogen-initiated HCC in the mouse shares features of pathogenesis with progressive human liver disease associated with hepatitis viruses and other risk factors that ultimately result in HCC and, therefore, mechanisms of hormonal modulation of the disease process delineated in the mouse will be relevant to the human situation. These studies will also lay the basis for future investigations into the potential for endocrine disruption of the gender differences in hepatocellular carcinogenesis. Furthermore, the gene products identified may yield new biomarkers useful in predicting exposure to endocrine disrupter chemicals and other environmental chemicals that have deleterious effects in the liver.
DESCRIPTION (provided by applicant): Cyanide (CN), a prototype mitochondrial toxicant, produces progressive degeneration in select brain areas. This selective vulnerability to cyanide is characterized in vivo by apoptotic cell death in cortex and necrotic degeneration in substantia nigra. In primary cultured cells, cyanide produces a unique, cytotoxic response in which cortical cells (CX) undergo apoptosis and mesencephalic cells (MC) undergo necrosis, paralleling the regio-specific responses observed in animals. A rapid rise in cytosolic free Ca2+ and generation of reactive oxygen species are initiators of both modes of cell death. Preliminary studies show that two mitochondrial proteins (BNIP3 and UCP-2) are sensors of the initiation signals and function as co-regulators of cell death to determine which cell death pathway is executed. Proposed studies will characterize the role of BNIP3 and UCP-2 as sensors/regulators of cyanide-induced apoptosis and necrosis using primary cultured rat CX and MC cells as comparative models. To examine BNIP3 and UCP-2 as regulators of cyanide-induced cell death, changes in their expression will be produced, followed by monitoring subsequent changes in apoptotic and necrotic markers. Transient transfection with cDNA will be used to increase expression and RNA interference will produce knock down. For characterizing the link between BNIP3 and UCP-2 as functional co-regulators, cells will be co-transfected (cDNA or RNA interference) to concurrently increase or decrease expression of these proteins to determine if the mode of death can be modified temporally or switched. Upstream and downstream signaling cascades that initiate and execute BNIP3/UCP-2-mediated apoptosis and necrosis will be compared in the CX and MC expression models. Signals that activate BNIP3/UCP-2 will be characterized and then linked to recruitment of execution pathways. This will include determining how BNIP3/UCP-2 activation executes cell death by monitoring cellular redistribution/activity of apoptotic and necrotic signaling factors, including Bcl-2 proteins, cytochrome c, caspases, ATP levels and mitochondrial membrane pore transition. The long-term goal of this project is to identify mechanisms underlying cyanide-induced cell death and understand why brain areas are selectively vulnerable to cyanide. This study will provide valuable insight into neurotoxicant-induced neurodegeneration and fundamental information on regulation of cell death in the CNS.
DESCRIPTION (provided by applicant): Altered brain iron (Fe) homeostasis has been shown in idiopathic Parkinson's disease (IPD) and in manganese (Mn)-induced Parkinsonism. The current proposal continues the central theme of our long-time research goal, i.e., to explore the role of brain barrier systems in metal-induced neurotoxicities. Divalent metal transporter-1 (DMT1) and metal transport protein-1 (MTP1) are two newly discovered metal transporters and function to transport metals across the cell membrane. In the Progress Report, we have demonstrated the presence of DMT1 and MTP1 in the choroid plexus, where the blood-cerebrospinal fluid (CSF) barrier (BCB) is located. We have also observed that Mn exposure increases DMT1 expression and mobilizes subcellular MTP1 in the BCB epithelia. However, the questions as to where the DMT1 and MTP1 are subcellularly co-localized in the BCB, how they function in concert to respond to divalent-metal fluxes on both sides of the BCB, by what mechanism Mn exposure alters the expression and function of both transporters, and how the dysregulation of DMT1 and MTP1 in the BCB by Mn exposure affects brain homeostasis of Fe and Mn, remain mysterious. Thus, to understand the structural functionality of DMT1 and MTP1 in the brain barrier and their dysfunction-associated neuronal disorders, we hypothesize that the altered expression of DMT1 and MTP1 in the choroid plexus following Mn exposure contributes to Mn- induced Fe metabolism disorder in the CSF. Our specific aims are: (1) to explore whether DMT1 and MTP1 control the direction of Fe transport at the BCB by investigating the subcellular location of DMT1 and MTP1 in choroidal epithelia, by blocking or inducing DMT1 and MTP1 expression to determine the direction of Fe and Mn transport at BCB, and by using siRNA technique to silence the genes encoding DMT1 and MTP1 to investigate Fe and Mn uptake and transport kinetics under DMT1 or MTP1 knock-down conditions; (2) to explore whether in vivo chronic Mn exposure distorts the expression of DMT1 and MTP1 in the BCB and selected regional blood-brain barrier and leads to increased fluxes of Fe between the blood and CSF, by using a rat chronic Mn exposure model and by a ventriculo-cisternal perfusion technique; and (3) to explore whether Mn exposure interferes the binding of iron-regulatory proteins to mRNAs of DMT1 and MTP1, since the stem-loop structure exists in 3'-untranslated regions (UTR) and 5'-UTR in DMT1 and MTP1 mRNA, respectively. Studies proposed in this application will define the inter-relationship between DMT1 and MTP1 in the BCB with regard to their subcellular locations, roles in transport of divalent metals at the BCB, and their regulation as affected by Mn exposure; will provide insight into the molecular mechanism by which Mn affects divalent Fe transport by brain barriers; and will ultimately provide a better understanding of Fe dysfunction-related neuronal diseases such as IPD.
DESCRIPTION (provided by applicant): Manganese (Mn2+) neurotoxicity resembles a number of aspects of the dopamine (DA) neuron degenerating disorder Parkinson's disease (PD). Both PD and Mn2+ toxicity is characterized by motor deficits and damage to substantia nigra and other basal ganglia nuclei, and dopamine or its metabolites are believed to contribute to the disorder. Furthermore, expression of the pre-synaptic protein alpha-synuclein, and the oxidative stress- induced protein parkin have been proposed to contribute to the pathogenesis of both disorders, and occupational exposure to Mn2+ has been invoked to predispose individuals to PD. Despite the initial characterization of the disorder over 150 years ago, and intensive research within the past several decades, the origin of the pathogenesis and the molecular determinants involved in Mn2+ neurotoxicity have yet to be fully elucidated. A significant hindrance in dissecting the molecular components of Mn2+-induced neurotoxicity is the high complexity of the vertebrate brain and lack of facile in vivo genetic models to determine and explore the mechanisms involved in the cell death. We have developed a novel pharmacogenetic model using the genetically tractable nematode C. elegans to dissect and characterize the molecular components involved in DA neuron degeneration (see Nass et al, PNAS, 2002; Nass and Blakely, Ann. Rev. Toxicol. Pharmacol., 2003). At the molecular level, the C. elegans nervous system is highly conserved both genetically and functionally with mammals, and all the genes responsible for DA biosynthesis, packaging, and reuptake are present and functional in the worm. We have shown that the nematode C. elegans DA neurons can be selectively damaged by exposure of whole animals to the parkinsonian-inducing neurotoxin 6- hydroxydopamine (6-OHDA) (see Nass et al, PNAS, 2002)2. We have also recently shown that a brief exposure to Mn2+ causes DA neuron cell death in the worm, that prior exposure to Mn2+ amplifies the 6- OHDA-induced DA neurodegeneration, and that RNA knockdown of the divalent metal transporter-1 (DMT- 1), a putative Mn transporter, partially protects against Mn toxicity in the worm. In our model system, the expression of the green fluorescent protein (GFP) in DA neurons will allow us a facile and powerful test to examine the role that DA, its metabolites, endogenous proteins, and neurotoxins play in Mn2+ - induced degeneration of DA neurons in vivo. These studies will also include a novel genome-wide screen to identify mediators and suppressors of Mn2+-induced toxicity.
DESCRIPTION (provided by applicant):
Skilled Support Personnel (SSP) are essential to site recovery, yet they are often under-trained in hazmat response and may lack knowledge of how to cope with specific incidents. Poor training and rusty skills lead to injury to themselves and others, as well as making the overall situation worse. Beyond full instructional courses, a continuum of training and real-time performance support is needed to enable these crews to work effectively. Mini-course modules for enroute and onsite training, on-the-job performance aids and simple checklists, and access to live expert mentors who support decision making or deliver mini lesson Just-in-Place(r) should all have a place in this training and performance support package. Just-in-Place(r) is our proprietary approach to delivering content and services, tied to context relevant factors such as: time, location, skills, tasks and who or what is nearby. The project team will meet this need by building the Viyant Hazmat Skilled Support Personnel Just in Place Performance Support System (Viyant Hazmat) in collaboration with established hazmat training organizations and service providers. Viyant Hazmat will enable mobile Just-in-Place training materials, performance aids, and checklists to be accessed at the job site and permit real-time collaboration with hazmat and medical experts at remote locations. Not only will SSPs be able to talk directly with experts, the experts' situational understanding will be increased by using web collaboration tools allowing the onsite SSP to send live images and video to remotely located experts. Viyant Hazmat comprises an integrated suite of computer hardware, ranging from wearable computers to cell phones, wireless communications, mixed reality software tools, content authoring tools, administration tools and support services that are optimized for improving safety and effective decision making. The specific target audience for this Phase I effort is demolition crews at Superfund, brownfields and disaster sites. Other hazmat audiences will be addressed during commercialization. Viyant Hazmat will be built on the Company's Viyant(tm) Standard Edition platform, which is currently being used to facilitate equipment repair, maintenance and installation training and operations for technicians at a distance. Our research will include: 1) prototyping the Viyant Hazmat software system; 2) conducting system usability and ergonomics testing with demolition workers with and without personal protective equipment; 3) developing sample mini training modules, performance aids and checklists based on existing training materials from partner service providers; 4) testing learning effectiveness of incident-specific training materials; and 5) field testing the system.
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