ALTERED CREATINE KINASE ENERGY METABOLISM IN HUMAN HEART FAILURE
G Robert, Professor
Johns Hopkins Universitycity: Baltimore country: United States (us)
Grant 5R01HL061912-12 from National Heart, Lung, And Blood Institute
Abstract: Heart failure (HF) is the final common pathway for most cardiovascular diseases and is associated with high mortality and morbidity. Because ATP is required for normal myocardial contractile function, it has been hypothesized that impaired ATP synthesis and/or delivery contribute to contractile dysfunction in HF patients. Our focus is on the creatine kinase (CK) reaction which generates ATP at the contractile elements, is the major cardiac energy reserve responsible for buffering ATP, and which is particularly critical because of the temporally varying energy demands of the beating heart and the spatial heterogeneity of the ATP generating and utilizing reactions within myocytes. We developed the first non-invasive means to measure the rate of ATP synthesis through myocardial CK in the human heart and observed significant reductions in human HF of sufficient magnitude to theoretically limit ATP delivery during the cardiac cycle. In other patients, the kinetics of ATP turnover through CK distinguished failing and non-failing hearts. This body of clinical evidence demonstrates a critical role for reduced CK capacity in human HF and is now supported by causal evidence that reduced CK contributes to dysfunction and remodeling in a murine model of HF. These recent, novel clinical and basic results from our laboratory provide the justification and rationale for studies to address important mechanistic and clinically-relevant issues concerning the prognostic and causal roles of reduced CK capacity in human HF as outlined in these specific aims. The first three aims examine the hypothesis that reduced CK capacity is a causal factor in progressive contractile dysfunction. They do so by testing whether it is an independent predictor of important clinical HF outcomes, including mortality, cardiac transplantation and HF hospitalization (first aim), that HF medications known to improve survival and clinical outcomes improve cardiac CK flux and energetics (second aim) and that reduced CK flux occurs when contractile dysfunction is first recognized in patients with malignant disease receiving cardiotoxic but also lifesaving chemotherapy (third aim). We will also examine two more mechanistic questions that can only now be tested in HF patients due, in part, to new higher field 3T MR scanners. These are whether spatial heterogeneity in CK flux and energetics across the myocardial wall contributes to dysfunction in HF patients and whether the temporal heterogeneity in ATP demand during the cardiac cycle is an important factor related to reduced CK buffering capacity on impaired function (fourth aim). The latter would have direct clinical relevance in terms of the importance of heart rate interventions in this patient population. We believe these proposed, truly translational studies address critical and timely questions of mechanistic and clinically-relevant importance. Our group is uniquely qualified and the timing is ideal to exploit recent observations and new technology to advance our understanding of the role of impaired ATP delivery through CK in human HF. The pumping action of the heart, like an engine, requires chemical fuel and the failing heart has been hypothesized to be low on fuel. The chemical fuel used by the heart is ATP and we recently developed the first means to measure the rate of ATP turnover in the human heart and find it is significantly reduced in heart failure. The proposed studies are important because they will determine whether that reduction in ATP rate predicts heart failure progression in death, and what the underlying mechanisms are that could be targeted for future heart failure therapy
Keywords: Address; Adrenergic beta-Antagonists; Affect; ATP Hydrolysis; ATP Synthesis Pathway; Attention; base; Beryllium; Bioenergetics; Buffers; Carbon; Cardiac; Cardiomyopathies; Cardiovascular Diseases; Cardiovascular system; Cessation of life; Chemicals; chemotherapy; Clinical; Clinical Data; clinical practice; Clinical Research; Clinical Treatment; clinically relevant; Creatine Kinase; Creatine Kinase MB Isoenzyme; Data; design; Dilated Cardiomyopathy; Disease; EFRAC; Elements; Energy Metabolism; Energy Transfer; Enzymes; Event; Exhibits; experience; Free Energy; Functional disorder; Funding; Future; gene therapy; Grant; Health; Heart; Heart failure; Heart Rate; Heart Transplantation; Heterogeneity; Hospitalization; Human; improved; inhibitor/antagonist; inorganic phosphate; insight; interest; Intervention; Kinetics; Laboratories; Left Ventricular Hypertrophy; Magnetic Resonance; Malignant - descriptor; Measures; Metabolic; Metabolism; Modeling; Morbidity - disease rate; Mortality Vital Statistics; Mus; Muscle Cells; Myocardial; new technology; novel; Outcome; Pathway interactions; patient population; Patients; Pharmaceutical Preparations; Phosphocreatine; Process; prognostic; Pump; Qualifying; Reaction; Relative (related person); Research; Role; Severities; Stress; Techniques; Technology; Testing; Time; translational study; Translations; Tyrosine; United States National Institutes of Health; Work
Relevance: The pumping action of the heart, like an engine, requires chemical fuel and the failing heart has been hypothesized to be low on fuel. The chemical fuel used by the heart is ATP and we recently developed the first means to measure the rate of ATP turnover in the human heart and find it is significantly reduced in heart failure. The proposed studies are important because they will determine whether that reduction in ATP rate predicts heart failure progression in death, and what the underlying mechanisms are that could be targeted for future heart failure therapy
Project start date: 1999-07-19
Project end date: 2014-07-31
Budget start date: 1-AUG-2011
Budget end date: 31-JUL-2012
PFA/PA: PA-07-070
5R01HL061912-12 (2011): $569497
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to G Robert
THE ROCHESTER CONFERENCE ON ORAL BIOLOGY POST GENOMICS FOR THE ORAL MICROBIOME
G Robert, Professor
University Of Rochestercity: Rochester country: United States (us)
Grant 1R13DE022234-01 from National Institute Of Dental & Craniofacial Research
Abstract: We request funds to support a conference entitled "The Rochester Conference on Oral Biology Post-genomics for the Oral Microbiome". The goal of this conference is to expand the use of post-genomic and systems biology approaches for the study of oral infectious disease and host-response. Oral microbial diseases occur on the teeth and soft tissues of the mouth. The microbial content of the mouth is substantial and oral infections are polymicrobial in nature. The ability of oral microbes to infect the mouth and maintain themselves in the face of competition from other bacteria, and resistance mechanisms of the human host, is the subject of ongoing research. The NIH Oral Microbiome project was undertaken to estimate the number and types of bacteria found in the human mouth, in health and disease. To date, the microbiome project has resulted in the identification of a large number of bacterial species in the oral cavity. Many oral species, identified from 16S rRNA sequences, have not yet been cultivated. Moreover, the majority of oral bacteria lack genetic systems and models of disease. Traditional approaches for evaluating specific genes in oral bacterial pathogenesis remains highly useful, where possible. However, there is a great need to find ways to accelerate our understanding of how the multitudes of oral micro-organisms interact with each other and with the human host. The proposed Conference will facilitate our goal by bringing together scientists who are working in the field of oral microbiology with scientists outside of the field who are using methods for mathematical modeling of polymicrobial diseases; proteomic approaches to evaluating microbial community metabolic networks; and genome-wide screens for bacterial genes involved with health or disease. The conference will draw upon established investigators; junior investigators; and, graduate students. The participating speakers are international in scope and will cover oral infectious disease from the perspective of caries, periodontal disease and host-inflammatory responses. Speakers from outside the oral research community, who work with other pathogens and on bacterial communities found in environmental circumstances outside of human hosts, will also participate. Collectively, the conference will foster an exchange of ideas, concepts, and methodologies with the goal of advancing our ability to treat, and ultimately prevent, oral infectious disease. This proposal seeks financial support for a conference of scientists who will meet to discuss new ways to prevent bacterial diseases in the mouth. The scientists attending the conference have knowledge and expertise about the study of complex bacterial diseases, like those in the human mouth. We expect that the conference will speed the development of new treatments and prevention of oral disease
Keywords: American; Area; Bacteria; Bacterial Genes; Bacterial Infections; bacterial resistance; Biochemical Pathway; Biological Models; Clinical; Collaborations; Commit; Communicable Diseases; Communities; Complex; Computing Methodologies; Critiques; Data; Dental caries; design; Development; Disease; Disease model; Emerging Technologies; Environment; experience; Financial Support; Fostering; Funding; Future; Genes; Genetic; Genome; genome wide association study; Genomics; Goals; graduate student; Health; Human; Human body; Human Microbiome; Image; Imaging technology; Immune response; Immunologist; infectious disease model; Inflammatory Response; International; Knowledge; Laboratories; Learning; malignant mouth neoplasm; mathematical model; meetings; Metabolic; Metagenomics; Methodology; Methods; Microbe; microbial; Microbial Biofilms; microbial community; microbial disease; microbial genome; Microbiology; microbiome; Modeling; Mouth Diseases; Nature; novel strategies; Oral; oral bacteria; oral biology; Oral cavity; oral infection; Oral Microbiology; oral microbiome; oral streptococci; Organism; pathogen; Pathogenesis; Periodontal Diseases; Physiology; prevent; Prevention; Proteomics; Recombinant DNA; Research; Research Personnel; research study; resistance mechanism; Resources; Ribosomal RNA; Role; Scientist; Site; Societies; soft tissue; Speed (motion); Streptococcus pneumoniae; symposium; System; Systems Biology; Technology; tool; Tooth Tissue; United States National Institutes of Health; Virulence; Virulence Factors; Work
Relevance: This proposal seeks financial support for a conference of scientists who will meet to discuss new ways to prevent bacterial diseases in the mouth. The scientists attending the conference have knowledge and expertise about the study of complex bacterial diseases, like those in the human mouth. We expect that the conference will speed the development of new treatments and prevention of oral disease
Project start date: 2011-06-07
Project end date: 2012-05-31
Budget start date: 7-JUN-2011
Budget end date: 31-MAY-2012
PFA/PA: PA-10-071
1R13DE022234-01 (2011): $20000
RETINAL CIRCUITRY FOR PRECISE CODING
G Robert
University Of Pennsylvaniacity: Philadelphia country: United States (us)
Grant 2R01EY016607-20 from National Eye Institute
Abstract: We propose to study the precision with which retinal circuits code different types of stimulus features. This will be important for understanding how retinal circuits inform the brain about tasks important for survival such as distinguishing a fleeing animal from one approaching. The retina´s ability to signal the visual world is limited by biological mechanisms, because the retinal output to the brain is limited by saturation and noise. To cope with this problem, the retina removes the background level using a variety of adaptation mechanisms, allowing ganglion cell signals to code for fine details. However the inevitable cost of these mechanisms is the addition of synaptic noise to the signal which limits fine details´ visibility. Although much is known about circuits presynaptic to ganglion cells, what is lacking beyond important details is an understanding of the rationale behind their signal processing mechanisms. For example, it is unknown how a ganglion cell´s presynaptic circuit shapes its neural code, the pattern of response that can inform about a stimulus, nor is it known what signal processing tradeoffs make necessary such circuit mechanisms as pooling of the receptive field center signal by convergence and gap junction coupling, and receptive field surround subtraction from amacrine and horizontal cell feedback. Using in vitro live recordings from characterized ganglion cells and horizontal cells and realistic computational models of them, we propose to test several hypotheses about the role of circuits presynaptic to the ganglion cell in its sensitivity and neural code. We hypothesize that a ganglion cell can simultaneously distinguish several stimuli that differ in contrast, size, or location, because these stimuli are conveyed by different neural codes. We will analyze the neural responses with an ideal observer, a computer program that discriminates using the likelihood rule between the responses to a pair of stimuli in a behaviorally-relevant task to measure the precision with which a neuron signals e.g. contrast or motion, and to measure the neural code. Using the ideal observer to analyze single and multiple recordings from retinal neurons, we will determine the sensitivity and neural code for discriminating multiple visual features. Next, we hypothesize that the retinal signal that relays the background level modulates the maintained synaptic release rate and signal-to-noise ratio (SNR) of the receptive field center, and that these are also modulated by the surround. Using live recordings and models, we will study how the ganglion cell´s presynaptic circuitry for center and surround control its SNR. Last, we hypothesize that reciprocal inhibition between starburst amacrine cells generates positive feedback to amplify the directional signal for the direction-selective ganglion cell. Using live recordings and models, we will test the hypothesis that reciprocal synaptic feedback helps the starburst amacrine network maximize sensitivity to direction of motion and reduce noise in the direction- selective ganglion cell. These proposed studies are new and important and will provide knowledge about circuit function relevant to a basic understanding of the brain, its behavior, and clinical testing of disease. The proposed studies of retinal circuitry will provide new knowledge about how the retina functions to reliably detect features of the visual environment. The use of ideal observer analysis allows comparing the performance of one or several neurons to the performance of a person looking at the same stimulus. The results provided by this method are relevant to public health because it will help scientists and eye doctors to determine the neural circuits that are responsible for vision in health and disease
Keywords: absorption; Amacrine Cells; Animals; Behavior; behavior test; Biological; Brain; cell type; Cells; Code; Computer Programs and Programming; Computer Simulation; computerized data processing; Contrast Sensitivity; coping; cost; Coupling; Detection; Dimensions; Discrimination (Psychology); Disease; Environment; Event; Eye; Feedback; ganglion cell; Gap Junctions; Health; horizontal cell; improved; In Vitro; insight; Joints; Knowledge; Lateral; Life; Light; Location; Measurement; Measures; Membrane Potentials; Methods; Metric; Modeling; Molds; Motion; Natural Selections; neural circuit; Neurons; Noise; novel; Output; paired stimuli; Pathway interactions; Pattern; Performance; Persons; Photoreceptors; Platelet Factor 4; Play; Positioning Attribute; presynaptic; prevent; Process; Property; public health medicine (field); receptive field; relating to nervous system; research clinical testing; Resolution; response; Retina; Retinal; Retinal Cone; retinal neuron; Role; Scientist; Shapes; Signal Transduction; Stimulus; Synapses; Testing; Vision; Visual; visual coding
Relevance: The proposed studies of retinal circuitry will provide new knowledge about how the retina functions to reliably detect features of the visual environment. The use of ideal observer analysis allows comparing the performance of one or several neurons to the performance of a person looking at the same stimulus. The results provided by this method are relevant to public health because it will help scientists and eye doctors to determine the neural circuits that are responsible for vision in health and disease
Project start date: 1991-09-30
Project end date: 2012-05-31
Budget start date: 1-JUN-2011
Budget end date: 31-MAY-2012
PFA/PA: PA-10-067
2R01EY016607-20 (2011): $392712
FUNCTION AND TARGETING OF A STABLE TRANSCRIPTION FACTOR COMPLEX IN LEUKEMIA
G Robert, Professor
Rockefeller Universitycity: New York country: United States (us)
Grant 1R01CA163086-01 from National Cancer Institute
Abstract: Transcription factors play critical roles in many types of cancer but, for a long time, were considered "undruggable" because of the lack of specific active sites that can be targeted by the types of small molecules that typify most current drugs. However, the use of short peptides has recently emerged as a promising strategy to target transcription factors that function through specific protein complexes, since distinct interaction patterns may confer a high degree of selectivity on the peptide. In this proposal, we seek to use this new strategy to target the leukemogenic fusion protein/transcription factor, AML1-ETO, that is most frequently involved in acute myeloid leukemia. We have found that, in leukemic cells, AML1- ETO resides in a stable protein complex containing multiple transcription factors and cofactors. Within this complex, the dimerized AML1-ETO directly interacts with a family of conventional transcriptional activators, E proteins, that are implicated in hematopoietic lineage developmental events. We also have found that the AML1-ETO dimerization domain (NHR2), which previously was shown to be critical for leukemogenesis, utilizes a distinct surface of the dimerized alpha-helixes to mediate the AML1-ETO interaction with a conserved motif in E proteins. This particular interaction pattern ideally allows the design of inhibitors to specifically disrupt the interaction and to manipulate the activities of AML1-ETO, thus providing a potential target for leukemia treatment. In this regard, and in further support of this NHR2-E protein interaction as a therapeutic target, a specific mutation that disrupts the NHR2-E protein interaction, but not NHR2 dimerization, has been shown to impair the capacity of AML1-ETO to enhance human hematopoietic stem cell self-renewal. Based on these biochemical and functional studies of the AML1-ETO-containing transcription factor/cofactor (AETFC) complex(es), we plan (i) to further identify and characterize the AETFC complex(es) by detailed mechanistic studies; (ii) to identify direct AML1- ETO target genes by genome-wide ChIP analyses; (iii) to clarify, through cell-based and cell-free in vitro transcription systems, the detailed mechanisms by which AML1-ETO and other components cooperate to (de)regulate transcription; (iv) to study (and validate) the biological functions of individual components and their interactions in leukemic cellular and mouse models; and (v) to design specific peptidomimetic inhibitors to manipulate the action of AML1-ETO in transcription and leukemogenesis. A number of chromosomal translocations result in leukemogenic fusion proteins that alter normal transcription programs. This study will provide a deeper understanding of the likely diverse functions and mechanisms of action of the leukemogenic fusion protein (AML1-ETO) that is most frequently involved in acute myeloid leukemia. This information, in turn, will be used to develop a peptidomimetic inhibitor that targets this leukemogenic fusion protein and offers a new therapeutic approach for related leukemias
Keywords: Active Sites; Affect; alpha helix; AML1-ETO fusion protein; base; Binding (Molecular Function); Binding Sites; Biochemical; Biological Assay; Biological Process; Bone Marrow; cancer type; CD34 gene; Cell model; Cells; Chimeric Proteins; Chromatin; Chromosomal translocation; cofactor; Complex; Consensus Sequence; design; Development; dimer; Dimerization; Dissection; DNA Binding Domain; DNA-Protein Interaction; E protein; Event; Family; Fetal Liver; Gene Activation; Gene Expression; Gene Expression Regulation; Gene Targeting; Genetic Transcription; genome-wide; Hematopoietic; Hematopoietic stem cells; Human; impaired capacity; In Vitro; Individual; inhibitor/antagonist; insight; Killings; leukemia; Leukemia, Myelocytic, Acute; Leukemic Cell; leukemogenesis; liver transplantation; Mediating; Modeling; Modification; Molecular Conformation; Molecular Profiling; mouse model; Mus; mutant; Mutation; novel; novel therapeutic intervention; novel therapeutics; overexpression; Patients; Pattern; Peptides; peptidomimetics; Pharmaceutical Preparations; Play; programs; protein complex; Proteins; Relative (related person); Repression; Role; self-renewal; small molecule; Surface; System; t(8;21)(q22;q22); Testing; therapeutic target; Time; Transcription Coactivator; transcription factor; Transcriptional Regulation
Relevance: A number of chromosomal translocations result in leukemogenic fusion proteins that alter normal transcription programs. This study will provide a deeper understanding of the likely diverse functions and mechanisms of action of the leukemogenic fusion protein (AML1-ETO) that is most frequently involved in acute myeloid leukemia. This information, in turn, will be used to develop a peptidomimetic inhibitor that targets this leukemogenic fusion protein and offers a new therapeutic approach for related leukemias
Project start date: 2011-09-14
Project end date: 2016-07-31
Budget start date: 14-SEP-2011
Budget end date: 31-JUL-2012
PFA/PA: PA-10-067
1R01CA163086-01 (2011): $351713
CELLGUARD-TD IMPROVES HUMAN TISSUE DIGESTION AND CELL SUBCULTURING
G Robert, Leading Professor
Cell Preservation Services, Inc.city: Owego country: United States (us)
Grant 1R43DK091952-01A1 from National Institute Of Diabetes And Digestive And Kidney Diseases
Abstract: Tissue digestion and cell extraction is a core element of a variety of bioprocessing methods ranging from isolation of the rare cancer stem cell for basic research to the purification of human islets or adipose-derived mesenchymal stem cells for transplant. While procedures vary, it is clear that each tissue digestion procedure subjects the intact tissue to cell stress that can result in a loss of cell viability and function. CPSI-Bioech is focused on improving the bioprocessing of a variety of human cell types at normothermic temperatures (20 to 370C) so that their pharmacological and cell therapy utility can be improved. To this end the Company is developing the CellGuard supplement series - reagents that when added to select bioprocessing procedures will protect and maintain cell viability and function. This, in turn, will lead to fewer post-graft or transfusion problems in the patient. This Phase 1 project proposes to develop CellGuard-TD - a supplement designed to enhance tissue digestion (TD) of human pancreatic tissue for islet isolation (CTDislet). Phase 1 will use a variety of molecular biology methods to develop a prototype CTDislet- the first supplement designed to improve pancreatic islets digestion through cell stress pathway modulation. CTDislet will be based on the well documented apoptosis-prone nature of human pancreatic islets, their low cell yield as a result of warm ischemia exposure, and the collagenase-induced damage to islets that results in a low number obtained from each pancreas. Phase 2 studies will be dedicated to a more in depth analysis and development of CTDislet as well as testing this new formulation at select islets procurement centers. A second aim of the Phase 2 studies will be the development of a "non-synthetic" CTDislet formulation comprised of cell stress inhibitors derived from botanical sources. The intent of this project is to develop a supplement that protects the fragile nature of human pancreatic islets during the isolation and handling process- a technology that will apply to native islets as well as islets differentiated from mesenchymal stem cells and other stem cell sources. The human health consequence of this research will be to improve the isolation, handling and transfusion of human islets used for treating diabetes. This project is designed to conduct research that will lead to the development of a series of products, termed CellGuard that will protect human cells during manipulation. A variety of human cell types are now being used to treat cancer, diabetes, and also serve as the building blocks of engineered tissues used in the biomedical field. Manipulating human cells such that they can be used for these purposes often compromises both cell function and viability. The CellGuard portfolio of products is designed to protect these cells when they are subjected to these harsh conditions on the path for clinical application. This particular project is designed to develop a CellGuard supplement that will improve the isolation and cell therapy application of human pancreatic islets leading to improved methods for treating diabetes
Keywords: Adipose tissue; Agreement; Apoptosis; Area; arm; Arrhythmia; base; Basic Science; Biological Preservation; Biological Sciences; Biology; bioprocess; Botanicals; Cancer stem cell; Cell Culture Techniques; Cell physiology; Cell Separation; Cell Survival; Cell Therapy; cell type; Cells; Cellular Stress; clinical application; cold temperature; collagenase; Culture Media; design; Development; Diabetes Mellitus; Digestion; Disease; Drug Formulations; Elements; Engineering; Environment; Event; experience; Foundations; Growth; Health; Human; Human Characteristics; Human Resources; human tissue; hypothermosol; implantation; improved; improved functioning; inhibitor/antagonist; Investigation; islet; Islet Cell; Islets of Langerhans; Lead; Legal patent; Letters; Malignant Neoplasms; Marketing; Medical Device; Mesenchymal Stem Cells; Methods; Molecular; Molecular Biology; neglect; novel; Pancreas; Pathway interactions; Patients; Pharmacologic Substance; Phase; phase 1 study; phase 2 study; Procedures; Process; Protocols documentation; prototype; Publications; Reagent; Research; Series; Solutions; Source; Stem cell transplant; Stem cells; Stress; System; Technology; technology development; Temperature; Testing; Therapeutic; Tissue Engineering; Tissues; Transfusion; Warm Ischemia; Work
Relevance: This project is designed to conduct research that will lead to the development of a series of products, termed CellGuard that will protect human cells during manipulation. A variety of human cell types are now being used to treat cancer, diabetes, and also serve as the building blocks of engineered tissues used in the biomedical field. Manipulating human cells such that they can be used for these purposes often compromises both cell function and viability. The CellGuard portfolio of products is designed to protect these cells when they are subjected to these harsh conditions on the path for clinical application. This particular project is designed to develop a CellGuard supplement that will improve the isolation and cell therapy application of human pancreatic islets leading to improved methods for treating diabetes
Project start date: 2011-09-20
Project end date: 2012-08-31
Budget start date: 20-SEP-2011
Budget end date: 31-AUG-2012
PFA/PA: PA-11-096
1R43DK091952-01A1 (2011): $184000
TROPHIC FACTOR SIGNALING AND MOTOR NEURON DEATH
G Robert
Childrens Hospital Of Philadelphiacity: Philadelphia country: United States (us)
Grant 2R01NS052325-06A1 from National Institute Of Neurological Disorders And Stroke
Abstract: This proposal focuses on the role of receptor tyrosine kinases in rendering motor neurons susceptible to neurodegenerative insults. TrkB is activated by its ligand, brain-derived neurotrophic factor. Several independent laboratories show that blocking TrkB activation in vitro can protect neurons from specific insults. Despite the heresy of these observations, we have seen the same phenomenon in vitro and in a specific in vivo circumstance. In specific aim #1 we will investigate the temporal aspect of this process by putting the mutant SOD mouse on a TrkBF616A background. TrkBF616A can be specifically antagonized by the orally active, blood brain barrier permeable agent, 1NMPP1. A second receptor tyrosine kinase of interest is the Insulin/Insulin-like growth factor receptor (IGF-R). In model organisms (and recently in mice) a conserved stress resistance pathway is evoked when the activity of the IGF-R is reduced. The FOXO3a transcription factor plays a key role in this process in worms and flies, but its function in vertebrates is unknown. In specific aim #2, we will determine if loss of FOXO3a ameliorates or exacerbates the mutant SOD mouse motor neuron disease. In the course of our investigations of FOXO3a, we found a marine sponge compound (Psammaplysene A, PA) that promotes nuclear localization of the transcription factor and is broadly neuroprotective in in vitro and in vivo models of neurodegeneration. While the evidence is strong that the compound works through FOXO3a, the precise molecular target is not known. In specific aim #3, several approaches will be employed to find the target and pathway through which PA acts. Overall this work attempts to find new disease modifiers that slow the progression of motor neuron disease. If successful, new therapeutic targets will emerge. It is not known why specific neuronal populations are vulnerable to the toxic effects of neurodegenerative stimuli. In this proposal we will examine the role played by two signaling pathways in rendering motor neurons susceptible to insults relevant to Lou Gehrig´s disease. In addition, we have identified a small molecule with remarkable neuroprotective activity. In this proposal we will determine the molecular target of this molecule and the signaling pathway through which it operates
Keywords: Aging; Amyotrophic Lateral Sclerosis; Animal Model; Blood - brain barrier anatomy; Brain; Brain-Derived Neurotrophic Factor; cancer cell; Cell Death; Cell Nucleus; Cessation of life; Dendrites; Development; disability; Disease; Disease Progression; Exercise; fly; Goals; Growth; Health; improved; In Vitro; in vivo; in vivo Model; insight; Insulin; Insulin-Like Growth Factor Receptor; interest; Investigation; Laboratories; Ligands; Longevity; Marines; Methods; Molecular; Molecular Target; Motor Neuron Disease; Motor Neurons; mouse model; Mus; mutant; Nerve Degeneration; Neurodegenerative Disorders; neuronal survival; Neurons; new therapeutic target; novel; Nuclear; Pathway interactions; Pharmaceutical Preparations; Play; Porifera; Process; public health medicine (field); public health relevance; Receptor Protein-Tyrosine Kinases; Research; research study; Resistance; Risk Factors; Role; Series; Signal Pathway; Signal Transduction; small molecule; Spinal Cord; Stimulus; Stress; Synaptic plasticity; Theft; Toxic effect; transcription factor; Vertebrates; Vulnerable Populations; Work
Relevance: Narrative It is not known why specific neuronal populations are vulnerable to the toxic effects of neurodegenerative stimuli. In this proposal we will examine the role played by two signaling pathways in rendering motor neurons susceptible to insults relevant to Lou Gehrig´s disease. In addition, we have identified a small molecule with remarkable neuroprotective activity. In this proposal we will determine the molecular target of this molecule and the signaling pathway through which it operates
Project start date: 2006-02-15
Project end date: 2016-01-31
Budget start date: 15-MAR-2011
Budget end date: 31-JAN-2012
PFA/PA: PA-10-067
2R01NS052325-06A1 (2011): $366406
CREATINE KINASE METABOLISM IN FAILING MURINE HEARTS
G Robert, Professor
Johns Hopkins Universitycity: Baltimore country: United States (us)
Grant 5R01HL063030-09 from National Heart, Lung, And Blood Institute
Abstract: This proposal aims to measure and manipulate in vivo cardiac flux through myocardial creatine kinase (CK) in mice to test the energy starvation hypothesis of heart failure (CHF). The requirement of ATP for normal cardiac contractile function is absolute and the CK reaction is the major energy reservoir of the heart. CK metabolites are reduced in CHF and predict outcomes. The first direct measures of ATP flux through CK in the human heart recently revealed dramatic 50-70% reductions in CK flux in CHF even before global [ATP] loss occurs. Despite this supporting evidence, conventional metabolic interventions have failed to augment CK pools or flux in failing hearts to directly test the energy starvation hypothesis. This application proposes the use of new transfection approaches, just developed by the investigators, to genetically over-express the factors most likely limiting CK flux in CHF and determine, in vivo, the energetic and functional consequences. The specific aims are 1.) to implement new clinical MR techniques in mouse studies for assessing in vivo cardiac CK metabolites, flux and function, 2.) to test the hypothesis that increasing CK expression in CHF increases in vivo cardiac CK flux and improves ventricular function, 3.) to test the hypothesis that increasing creatine transport protein expression will increase myocardial creatine, CK flux, and mechanical function in CHF, 4.) to test the hypothesis that conditional CK gene deletion will exacerbate the development of CHF and that CK rescue will provide protection. This proposal uniquely brings together novel non-invasive tools to measure in vivo cardiac CK flux, new technology to perform genetic CK manipulations, relevant animal models that capitulate characteristics of human CHF, and finally, sophisticated means to assess the functional consequences. These mouse studies offer interventions not possible in human CHF and promise new insights for this prevalent, growing disease. Lay summary Heart failure is an important and growing cause of morbidity and mortality in the United States. Our recent observations in human heart failure guide these studies that currently can only be performed in mice. We will use state-of-the-art techniques to increase energy metabolism in failing mouse hearts and see if that improves the contraction of the heart and reduces heart failure
Keywords: Adult; Animal Model; Animals; ATP Synthesis Pathway; Attenuated; base; Biochemistry; Cardiac; Carrier Proteins; Chemicals; CKB gene; Clinical; Creatine; Creatine Kinase; Creatine Kinase MB Isoenzyme; Development; Disease; driving force; Energy Metabolism; enzyme activity; Exhibits; functional decline; Functional disorder; Gene Deletion; Genetic; genetic manipulation; Genetic Techniques; Heart; Heart failure; hemodynamics; Human; Human Characteristics; Imaging Techniques; improved; improved functioning; in vivo; inorganic phosphate; insight; Intervention; Ischemia; Knock-out; Knowledge; Magnetic Resonance Imaging; Measures; Mechanics; meetings; Metabolic; Metabolism; Methods; MM form creatine kinase; Modeling; Morbidity - disease rate; Mortality Vital Statistics; mouse model; Mus; Muscle; Muscle Cells; Myocardial; Myocardial Contraction; new technology; novel; Outcome; Phosphocreatine; Plasmids; pressure; programs; protein expression; Protein Isoforms; Reaction; Research Personnel; response; Spectrum Analysis; Speed (motion); Starvation; Stress; Techniques; Testing; Time; tool; Transact; Transfection; Transgenic Mice; Translations; United States; Ventricular Function; Work
Project start date: 2000-04-01
Project end date: 2012-03-31
Budget start date: 1-APR-2011
Budget end date: 31-MAR-2012
5R01HL063030-09 (2011): $410000
RETINAL CIRCUITRY FOR ROBUST DIRECTION SELECTIVITY
G Robert
Oregon Health And Science Universitycity: Portland country: United States (us)
Grant 1R01EY022070-01 from National Eye Institute
Abstract: This project proposes to study mechanisms of synaptic processing within specific ganglion cell types in the mammalian retina, both by direct neurophysiological recording and through the use of realistic computer models. Our visual system functions under a wide range of light conditions from night to day, and the retina adapts to prevent saturation, so that the output is largely invariant to changes in the illumination level. The synaptic mechanisms that accomplish adaptation and signal transmission introduce noise, which, coupled with the limited dynamic range of neurons, reduces the fidelity of the visual signal. To cope with this problem, the retina segments the visual world using different types of ganglion cells that each code specific visual features with high fidelity. This project focuses on a specific type of retinal ganglion cell that signals directional motion, called the direction-selective ganglion cell (DSGC). Using a live in-vitro isolated rabbit retina, we will record responses of neurons to light stimuli, and construct computational models of the responses to determine the biophysical mechanisms present. The study comprises three sections. Aim 1 examines the function of the starburst amacrine cell (SBAC), essential for generating the direction selective signal for the DSGC. This aim tests several hypotheses relating to specific biophysical mechanisms intrinsic to the cell, such as voltage-gated channels, that generate its directional output. A realistic computer model of the SBAC will help to determine which mechanisms are present. Aim 2 tests the hypothesis that inhibition between adjacent cells within the network of SBACs is crucial for amplifying directional signals. The experimental results will be used to develop and test a computational model, derived from the results of Aim 1 that contains several SBACs with their network interactions. Aim 3 examines noise and precision in the spiking output of the direction-selective ganglion cell, and will account for its spiking properties using a computational model based on physiological results from all three Aims. The final model will represent a detailed and essentially complete representation of directional signaling in the mammalian retina. Overall, the proposed research will improve our understanding of the complex circuitry of the adult retina; the knowledge gained will inform continuing efforts to develop treatments and visual prosthetic devices that restore vision loss from a range of eye diseases. The overall goal of this research project is to understand how specific nerve cells in the eye, the retinal ganglion cells, detect motion in our environment and relay that information to the brain. Improving our understanding of the healthy eye will inform and facilitate the continued development of novel therapeutic treatments for human eye disease
Keywords: Accounting; Adult; Amacrine Cells; area striata; base; Blindness; Brain; cell type; Cells; Chloride Ion; Chlorides; Code; Complex; Computer Simulation; Contracts; coping; Coupled; Dendrites; Development; Elements; Environment; Eye; Eye diseases; Feedback; ganglion cell; Goals; Human; Image; improved; In Vitro; Knowledge; Life; Light; light intensity; Lighting; Modeling; Morphology; Motion; neuromechanism; Neurons; neurophysiology; Noise; novel therapeutics; Oryctolagus cuniculus; Output; Physiological; postsynaptic; Potassium Channel; presynaptic; prevent; Process; Property; Prosthesis; Publications; Research; Research Project Grants; response; Retina; Retinal; Retinal Ganglion Cells; Signal Transduction; Stimulus; Synapses; Testing; Training; transmission process; Trees; Visual; Visual system structure; voltage; voltage gated channel; Work
Relevance: The overall goal of this research project is to understand how specific nerve cells in the eye, the retinal ganglion cells, detect motion in our environment and relay that information to the brain. Improving our understanding of the healthy eye will inform and facilitate the continued development of novel therapeutic treatments for human eye disease
Project start date: 2011-12-01
Project end date: 2015-11-30
Budget start date: 1-DEC-2011
Budget end date: 30-NOV-2012
1R01EY022070-01 (2012): $406000
LETROZOLE THERAPY FOR IVF/ET: EFFECTS ON MULTIPLE PREGNANCY AND TREATMENT BURDEN
G Robert
University Of Texas Hlth Sci Ctr San Antcity: San Antonio country: United States (us)
Grant 5U10HD055942-05 from Eunice Kennedy Shriver National Institute Of Child Health & Human Development
Abstract: This application is written in response to RFA-HD-06-008 to participate as a Clinical Site in an ongoing multicenter cooperative program designed to conduct clinical studies investigating problems in reproductive medicine. The application directly addresses the mission of the Reproductive Sciences Branch to ensure the birth of healthy, wanted babies through studies on human fertility and infertility. The long term objectives of the proposed project are to reduce the incidence of multiple pregnancy associated with assisted reproductive technology (ART), to reduce the incidence of excess embryo production and embryo cryopreservation associated with ART, and to reduce the burden on patients of ART therapy. Over 100,000 cycles of ART treatment are performed each year in the US, and ART is now responsible for 1% of all births. Advances in ART have increased its efficacy, but have also led to a large increase in the incidence of multiple pregnancies. The public health impact of ART associated multiple pregnancies cannot be overstated. The majority of ART cycles in the US employ complex treatment regimens entailing self administration of multiple parenteral drugs and intensive outpatient monitoring. These regimens facilitate the creation of multiple embryos, which promotes the transfer and cryopreservation of excess embryos. The proposed project aims to address the shortcomings of conventional ART therapy by examining via a randomized clinical trial design an alternative ovarian stimulation regimen based on the oral administration of the aromatase inhibitor letrozole and minimal monitoring. The specific aims of the project are to 1) compare the incidence of multiple pregnancies in letrozole ART cycles with the incidence in conventional ART, 2) determine the incidence of embryo cryopreservation in the two treatment regimens, 3) compare the cumulative live born delivery rate per patient derived from two cycles of letrozole ART compared to one cycle of conventional ART, 4) examine the cumulative cost of two cycles of letrozole ART compared to the cost of a single cycle of conventional ART, and 5) compare self-reported levels of treatment burden and participant concerns between the two regimens. It is hypothesized that the cost savings and reduced treatment burden associated with letrozole therapy compared to conventional ART will allow patients to undergo two cycles of letrozole based ART, which will result in a similar live born delivery rate, a significantly lower incidence of multiple pregnancy and a significantly lower incidence of embryo cryopreservation. The relevance of the project derives from its potential to reduce multiple pregnancies with their attendant costs and risk of death and disability. The proposed examination of treatment costs is relevant given the problem of access to ART care, which limits appropriate utilization in the US. Finally, the project addresses relevant social and ethical concerns regarding embryo cryopreservation
Keywords: Address; Adverse event; Advisory Committees; Ambulatory Monitoring; Area; Aromatase Inhibitors; Assisted Reproductive Technology; Award; base; Birth; Caring; Cessation of life; Clinical Research; clinical research site; Clinical Trials Design; Collection; Complex; cost; Cost Savings; Cryopreservation; Data; Data Analyses; Data Coordinating Center; Data Reporting; design; Development; disability; Embryo; Enrollment; Ensure; Ethics; Fertility; Human; Incidence; Infertility; Letrozole; Life; Measures; meetings; Mission; Monitor; Multiple Pregnancy; Oral Administration; Ovarian Stimulations; Participant; Patient Self-Report; Patients; Pharmaceutical Preparations; Policies; Procedures; Production; programs; Protocols documentation; public health medicine (field); Publications; quality assurance; Randomized Clinical Trials; Regimen; Reporting; reproductive; Reproductive Medicine; Research; Research Design; response; Risk; Science; Self Administration; social; Study Subject; symposium; TimeLine; transmission process; Treatment Cost; Treatment Protocols; United States National Institutes of Health; Voting; Writing
Project start date: 2007-09-07
Project end date: 2012-07-31
Budget start date: 1-AUG-2011
Budget end date: 31-JUL-2012
PFA/PA: RFA-HD-06-008
5U10HD055942-05 (2011): $274799
REDUCING VIOLENCE AGAINST WOMEN WITH ALCOHOLIC PARTNERS
G Robert, Senior Research Scientist
State University Of New York At Buffalocity: Buffalo country: United States (us)
Grant 5R01AA016799-04 from National Institute On Alcohol Abuse And Alcoholism
Abstract: This application builds on the investigators´ prior work assessing and training more effective coping skills in women with an alcoholic partner who is not in treatment. To date, that work has focused largely on the effects of Coping Skills Training (CST) and an alternate, 12-step Facilitation (TSF) treatment on the woman´s own functioning and, secondarily, on the partner´s drinking. However, exploratory analyses suggest that CST may be particularly effective (relative to TSF) in reducing partner physical violence against the woman, reducing violent-partner drinking over time, and eliminating the positive relationship between partner drinking and violence during follow-up. In this application, we use improved relationship-violence methodology to (a) test the replicability of these findings, (b) evaluate the effects of the treatments on the woman´s own violence toward her partner-heretofore not assessed, and (c) explore the constructs and putative causal pathways operating in a heuristic model of alcoholic partner and spouse negative affect, partner alcohol involvement, aversive marital behaviors, and spouse self-control (coping) skills. With respect to the latter, we will explore whether an increase in the woman´s coping skill in CST, relative to TSF, will moderate (buffer) the effects of partner alcohol involvement and partner violence on her own negative affect. Skill level also is thought to further moderate the effect of the woman´s negative affect on her own violent relationship behaviors. A reduction in the woman´s own violence is hypothesized to reduce partner violence and the partner´s own negative affect. A reduction in the latter then is thought to account for a reduction in his drinking and a further reduction in his violence against the woman. The above a priori and exploratory aims will be evaluated in a 2- group CST vs. TSF longitudinal design. One hundred and fifty women with a physically-violent alcoholic partner not currently in treatment will be randomly assigned within therapy groups to either CST or TSF. All participants will be followed at 90-day intervals for a period of 12 months posttreatment during which both the woman´s and partner´s negative affect and aversive marital behaviors are assessed, partner drinking measured, and the woman´s coping skill acquisition evaluated. Estimates suggest that nearly half of all women with alcoholic partners experience some partner physical violence. Although these women typically have been advised by family, friends, and others to leave the relationship, many remain. Helping these individuals improve their own functioning and reduce the violence they experience may have a greater public health benefit than simply advising them to leave the partner or referring them to 12-step groups
Keywords: Accounting; Acculturation; Address; Affect; Aggressive behavior; alcohol involvement; Anger; Antisocial Personality Disorder; Anxiety; Behavior; Buffers; clinically significant; cohort; Coping Skills; design; drinking; Evaluation; experience; Family; follow-up; Frequencies (time pattern); Friends; Goals; Group Therapy; Health Benefit; heuristics; improved; Incidence; indexing; Individual; Injury; Left; Link; longitudinal design; Low Prevalence; Measurement; Measures; Mediating; Methodology; Modeling; Outcome; Participant; partner violence; Pathway interactions; Population; Prevalence; problem drinker; psychologic; public health medicine (field); Randomized; Recording of previous events; relationship violence; Relative (related person); Reporting; Research Personnel; Self-control as a personality trait; skill acquisition; skills; skills training; Source; Spouses; Stress; Testing; Time; Training; treatment effect; Violence; violence against women; violent relationship; Woman; Work
Project start date: 2008-05-01
Project end date: 2013-04-30
Budget start date: 1-MAY-2011
Budget end date: 30-APR-2012
PFA/PA: PA-07-066
5R01AA016799-04 (2011): $543150
MECHANISMS OF P53 TARGET GENE ACTIVATION IN DNA DAMAGE RESPONSES
G Robert, Professor
Rockefeller Universitycity: New York country: United States (us)
Grant 5R01CA129325-05 from National Cancer Institute
Abstract: The tumor suppressor p53, a classical transcription factor, is activated by diverse signals associated with various cellular and genotoxic stress responses and, through its selective activation of cognate target genes, induces either cell cycle arrest (allowing DNA repair) or apoptosis. The broad objective is to understand the underlying mechanisms and, ultimately, the basis for differential activation of p53 target genes and cell fate decisions that result in repair or elimination of potentially malignant cells with damaged DNA. Recent studies indicate that the function of p53 is mediated by a number of transcriptional co-activators that act either by effecting modifications (acetylation, methylation, phosphorylation, ubiquitination) of nucleosomal histones or p53 itself (e.g. pSOO/CBP, GCN5, TIP60, PRMT1, CARM1, MLL1), or by facilitating direct communication with the basal transcriptional machinery (e.g. Mediator), or by unknown mechanisms (e.g. ASPP, JMY, p63, p73). In particular, cellular assays and biochemical assays reconstituted with purified factors and either DNA or recombinant chromatin templates have established both independent and ordered cooperative functions of histone acetyltransferases and methyltransferases, DNA damage-induced interactions of cofactors with p53, and DNA damage-induced association of multiple coactivators on a single p53 target gene. In a major extension of this work, using both in vitro and in vivo assays, the specific aims are (i) to employ defined cell free systems, with purified factors and DNA templates, to detail the function and mechanism of action of p53 co-activators that facilitate direct (post chromatin remodeling) activation of the general transcription machinery, (ii) to establish defined cell free systems, with purified factors and recombinant chromatin templates, to investigate the function and mechanism of action of p53 co-activators that effect covalent histone modifications and chromatin remodeling, (iii) to investigate the role of DNA damage-induced covalent modifications of p53 in the function of cofactors that directly (Mediator) or indirectly (chromatin modifying factors) effect target gene activation, (iv) to employ cell-based assays (ChIP, RNA interference, gene knockout/knockin) to establish the in vivo accumulation and function of co-activators on p53 target genes in response to DNA damage, and (v) to assess variations in cofactor usage as a function of different genotoxic agents, different cell types, and different p53 target genes (apoptotic versus cell cycle arrest), as well as a possible co-activator redundancy that may contribute to biological robustness for any given DNA damage response. Cancer represents a major health problem and over half of human cancers have defects in p53 or ,in some cases, factors that regulate p53, resulting in abnormal growth of cells with damaged DNA and, ultimately, the emergence of malignant cells. Understanding the mechanisms by which p53 target genes are controlled may facilitate development of ways to counter the adverse effects of altered p53 function
Keywords: Acetylation; Adverse effects; Apoptosis; Apoptotic; base; Biochemical; Biological; biological adaptation to stress; Biological Assay; cancer cell; Cell Cycle Arrest; cell growth; cell type; Cell-Free System; Cells; Cellular Assay; Cellular Stress; Chromatin; chromatin remodeling; coactivator-associated arginine methyltransferase 1; cofactor; Communication; Defect; Development; DNA; DNA Damage; DNA Repair; EP300 gene; Gene Activation; Gene Targeting; Genetic Transcription; Genotoxic Stress; Health; histone acetyltransferase; histone methyltransferase; histone modification; Histones; HTATIP gene; Human; In Vitro; in vivo; knockout gene; Malignant Neoplasms; Mediating; Mediator of activation protein; Methylation; Modification; Mutagens; PCAF gene; Phosphorylation; Protein p53; Recombinants; reconstitution; repaired; response; RNA Interference; Role; senescence; Signal Pathway; Signal Transduction; Transcription Coactivator; transcription factor; TRAP Complex; Ubiquitination; Variant; Work
Project start date: 2007-08-01
Project end date: 2012-05-31
Budget start date: 1-JUN-2011
Budget end date: 31-MAY-2012
5R01CA129325-05 (2011): $799836
Sponsored Links Excellgen http://Excellgen.com
RISE PROGRAM OPTION I: UNIVERSITY OF PUERTO RICO AT CAYEY
G Robert, Director
University Of Pr Cayey University Collcity: Cayey country: United States (us)
Grant 2R25GM059429-13 from National Institute Of General Medical Sciences
Abstract: The RISE program continues to transform the academic environment at UPR-Cayey by focusing on the preparation of students to become scientists. As part of this long term goal of increasing the number of Hispanics studying for the Ph.D., this proposal is concentrating on helping students in four areas, 1- their lack of awareness for career opportunities and of their own potential, 2- their limited research skills and experience, 3- limited quantitative and qualitative skills, and 4- limited English language skills. These were identified and interventions will be monitored by an external evaluator working with the activity coordinators, program director, and the RISE Advisory Committee. To address these needs, five developmental activities are prescribed- visiting scientists, research skills, discovery chemistry, language skills, and research experiences. Reaching the entire science community, visiting alumni scientists provide seminars and workshops in English to improve attendees´ awareness of new techniques and discover science careers, and to serve as role models. Improving the skills of motivated students will be accomplished during their first year by having activities related to research techniques, scientific literacy, and language skills. RISE students will take Discovery Chemistry to quantitative and qualitative skills. Research experiences for twelve students will be offered during the academic year in Puerto Rico with active investigators at four collaborating institutions. Additional research experiences will be offered to over 60 students during summer internships at 20 collaborating institutions in the USA. Each component enables students to develop their potential by interacting with a different group of experts 1) research and language skills with faculty mentors; 2) motivational visiting alumni scientists; 3) inquiry methodology by chemistry faculty and 4) research lab experience with either on- island research mentors or with off-island scientists located in the USA. Each expert group challenges the students as they develop utilizing the appropriate environment and level of expertise to produce UPRC alumni who will be biomedical research scientists. During the last 5 years (2005-09), 32 RISE alumni completed Ph.D.s from major research universities; a 100% increase over the previous five-year period. By doubling the number of Ph.D. alumni, RISE will impact the community. These UPRC scientists will serve to meet the health disparity needs of the Hispanic population in Puerto Rico and the nation by working to discover the causes and remedies of diseases. The RISE program continues to transform the academic environment at UPR-Cayey by focusing on the preparation of students to become scientists. These UPRC scientists will serve to meet the health disparity needs of the Hispanic population in Puerto Rico and the nation by working to discover the causes and remedies of diseases
Keywords: Address; Advisory Committees; Area; Awareness; base; Bibliography; Biomedical Research; career; Chemistry; Communities; Comprehension; Data; design; Development; Discipline; Disease; Doctor of Philosophy; Educational process of instructing; Educational workshop; English Language; Environment; experience; Exposure to; Faculty; Failure (biologic function); Goals; health disparity; Hispanics; improved; innovation; Institution; interdisciplinary approach; Internships; Intervention; Investigation; Island; Language; Learning; lectures; Life; literacy; Literature; Measurable; meetings; member; Mentors; Meta-Analysis; Methodology; Mission; Monitor; Motivation; Oral; posters; Preparation; Process; programs; publication formats; Publications; Puerto Rico; Reading; Reporting; Research; Research Activity; Research Personnel; Research Project Grants; Research Proposals; Research Technics; role model; Rotation; Schools; Science; Scientist; skills; Students; symposium; Teaching Method; Techniques; Testing; Thinking, function; Universities; Visit; Work; Writing
Relevance: The RISE program continues to transform the academic environment at UPR-Cayey by focusing on the preparation of students to become scientists. These UPRC scientists will serve to meet the health disparity needs of the Hispanic population in Puerto Rico and the nation by working to discover the causes and remedies of diseases
Project start date: 1999-05-01
Project end date: 2016-07-31
Budget start date: 1-AUG-2011
Budget end date: 31-JUL-2012
PFA/PA: PAR-10-004
2R25GM059429-13 (2011): $329986
INTEGRATED POLYMER MICROFLUIDIC DEVICE FOR MULTIPLEX DIAGNOSIS OF VIRAL INFECTION
G Robert
Geneva Foundationcity: Tacoma country: United States (us)
Grant 1R01AI096215-01 from National Institute Of Allergy And Infectious Diseases
Abstract: The rapid and accurate diagnosis of infection is critical for managing potential exposures to highly virulent pathogens, whether occurring from an act of bioterrorism or a natural event. Antibodies are very sensitive serum biomarkers that can be exploited to identify recent or past exposures to infectious agents, and antibody analysis methods are especially important for samples containing negligible amounts of detectable virus or genetic material from the pathogen. Commonly practiced methods for measuring antibody responses in the clinic are generally encumbered by a focus on single pathogens, several time-consuming performance steps, slow turn-around time for data acquisition, issues of specificity and sensitivity, and variable results. We propose to develop a simple microfluidic device that will integrate many complex tasks required for antibody- based diagnosis, from processing of finger-prick blood samples to identifying exposures to specific infectious agents, taking advantage of the unique physical properties of micro- and nanofluidics to enhance assay performance. Our studies will focus on point-of-care diagnosis of infections caused by two pathogen groups with the greatest potential impact on public health arboviruses (Japanese Encephalitis, Chikungunya, Dengue, Yellow Fever and others) and filoviruses (Ebola, Marburg). We envision that the microarray format of the microfluidic device can be rapidly expanded to include tests for an extensive number of other biological agents. The collaborative team will consist of investigators from the Biosensor and Protein Microarray Laboratory from the Research Institute of Infectious Diseases, and the MicroElectroMechanical Systems (MEMS) and Microfluidics Laboratory of the University of Maryland. The project team includes unique expertise in infectious disease research, MEMS, microfluidic systems, and microarray-based technologies for the high-throughput detection of human antibody interactions with signature proteins of pathogens. Sera from clinical and veterinary cases of infection will be used for extensive evaluation of the device. Our approach will require only nanoliter quantities of serum for analysis and will lead to the development of a low cost, disposable device for rapid and sensitive detection of pre-symptomatic, symptomatic or convalescent biomarkers of infectious diseases. The proposed research will result in a simple, low cost device for the rapid diagnosis of infections caused by many viruses that are important to public health. This small, disposable plastic device will be targeted for practitioners with very little experience in laboratory methods
Relevance: The proposed research will result in a simple, low cost device for the rapid diagnosis of infections caused by many viruses that are important to public health. This small, disposable plastic device will be targeted for practitioners with very little experience in laboratory methods
Project start date: 2011-08-19
Project end date: 2016-07-31
Budget start date: 19-AUG-2011
Budget end date: 31-JUL-2012
PFA/PA: RFA-AI-10-017
1R01AI096215-01 (2011): $1088801
EYE MOVEMENT: PHYSIOLOGICAL ORGANIZATION
G Robert
New York University School Of Medicinecity: New York country: United States (us)
Grant 5R01EY002007-32 from National Eye Institute
Abstract: The purpose of this research is to study the ontogeny of oculomotor neuron physiology and behavior by exploiting the developmental genetics of zebrafish. To achieve this objective, we will investigate the physiology and genetic specification of four neuronal subtypes that contribute to the production of horizontal and torsional eye movement. The proposed research will focus on quantifiable behaviors and physiological properties of oculomotor neurons and circuits to explore the role of Hox paralog group 4 genes (hoxa4a, Hoxb4a, hoxc4a, hoxd4a) in the patterning and differentiation of three nuclei specific for horizontal eye motion that originate from hindbrain rhombomeres 7 and 8. The first nucleus, PNI, performs a neural integration to provide an eye position signal essential for horizontal fixation and vestibuloocular reflex performance. The second, VNI, encodes eye velocity and provides the major input signal to the vestibulocerebellum, and thus is instrumental in all oculomotor plasticity paradigms. A third nucleus, the inferior olive (IO), provides climbing fiber input to the cerebellum necessary for eye movement stability. In addition, the physiology and development of the tangential nucleus (TAN) will be studied. TAN is responsible for gravitoinertial compensatory eye reflexes and develops in rhombomere 5 under control of Hox paralog group 3 genes. Otolith-induced torsional eye motion will provide an assay for cross-regulatory effects between Hox4 and Hox3 genes. The first aim of the project will characterize the electrophysiology, morphology, and behavior of identified hindbrain oculomotor neurons endogenously labeled with reporter proteins driven by specific Hox gene regulatory sequences. The second aim will use targeted misexpression of each Hox4 paralog to produce changes in neuronal structure / function and eye movements (as documented in Aim 1) to test the contributions of Hox4 genes to specification of PNI, VNI, IO and TAN neurons. The third aim will use perturbation of retinoic acid-sensitive regulatory pathways to manipulate hindbrain segmentation and neuronal specification to identify additional genes required for origin, migration and function of the four oculomotor nuclei. Disruptions in the genetic regulatory cascades specifying these oculomotor subgroups will be directly linked to morphological and electrophysiological alterations in their task specific neural networks as reflected in behavioral sequelae. The overall objective of the proposed work is to analyze oculomotor neurons and behaviors that have been functionally conserved throughout vertebrate evolution to establish a basis for understanding the developmental genetic underpinnings of human oculomotor behavior and eye movement disorders
Keywords: Affect; Automobile Driving; base; Behavior; Behavioral; Biological Assay; Biological Neural Networks; C.I. Solvent Yellow 56; Cell Nucleus; Cells; Cerebellum; Development; developmental genetics; DNA; Electrophysiology (science); Enhancers; Evolution; Exhibits; Eye; Eye Movements; eye velocity; Fiber; Genes; Genetic; Growth; hindbrain; Human; Image; immunocytochemistry; Immunohistochemistry; In Situ Hybridization; Individual; Inferior; Injection of therapeutic agent; islet; Label; Larva; Link; Location; loss of function; member; Microscopy; migration; Morphology; Motion; neuron development; Neuronal Differentiation; Neurons; Ocular Motility Disorders; oculomotor; oculomotor behavior; Oculomotor nucleus; Oligonucleotides; Olives - dietary; otoconia; paralogous gene; Pattern; Performance; Phenotype; Physiological; Physiology; Play; Population; Positioning Attribute; Production; programs; Property; Proteins; Reflex eye movement; Regulation; Regulator Genes; Regulatory Element; Regulatory Pathway; relating to nervous system; Reporter; Research; Research Personnel; RNA; Role; sample fixation; Signal Transduction; Specific qualifier value; Specimen; Structure; Subgroup; System; Testing; Time; Torsion (malposition); Tracer; transcription factor; Transgenic Organisms; Tretinoin; Up-Regulation (Physiology); vestibulo-ocular reflex; Visual; Work; Zebrafish
Project start date: 1976-09-30
Project end date: 2012-03-31
Budget start date: 1-APR-2011
Budget end date: 31-MAR-2012
5R01EY002007-32 (2011): $402732
SIS MULTICENTER STUDY OF DURATION OF ANTIBIOTICS FOR INTRAABDOMINAL INFECTION
G Robert, Professor Of Surgery
University Of Virginia Charlottesvillecity: Charlottesville country: United States (us)
Grant 5R01GM081510-05 from National Institute Of General Medical Sciences
Abstract: The optimum duration of antibiotic therapy for intraabdominal infection remains unknown and has been identified by the Surgical Infection Society as a high priority for clinical research. The ultimate objective of our research is to optimize (and reduce) the duration of antibiotic therapy for intraabdominal infection throughout the world. The hypothesis to be tested is that four days of therapy for intraabdominal infection will lead to similar outcomes and a shorter duration of therapy when compared to a course based on the resolution of physiologic parameters in the setting of adequate operative or percutaneous intervention. This proposal is for a multicenter, randomized, double-blind (until the fourth day of therapy), non-inferiority clinical trial comparing a predetermined four days of antibiotic therapy to antibiotic therapy terminated one day after normalization of white blood cell count (= 11,000/ul) and normalization of systemic temperature (< 38.0¿ C) for one whole calendar day (and a maximum of 10 days of antibiotic therapy) in the setting of complicated intraabdominal infection treated with adequate source control. Inclusion criteria include age = 16 years, ability to obtain informed consent from the patient or surrogate, presence of an intraabdominal infection requiring any duration of hospitalization and managed with open, laparoscopic, or percutaneous intervention, and, adequate source control in the opinion of the local investigator and Principal Investigator. 1,120 patients will be enrolled to ensure adequate power to assess equivalence of the two arms. The primary endpoint will be percentage failure conditioned by assigned duration of antibiotic therapy (intent to treat analysis). Failure will be defined as need for reintervention (surgical or percutaneous), surgical site infection, or death within 30 days of the original intervention for intraabdominal infection. In addition, multiple secondary endpoints will be assessed, including duration of antibiotic therapy and the incidence of infection at non-abdominal and non-surgical wound sites, particularly with antibiotic-resistant pathogens. The ultimate objective of our research is to change practice throughout the world, specifically by shortening the duration of antibiotic therapy for intraabdominal infections and thus decreasing resource utilization and decreasing the selection of antibiotic-resistant pathogens
Keywords: 16 year old; Abbreviations; Abdomen; Abdominal Infection; Aftercare; Americas; Aminoglycosides; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; antimicrobial; Antimicrobial Resistance; arm; bacterial resistance; base; Blinded; Calendar; Carbapenems; Case Report Form; Cephalosporins; Cessation of life; Clinical; clinical practice; Clinical Research; Clinical Trials; Clinical Trials Data Monitoring Committees; Clostridium difficile; cohort; Communicable Diseases; Data; Death Rate; design; Double-Blind Method; Enrollment; Ensure; Enterococcus faecalis; Enterococcus faecium; Expenditure; Failure (biologic function); Fever; Fluoroquinolones; Funding; fungus; Goals; Gram-Positive Cocci; Hospital Mortality; Hospitalization; Incidence; inclusion criteria; Infection; Informed Consent; inhibitor/antagonist; Institutional Review Boards; Intervention; Intra-abdominal; Laboratories; Lactamase; Lead; Leukocytosis; Methicillin; methicillin resistant Staphylococcus aureus (organism); Microbiology; Multicenter Studies; Operative Surgical Procedures; Organism; Outcome; Oxacillin; pathogen; Patients; Penicillins; Peripheral; Pharmaceutical Resources; Physicians; Physiological; pragmatic trial; Principal Investigator; programs; prospective; Randomized; randomized trial; Recurrence; Research; Research Personnel; Resistance; Resolution; Resources; Risk; Rods (Retina); Safety; secondary infection; Site; Societies; Source; standard of care; Staphylococcus aureus; Surgeon; Temperature; Testing; therapy duration; Time; treatment duration; trial comparing; United States; United States Food and Drug Administration; Vancomycin resistant enterococcus; White Blood Cell Count procedure; wound
Project start date: 2007-09-11
Project end date: 2012-08-31
Budget start date: 1-SEP-2011
Budget end date: 31-AUG-2012
5R01GM081510-05 (2011): $477151
SURGICAL INFECTIOUS DISESES AND TRANSPLANTATION TRAINING GRANT
G Robert, Professor Of Surgery
University Of Virginia Charlottesvillecity: Charlottesville country: United States (us)
Grant 5T32AI078875-03 from National Institute Of Allergy And Infectious Diseases
Abstract: The number of surgeons trained to perform research in humans, including translational research, is inadequate when considering that close to half of all patients hospitalized with acute illness either require an operation or are cared for by surgeons. Many of these patients suffer from infections or have received organ transplants and are managed almost exclusively by surgeons and are of interest to the NIAID. The two objectives of this application are 1) To increase the number of surgeons who perform meaningful translational research in the future by systematically training research residents in the methods of multiple levels of clinical research, particularly in the area of infection complicating acute illness or transplantation, and 2) To increase the number of minority surgeons in academic practice who will be able to improve the health of historically underserved patient populations with increased rates of death. Surgical residents with two or three years of clinical training will be admitted to the program, with a total of four trainees at any given time. Students will usually enter a dedicated, two to three year training period that will include a core clinical project related to infectious diseases affecting acutely ill or transplanted patients, and coursework in the University of Virginia Department of Public Health Sciences that will result in a Masters Degree in Public Health or Clinical Research. Alternatively, a small number of students may elect a core laboratory research experience related to surgical infections or transplantation that will, with the addition of classes and small group seminars in the Department of Public Health Sciences, equip them for an academic career in translational research. Through these techniques, a cohort of young, well-trained surgeons will be developed who will have the tools to perform high quality, ethical human research specifically centered on that interface of clinical and basic science that is translational research. RELEVANCE Only a small number of surgeons perform research specifically aimed at improving the outcomes of these patients by safely introducing new scientific breakthroughs to their care. We propose to train young surgeons, including those from traditionally underrepresented minorities, to be able to perform this research throughout their careers and increase the survival of patients suffering from severe surgical diseases
Keywords: Grant; Operative Surgical Procedures; Training; Transplantation
Project start date: 2009-08-01
Project end date: 2014-05-31
Budget start date: 1-JUN-2011
Budget end date: 31-MAY-2012
PFA/PA: PA-08-226
5T32AI078875-03 (2011): $222704
G Robert
University Of Pennsylvaniacity: Philadelphia country: United States (us)
Abstract: The main goal of this core is to identify a panel of surrogate protein markers measurable in human serum that, in combination with other clinical factors, predicts long-term dysfunction following mild TBI. A second goal is to use surrogate markers for axonal degeneration to promote studies of the underlying molecular mechanisms and provide further validation for experimental models used in this proposal. Thus far, no individual marker has consistently demonstrated the ability to predict long-term functional impairment following mild TBI. To identify new markers for TBI and develop a marker panel, my laboratory initiated the first global analysis of protein release from degenerating neurons. We identified 14-3-3(3, 14-3-3^, a hypophpsphorylated form of neurofilament H, a calpain-derived cleavage product of a-spectrin, and a caspase cleavage product of aspectrin as highly abundant neuron-enriched proteins released from dying neurons. The validity of this approach was established by immunodetection of these proteins in CSF and serum following TBI or cerebral ischemia in rats, as well as severe TBI or surgically-induced circulation arrest in humans. To extend this research to mild human TBI, the specific aims of the core are to (1) determine relationships between surrogate marker protein release, sodium channel dysfunction and axonal degeneration in a neuronal culture model for axonal injury (Project 3); (2) analyze our novel surrogate marker panel in a pig model of mild TBI, and relate changes to axonal histopathology and sodium channel dysfunction (Project 2); (3) evaluate serum elevations for the same marker panel in mild human TBI, and compare their levels with cognitive, neurological, and radiological measures of dysfunction (Project 1). A surrogate marker-based method for the prognosis of mild TBI, along with a simple quantitative means of assessing experimental neuroprotective therapies, would have enormous benefit for the management and treatment of mild TBI
Keywords: Acute; Antibodies; attenuation; Axon; axonal degeneration; base; biomarker; Blood Circulation; Brain; Brain Injuries; Calpain; candidate marker; caspase; Cerebral Ischemia; Cerebrospinal Fluid; Clinical; clinical application; Cognitive; Dependence; Deubiquitinating Enzyme; Diffuse Axonal Injury; Experimental Models; Family suidae; Fostering; functional disability; Functional disorder; Goals; Histopathology; Human; In Vitro; Individual; injured; Injury; Laboratories; Libraries; Measurable; Measures; Methods; Modeling; Modification; Molecular; Molecular Weight; Nerve Degeneration; Nervous system structure; Neurobiology; neurofilament; Neurofilament-H; Neurologic; Neurons; novel; novel marker; outcome forecast; Patients; pre-clinical; Protein Analysis; Proteins; Proteomics; Rattus; Research; research clinical testing; Running; Sensitivity and Specificity; Serum; Serum Markers; Sodium Channel; Spectrin; Stretching; Surrogate Endpoint; Surrogate Markers; Techniques; Testing; Traumatic Brain Injury; Validation
Budget start date: 1-AUG-2011
Budget end date: 31-JUL-2012
5P01NS056202-04_9002 (2011): $133836
BRAINSTEM AND CEREBELLAR CONTROL OF HORIZONTAL EYE MOVEMENT
G Robert
New York University School Of Medicinecity: New York country: United States (us)
Abstract: The broad goal of the research is to better understand cerebellar function by investigating the neuronal signal processing underlying compensatory eye movements. For the most part the experimental method will be manipulation of the extracellularly recorded activity of cortical interneurons and Purkinje cells in the cerebellar flocculus through presentation of natural visual and vestibular stimuli to the rabbit. A central tenet is that to understand how the cerebellum functions as a ´neuronal machine´, it is necessary to know, from a systems point of view, the signal content and signal processing of cerebellar cortical interneurons (granule-, Golgi-, basket/stellate-, and unipolar brush cells). Presently, this knowledge is hardly available, particularly for an awake, behaving animal. Consequently, one focus is investigation of the signal content and processing of floccular interneurons in the awake rabbit presented with natural stimuli. The various types of cerebellar cortical interneurons can now be identified using our recent finding the each type of cerebellar cortical interneuron has a characteristic pattern of spontaneous activity. A second focus is the signal content and synchrony of climbing fiber activity, recorded as floccular Purkinje cell complex spikes. In Specific Aim 1 the relations between the vestibularly and visually induced modulation of floccular interneuronal activity and aspects of eye movement behavior (kinematics, vestibulo-ocular response gain, and vergence) will be investigated in the rabbit. Specific Aim 2 will use local cortical injections of the GABA-A receptor blocker gabazine to examine the role of Golgi cell inhibition in producing the high frequency burst activity characteristic of rabbit floccular granule cells. In Specific Aim 3 the signal content of the mossy fiber input to the rabbit flocculus arising from the brainstem vestibular and prepositus hypoglossi nuclei will be determined so as to help assess what signal transformations occur at mossy fiber-granule cell synapses. In Specific Aim 4 the contribution of the prepositus hypoglossi nuclei to the vestibularly induced floccular complex spike modulation will be studied using ablative and chemical lesions. Specific Aim 5 deals with .the role of complex spike synchrony in motor behavior. Synchrony during vestibular stimulation will be compared to that occurring without stimulation to evaluate the hypothesis that synchrony will be altered during vestibular stimulation because of the modulation of the GABA input from the prepositus hypoglossi nuclei to the dorsal cap of the inferior olive. Understanding the signal processing of cerebellar cortical interneurons and climbing fiber messages is relevant to comprehending the cerebellum´s role both in the moment-to-moment control of movement and posture and in learning adaptive motor behaviors. Cerebellar dysfunctions are often due to genetic dysfunctions, and knowledge of signal processing in the rabbit cerebellum has already been shown to be transferable to mouse models of gene-based dysfunctions. . ¿ ; -
Keywords: Acceleration; Animals; Aspirate substance; awake; base; Behavior; Brain Stem; Brush Cell; Cell Nucleus; Cells; Cerebellar Diseases; Cerebellum; Characteristics; Chemicals; Complex; computerized data processing; Coupling; Cytoplasmic Granules; Dorsal; Electric Stimulation; Evaluation; Eye Movements; Fiber; Frequencies (time pattern); Functional disorder; GABA-A Receptor; gabazine; gamma-Aminobutyric Acid; Genes; Genetic; Goals; Golgi Apparatus; granule cell; Inferior; Injection of therapeutic agent; Interneurons; Investigation; kinematics; Knowledge; Learning; Lesion; Measures; Membrane; Methods; mossy fiber; Motor; mouse model; Movement; Neurobiology; Neurons; Olives - dietary; Oryctolagus cuniculus; Output; Pattern; Phase; Photic Stimulation; Posture; Principal Investigator; Process; programs; Property; Purkinje Cells; Rattus; relating to nervous system; Research; response; Role; Signal Transduction; Source; spatiotemporal; stellate cell; Stimulus; Synapses; System; Techniques; Testing; Time; visual-vestibular
Budget start date: 1-MAY-2011
Budget end date: 30-APR-2012
5P01NS013742-33_0002 (2011): $395713
PROBING LIGHT RESPONSES OF ON BIPOLAR AND AII AMACRINE CELLS WITH CALCIUM IMAGING
G Robert, Research Professor
University Of Pennsylvaniacity: Philadelphia country: United States (us)
Grant 1R21EY021308-01 from National Eye Institute
Abstract: Retinal bipolar cells are the key link between photoreceptors and ganglion cells. One bipolar cell type, the rod bipolar cell, transmits the dim light signal at night, while about 10 types of cone bipolar cells transmit the detailed information of the visual image in daylight. Because the visual image contains information from various features (contrast, spatial, temporal, color, etc.), each cone bipolar type extracts certain features and transmits them optimally. The largest class of bipolar cells, the ON class, conveys positive contrast with responses that are mediated by a transduction cascade. When whole-cell patched, their light responses runs down rapidly. Consequently, information about the physiological properties of different ON cone bipolar cell types is scarce. Recently, a new calcium indicator protein (GCaMP3) was developed, and it can specifically be targeted to ON bipolar cells (under control of mGluR6 promoter) or to the closely connected AII amacrine cells (under control of mGluR1 promoter). We here propose to image this indicator with two-photon microscopy and combined it with electrophysiology to investigate the physiology and visual contribution of these cells. Aim 1 will investigate the rod bipolar cell´s adaptation mechanism that critically depends on calcium accumulation to lower the response gain. Retinas will be stimulated with ascending light intensities and calcium signal will be recorded in rod bipolar dendrites and axon terminals. Input-output functions will determine the amount of calcium that causes adaptation. The source of calcium will be determined by either emptying calcium stores, blocking intracellular calcium channels, or blocking TRPM1 transduction channels. Aim 2 will determine the physiological differences among the types of ON cone bipolar cells in two ways. First, the retina will be stimulated with flashing or temporally modulated sinusoidal light with varying intensities, and the calcium responses of different cone bipolar types will be recorded by imaging axon terminals that reside in all ON layers of the inner plexiform layer. Second, an AII cell will be depolarized, and the strength of its coupling to the cone bipolar types will be measured by calcium imaging. In order to reveal the cell type identity of the imaged terminals, at the end of the recording session, dye will be injected into multiple cells with a microelectrode. Aim 3 will measure the dynamics of coupling and noise within the AII network under different light intensities using two complementary methods. First, AII amacrine cells will be infected with channelrhodopsin fused to GFP; an AII cell will be patched with whole cell configuration; channelrhodopsin at various distances from the patched cell will be stimulated; and the resulting voltage in the cell will be recorded. Second, AII amacrine cells will be infected with GCaMP3; current will be injected into a cell that is whole-cell patched; and the resulting calcium response in neighboring AII cells will be measured. These experiments will be repeated after blocking gap junctions and/or Na+ channels. The proposed experiments will greatly facilitate our understanding of retinal circuits and parallel processing and they will help apply this knowledge to efforts in restoring vision. Our goal of imaging light-evoked calcium responses in the ON bipolar cells and the AII amacrine cells will have a substantial impact on the field because these recordings are still novel and they promise to pave the way for efficient recordings from specific cell compartments in the retina. These will yield important new information relatively fast, and will gain greater understanding of the principle of visual processing in night and day vision. This understanding in turn will help design more optimal approaches for the ever developing tools of genetic therapy
Keywords: Address; Amacrine Cells; Amacrine Cells of Retina; Axon Terminals; Benzoic acid, 2-((2, 6-dichloro-3-methylphenyl)amino)-; biological signal transduction; Blood Coagulation Factor IV; Ca Release Channel-Ryanodine Receptor; Ca++ element; Calcium; Calcium Channel; Calcium Channel Antagonist Receptor; calcium indicator; Calcium Ion Signaling; Calcium Signaling; Calcium-Ryanodine Receptor Complex; Cell Communication and Signaling; Cell Signaling; cell type; Cells; Coagulation Factor IV; Color; Coloring Agents; Communicating Junction; computerized data processing; Cone; cone cell; Cones (Eye); Cones (Retina); Coupled; Coupling; data processing; Dendrites; design; designing; Dyes; Electrical Synapse; Electrodes, Miniaturized; Electrophysiology; Electrophysiology (science); experiment; experimental research; experimental study; Factor IV; Fluorescence Agents; Fluorescent Agents; fluorescent dye/probe; Fluorescent Dyes; Frequencies (time pattern); Frequency; gangliocyte; ganglion cell; Gap Junctions; gene product; gene therapy; Gene Transfer Clinical; Gene Transfer Procedure; Gene-Tx; Genetic Intervention; genetic therapy; Goals; Heparin; Heparinic Acid; Image; imaging; Inner Plexiform Layer; Intervention, Genetic; Intracellular Communication and Signaling; Ion Channels, Calcium; Knowledge; Light; light intensity; Link; Low-resistance Junction; Maps; Measures; Meclofenamate; Meclofenamic Acid; Mediating; Methods; Microelectrodes; Microscopy; Molecular Biology, Gene Therapy; Morphology; Nerve Cells; Nerve Endings, Presynaptic; Nerve Unit; Neural Cell; Neurocyte; Neuromodulator; neuronal; Neurons; Neurophysiology / Electrophysiology; Nexus; Nexus Junction; Noise; novel; Ocular Physiology; Output; parallel processing; Photons; Photoradiation; Photoreceptor Cell; Photoreceptors; Photoreceptors, Cone; Photosensitive Cell; Physiologic; Physiological; Physiology of the Eye; Presynaptic Terminals; Process; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Property; Property, LOINC Axis 2; Proteins; public health relevance; receptor; Receptor Protein; Receptors, Calcium Channel Blocker; research study; response; Retina; Retinal; Retinal Cone; Rod; rod cell; Rod Photoreceptors; Rods (Eye); Rods (Retina); Running; Ryanodine; Ryanodine Receptor; Ryanodine Receptor Calcium Release Channel; Ryanodine Receptors; Ryanodol, 3-(1H-pyrrole-2-carboxylate); Sight; signal processing; Signal Transduction; Signal Transduction Systems; Signaling; Source; stem; Stimulus; Stratification; Synapses; Synaptic; Synaptic Boutons; Synaptic Terminals; Testing; Thapsigargin; Therapy, DNA; tool; two-photon; V (voltage); VDCC; Vision; Visual; visual information; Visual Physiology; visual process; visual processing; Visual Receptor; voltage; Voltage-Dependent Calcium Channels
Relevance: Our goal of imaging light-evoked calcium responses in the ON bipolar cells and the AII amacrine cells will have a substantial impact on the field because these recordings are still novel and they promise to pave the way for efficient recordings from specific cell compartments in the retina. These will yield important new information relatively fast, and will gain greater understanding of the principle of visual processing in night and day vision. This understanding in turn will help design more optimal approaches for the ever developing tools of genetic therapy
Project start date: 2011-01-01
Project end date: 2012-12-31
Budget start date: 1-JAN-2011
Budget end date: 31-DEC-2011
PFA/PA: PA-10-069
1R21EY021308-01 (2011): $231564
G Robert
Washington Universitycity: Saint Louis country: United States (us)
Grant 2R01GM047909-16 from National Institute Of General Medical Sciences
Abstract: Project Summary/ Cytochromes are heme proteins essential for the aerobic and anaerobic growth of most organisms, including human pathogens. Recently it has become clear that dedicated assembly factors are crucial for cytochrome biogenesis. The biogenesis of c-type cytochromes occurs by one of three pathways, systems I, II, or III. System I has eight (CcmABCDEFGH) and system II has two (CcsBA) dedicated assembly factors (membrane proteins), while system III uses a single enzyme called cytochrome c heme lyase. Because only prokaryotes, plants, and protozoa use systems I and II, these pathways represent potential targets for antimicrobial agents. The c-type cytochromes possess heme that is covalently ligated to the apocytochrome at two cysteines. The cysteines and heme (to Fe2+) must be reduced for attachment to occur. Moreover, all cytochromes c are assembled and function outside of the inner membrane. This study examines how proteins in systems I and II deliver heme, synthesized inside the cell, and attach it to the secreted unfolded apocytochrome c. The proposal takes advantage of our previous success in purifying all proteins of systems I and II from recombinant Escherichia coli. For most of these purified components, endogenous heme has been trapped, facilitating analyses of heme transport, red-ox control, and attachment mechanisms. Three aims are proposed, the first two for system I, and the third for system II. System I is described in two steps. Step 1 is the CcmABCD-mediated synthesis and release of periplasmic holoCcmE (heme in the oxidized Fe3+ state). Aim 1 analyzes this step, establishing residues in CcmC and CcmE that directly interact with heme and the chemical mechanisms to form the oxidized holoCcmE. The holoCcmE release step is reconstituted with purified CcmABCDE proteins. Step 2, analyzed in Aim 2, includes the CcmF/H-mediated reduction of holoCcmE (to Fe2+) and ligation of this heme to apocytochrome. The mechanism of reduction in vivo (e.g., qui-based) is analyzed, as well as residues in CcmF for interacting with CcmH, holoCcmE, and a novel b heme we discovered. Aim 3 analyzes the recombinant system II CcsBA integral membrane protein that we demonstrated has heme export and cytochrome c synthetase functions. The following hypothesis will be tested CcsBA binds heme via two conserved histidines in a "channel" and protects the heme from oxidation as it moves to an external heme binding site. The external apocytochrome c binding site and attachment to heme will be studied in vitro. Prokaryotes have evolved hundreds of different cytochromes c, ranging from the mitochondrial-like cytochrome c with a single heme to extraordinary c-type cytochromes with over ten heme molecules attached to a single polypeptide. Results here will unravel the mechanisms underlying heme transport, heme red-ox control, and heme attachment to all prokaryotic and plant c-type cytochromes. PojectNaratie ranismshavecmmnmechanismst cnver chemicalfosdtfsinto nrgy,forexample hemiohconra aerobicresiratorcyai.However,to asemblea ubiquious cmpnen fthesehains caledctchrmec, prkaroesandceraineuaroesusequiedifferen pahwcasle( ytem andI)thanhumans(systemIId).e rsaUndin ssemsIandIIa ehe e lvlropose herewl neablethediscver fhemial (anibitics)tha pecificall are heprkaroicsse,tmhsu impatingsuchdiseae as tuberuloi,uler, an any resiratoryan nfammatoryilnesses
Keywords: ing; Address; adduct; Aerobic; antimicrobial drug; base; Binding (Molecular Function); Binding Sites; Bioenergetics; Biogenesis; Biological Assay; Cell membrane; Cells; Chemicals; Chloroplasts; cofactor; Complex; Cysteine; cytochrome c; Cytochrome c Group; cytochrome C synthetase; Cytochromes; Electron Transport; Engineering; Enzymes; Escherichia coli; Eukaryota; Figs - dietary; frontier; Growth; Heme; Hemeproteins; Histidine; Human; Hydroquis; In Vitro; in vivo; Integral Membrane Protein; Intracellular Transport; Ligands; Ligase; Ligation; Mediating; Membrane; Membrane Proteins; Mitochondria; Modeling; Molecular; novel; One-Step dentin bonding system; Organism; oxidation; Oxidoreductase; pathogen; Pathway interactions; periplasm; Plants; polypeptide; Prokaryotic Cells; Protein C; Proteins; Protozoa; Quis; Recombinants; reconstitution; success; System; Testing; Transmembrane Domain
Relevance: PojectNaratie ranismshavecmmnmechanismst cnver chemicalfosdtfsinto nrgy,forexample hemiohconra aerobicresiratorcyai.However,to asemblea ubiquious cmpnen fthesehains caledctchrmec, prkaroesandceraineuaroesusequiedifferen pahwcasle( ytem andI)thanhumans(systemIId).e rsaUndin ssemsIandIIa ehe e lvlropose herewl neablethediscver fhemial (anibitics)tha pecificall are heprkaroicsse,tmhsu impatingsuchdiseae as tuberuloi,uler, an any resiratoryan nfammatoryilnesses
Project start date: 1994-09-01
Project end date: 2015-07-31
Budget start date: 1-AUG-2011
Budget end date: 31-JUL-2012
PFA/PA: PA-10-067
2R01GM047909-16 (2011): $342000
HIGH THROUGHPUT ASSAY FOR DETECTING PROTEIN MODIFICATIONS IN CELL LYSATES
G Robert, President
Bellbrook Labs, Llccity: Madison country: United States (us)
Grant 1R43GM101771-01 from National Institute Of General Medical Sciences
Abstract: Post translational modifications (PTMs) such as phosphorylation, glycosylation and methylation play a central and ubiquitous role in cellular signal transduction, and the enzymes that catalyze these reactions such as kinases and methyltransferases are the targets of intense drug discovery efforts for virtually every therapeutic area, with the most intense focus on cancer. Specific detection of posttranslational modifications (PTMs) in cell extracts is a fundamental technical challenge facing drug discovery and proteomics researchers working in this area. Though many site-specific antibodies are available, they often are not selective enough to differentiate between closely related PTM sites, leading to unreliable results. Moreover, the immunocytochemical or Western blot methods used to detect PTMs are difficult to perform in an automated, high throughput fashion, which discourages their use for identifying potential drug molecules using high throughput screening (HTS). To overcome these technical barriers, we propose to develop a generic platform for rapid, in vitro development of assays for highly specific, homogenous detection of PTMs that leverages the extensive pool of available PTM antibodies. Phase I feasibility will include a) increasing the specificity of PTM immunodetection methods using peptides from phage display libraries that specifically recognize antibody-phosphoprotein complexes and b) demonstrating homogenous detection of the trivalent immune complex using a new single label method called Quenched Resonance Energy Transfer. In Phase II, BellBrook will develop panels of "High Throughput-PTM" assays for many of the most therapeutically relevant kinase and methyltransferase pathways and commercialize them as HTS cellular assay kits with a simple lyse-and-detect format. These products could have a significant impact on drug discovery for cancer and other diseases by enabling large scale screening for kinase and methyltransferase inhibitors in the physiological context of intact cells using the most immediate endpoint of target enzyme activity a specific PTM. Additionally, we will investigate how the specificity and detection enhancements of the High Throughput-PTM platform can be applied to improve proteomic methods, such as histochemical detection of PTMs, and thus accelerate efforts to map PTMs in healthy and diseased tissues. The function of most proteins in the cell is regulated by covalent modifications, and aberrant modifications underlie many disease pathologies, especially cancer. We propose to develop detection methods to accelerate the identification of drug molecules that prevent specific aberrant protein modifications without affecting normal ones
Keywords: Affect; Amino Acids; Antibodies; Antigen-Antibody Complex; Antigens; Area; assay development; base; Binding (Molecular Function); Biochemical; Biological Assay; Cell Extracts; Cell physiology; Cells; Cellular Assay; Complex; Cytolysis; Detection; Disease; drug discovery; Energy Transfer; Environment; enzyme activity; Enzymes; Epidermal Growth Factor Receptor; Epitope Mapping; Event; Fluorescence; gel electrophoresis; Generations; Generic Drugs; glycosylation; high throughput screening; Immunoblotting; immunocytochemistry; improved; In Vitro; inhibitor/antagonist; Internet; Label; Lanthanoid Series Elements; Liquid substance; Malignant Neoplasms; Maps; Measures; Methods; Methylation; Methyltransferase; Modeling; Modification; mutant; Noise; Pathology; Pathway interactions; Peptide Phage Display Library; Peptides; Pharmaceutical Preparations; Phase; Phospho-Specific Antibodies; Phosphopeptides; Phosphoproteins; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Play; Post-Translational Modification Site; Post-Translational Protein Processing; prevent; Proteins; Proteomics; Protocols documentation; Reaction; Research Personnel; Role; Screening procedure; Signal Transduction; Site; Solid; Specificity; Therapeutic; Time; Tissue Sample; Tissues; tool; Western Blotting; Work
Relevance: The function of most proteins in the cell is regulated by covalent modifications, and aberrant modifications underlie many disease pathologies, especially cancer. We propose to develop detection methods to accelerate the identification of drug molecules that prevent specific aberrant protein modifications without affecting normal ones
Project start date: 2011-09-26
Project end date: 2012-09-25
Budget start date: 26-SEP-2011
Budget end date: 25-SEP-2012
PFA/PA: PA-10-050
1R43GM101771-01 (2011): $266465
Sponsored Links Excellgen http://Excellgen.com
GENDER - SENSITIVE TREATMENT IN MIXED - GENDER PROGRAMS
G Robert
Westat, Inc.city: Rockville country: United States (us)
Grant 5R01DA020082-05 from National Institute On Drug Abuse
Abstract: Over the past two decades, gender-sensitive services have emerged in response to the multidimensional profile of problems that women display upon admission to substance abuse treatment. This has been accompanied by considerable research on women in both mixed-gender treatment settings and specialized women-only settings, and some evidence has accumulated to suggest that women admitted to women-only programs have better retention and better outcomes relative to traditional mixed-gender programs. However, most women in the United States are treated in nonspecialized mixed-gender settings and little empirical research has measured the degree to which gender-sensitive programming occurs in such settings, related it to treatment outcomes, or assessed whether the value added offsets the costs. The present study will 1) Assess naturally occurring variation in gender-sensitive programming for women across traditional, mixed- gender short-term residential treatment programs in Washington State; 2) Determine value-added from gender-sensitive programming, in terms of proximal and distal client outcomes for women; 3) Determine which components or practices of gender-sensitive programming in mixed-gender settings appear promising; and 4) Determine whether such programming is cost effective and cost beneficial. The target population is women 18 years old and over admitted to one of 15 short-term residential mixed-gender programs in Washington from April 1, 2005 through March 31, 2009. The principal independent variable is program-level gender-sensitivity, which will be established through interviews with multiple key informants at each program site using semistructured protocols. Distal outcomes (repeat treatment episodes, employment rates and earned income, dependence on unearned income supports, and involvement with the criminal justice and child welfare systems) will be tracked for 2 years pre- and 2 years postadmission from administrative data sources
Keywords: 18 year old; Address; Admission activity; Advisory Committees; Aftercare; Alcohol abuse; Alcohols; Area; Characteristics; Child Welfare; Client; Clinical Trials Network; Collaborations; cost; cost effective; cost effectiveness; Cost Effectiveness Analysis; Costs and Benefits; Criminal Justice; Data; Data Sources; Dependence; Development; disability; Distal; Drug abuse; Effectiveness; Elements; Eligibility Determination; Empirical Research; Employment; Enrollment; Environment; Evaluation Research; Evidence based practice; Funding; Gender; Goals; Health; Health Services; Health Services Research; improved; Income; Indigenous; informant; innovation; instrument; instrumentation; interest; Intervention; Interview; Knowledge; Link; Measures; Medical; meetings; member; Mental Health Services; Methamphetamine; method development; Methodology; Methods; Modeling; National Institute of Drug Abuse; Natural experiment; Outcome; Outpatients; Parenting behavior; Performance; Persons; Pilot Projects; Population; Process; programs; Protocols documentation; Recommendation; Records; Relative (related person); Reporting; Research; research and development; Research Infrastructure; Research Personnel; Research Support; Residential Treatment; response; Services; Site; social; Social Health Services; Social Welfare; Specific qualifier value; Statutes and Laws; Stress; Substance abuse problem; substance abuse treatment; System; Target Populations; treatment as usual; Treatment outcome; treatment program; treatment site; United States; Variant; Washington; Woman
Project start date: 2007-09-07
Project end date: 2012-08-31
Budget start date: 1-SEP-2011
Budget end date: 31-AUG-2012
PFA/PA: PA-01-097
5R01DA020082-05 (2011): $546675
PATHOGENIC MECHANISMS OF PRESENILIN MUTATION
G Robert
University Of Pennsylvaniacity: Philadelphia country: United States (us)
Grant 5R01AG017138-09 from National Institute On Aging
Abstract: Mutations in the genes encoding presenilins 1 and 2 (PS-1 and PS-2) and the ¿-amyloid precursor protein (APP) are the leading cause of familial Alzheimer´s disease (FAD). Overexpression of these gene mutations in transgenic mice recapitulates pathological and behavioral features of AD. It has supported the hypothesis that aggregation of the amyloid A¿42 protein is an important trigger for disease onset and progression, and fostered the development of therapeutic strategies based on reducing A¿42 aggregates. However, the utility of transgenic models for investigating AD pathogenesis and evaluating candidate therapies is constrained by the dependence on ectopic overexpression. As an alternative, we introduced mouse lines in which gene targeting was used to "knock-in" disease-causing mutations into their endogenous genes. Our studies established the only mouse model for AD-type amyloid, tau, and neuroinflammatory pathologies that does not rely on ectopic overexpression, and identified neurobiological and molecular mechanisms central to AD pathogenesis. The proposed research would extend these findings by delineating at the molecular level a key mechanism for A¿42 overproduction, defining neurobiological roles for amyloid, tau, and neuroinflammatory pathologies in impairing forms of adaptive plasticity in hippocampal circuits known to be both important for long-term memory and severely impacted in AD, and identifying therapies that reverse these pathologies and rescue the plasticity deficits. Specific Aim 1 will identify structural and functional changes caused by mutant PS-1 in the mouse brain ?-secretase, the protease that forms the amyloid A¿42 protein, and test the hypothesis that mutant PS-1 confers a pathogenic conformation on the protease. Specific Aim 2 will test the hypothesis that the amyloid and tau pathologies impair synaptic plasticity in the entorhino-hippocampal perforant pathway, and evaluate multiple pharmacologic strategies for reversing the pathology and impaired plasticity. Specific Aim 3 will test the hypothesis that mutant PS-1 hinders neurogenesis in the adult hippocampus through amyloid-triggered neuroinflammation, and determine its reversibility. The proposed research will advance our understanding of molecular and neurobiological mechanisms of FAD-linked gene mutations and evaluate several therapeutic strategies aimed at reducing neuroplasticity deficits in a faithful mouse genetic model of FAD. "Pathogenic mechanisms of presenilin mutation" The proposed research will use a novel and faithful mouse genetic model to advance our understanding mechanisms by which certain gene mutations cause inherited Alzheimer´s disease. It will promote basic understanding of the neurobiological mechanisms underlying the cognitive and behavioral syndrome of AD, and evaluate multiple candidate therapeutic strategies that target these neurobiological processes. This translational research in a disease-relevant preclinical model is crucial to the development of therapies aimed at delaying the onset and slowing the progression of AD with a high likelihood of success in the clinic
Keywords: Adult; Adverse effects; Age; age related; Aging; Alzheimer`s Disease; Amyloid; Amyloid beta-Protein Precursor; Amyloid deposition; amyloid pathology; Antibodies; Attenuated; base; Behavioral; Brain; cell type; Cerebral hemisphere hemorrhage; Clinic; Cognition; Cognitive; Complex; Cues; Data; Defect; dentate gyrus; Dependence; Development; Disease; Disease Progression; disease-causing mutation; familial Alzheimer disease; Fostering; functional disability; Functional disorder; Gene Mutation; Gene Targeting; Gene Transfer Techniques; Genes; Genetic Models; Genotype; Gliosis; Goals; Growth; Hippocampus (Brain); Human; Immunization; Impaired cognition; Impairment; In Vitro; Induced Mutation; Inflammatory; Inherited; inhibitor/antagonist; Intervention; Knock-in Mouse; Laboratory Study; Light; Link; long term memory; Long-Term Potentiation; Mediating; Memory; Meningoencephalitis; Microglia; Minocycline; Modeling; Molecular; Molecular Conformation; Moods; mouse model; Mus; mutant; Mutant Strains Mice; Mutation; nerve stem cell; neurobiological mechanism; Neurobiology; neuroblast; neurogenesis; neuroinflammation; Neuronal Plasticity; Neurons; neuropathology; novel; Onset of illness; overexpression; Pathogenesis; Pathology; Peptide Hydrolases; Perforant Pathway; Physiological; Physiology; Population; pre-clinical; Pre-Clinical Model; presenilin; presenilin-1; Process; protein aggregation; Protein Overexpression; Proteins; Regulation; relating to nervous system; Relative (related person); Research; Role; secretase; stem; Stem cells; stoichiometry; Structure; success; Synapses; Synaptic plasticity; Syndrome; System; tau mutation; tau Proteins; Tauopathies; Testing; Therapeutic; Therapeutic Agents; therapeutic development; therapy development; Transgenes; Transgenic Mice; Transgenic Model; Translational Research
Project start date: 2001-02-01
Project end date: 2013-02-28
Budget start date: 1-MAR-2011
Budget end date: 29-FEB-2012
PFA/PA: PA-07-070
5R01AG017138-09 (2011): $260192
C-O AND C-N BOND FORMATION USING METAL-BASED REAGENTS
G Robert, Gerald E. K. Branch Distinguished Profes
University Of California Berkeleycity: Berkeley country: United States (us)
Grant 5R01GM025459-31 from National Institute Of General Medical Sciences
Abstract: Metal-mediated oxidation reactions play an important role in biological processes and in abiological reactions used in organic synthesis. The function of a large proportion of these reactions is the transfer of groups containing heteroatoms such as nitrogen, oxygen, phosphorus and sulfur to organic molecules. Many of these processes have been postulated to proceed via metal complexes that contain reactive metal-heteroatom multiple bonds. However, exploration of the chemistry of these functionalities, extending and applying their reactions to transformations useful in organic synthesis, and understanding the mechanisms of their reactions, lag substantially behind what is know about analogous processes involving metal-carbon multiple bonds (carbene complexes). As a result, there is a need for development of new chemistry based on isolable complexes that bear metal-heteroatom multiple bonds. The overall goal of this proposal is the development of systematic methods for the synthesis of complexes containing metal-heteroatom multiple bonds, the development of new stoichiometric and catalytic reactions in which these bonds play a central role that can be applicable to organic synthesis, and the study of the scope and mechanisms of these reactions. A significant part of the project will be devoted to the synthesis of chiral, enantioresolved complexes that can be used in the development of stereoselective transformations leading to enantioenriched organic compounds with new bonds between carbon and heteroatoms. Specific targets for the proposed grant period will be the development of (1) stereoselective, catalytic and stoichiometric carboamination of alkynes; (2) catalytic diimination of unsaturated substrates; (3) highly regio- and stereoselective allylic substitutions of allyl halides and ethers; (4) methods for desymmetrization of meso-epoxides; (5) reactive and enantioselective catalysts for the hydroamination of alkenes; (6) catalytic hydrophosphination of alkenes and alkynes; (7) exploration of the addition of organic C-H bonds to metal- heteroatom multiple bonds
Keywords: Affect; Alkenes; Alkynes; Area; base; Bears; Biological; Biological Function; Biological Process; C element; carbene; Carbon; catalyst; Chemistry; Classification; Complex; Cytochrome P-450; Cytochrome P-450 Enzyme System; Cytochrome P450; design; designing; Development; Epoxides; Epoxy Compounds; Ethers; Goals; Grant; Hydrogen Bonding; improved; Investigation; Learning; Mediating; metal complex; Metals; Methane; Method LOINC Axis 6; Methodology; Methods; methylene; methylene radical; Mono-S; MonoS; N element; N2 element; Nitrogen; O element; O2 element; Olefins; Organic Synthesis; oxidation; Oxygen; Oxygenases; P element; P450; Phosphorus; Play; Process; Reaction; Reagent; Research; Research Design; Role; S element; Science of Chemistry; social role; study design; Study Type; Sulfur; System; System, LOINC Axis 4; Systematics; Transition Elements; transition metal; Ursidae; Ursidae Family
Project start date: 1978-08-01
Project end date: 2011-06-30
Budget start date: 1-JAN-2009
Budget end date: 30-JUN-2011
5R01GM025459-31 (2009): $321020
RELAXIN FAMILY PEPTIDES AND HEPATIC FIBROSIS
G Robert, Assoc Professor
University Of Nebraska Medical Centercity: Omaha country: United States (us)
Grant 5R01AA015509-03 from National Institute On Alcohol Abuse And Alcoholism
Abstract: Hepatic fibrosis results from chronic liver injury induced by alcohol abuse, hepatitis and other causes that progresses to cirrhosis and liver dysfunction. The hormone relaxin has shown promise as an antifibrotic agent in a number of tissues, including the liver, and relaxin has antifibrotic effects on hepatic stellate cells, the major source of fibrillar collagen in liver disease. Little is known about the expression of relaxin receptors and their ligands during the progression of hepatic fibrosis. The objective is to define the contribution of relaxin family hormones to the pathogenesis and treatment of fibrotic liver disease by testing the hypothesis that interaction of relaxin ligands with specific receptors contributes to protection against hepatic fibrosis. To test this hypothesis, three specific aims are proposed Specific Aim #1. Determine the therapeutic efficacy of using relaxin peptide hormones alone and in combination to treat experimental liver disease. Specific Aim #2. Determine the role of relaxin receptors in the progression and resolution of experimental liver disease using relaxin receptor-null mice. Specific Aim #3. Define the relationship between relaxin receptor signaling and the antifibrotic action of peroxisome proliferator-activated receptor gamma. To achieve these aims, relaxin peptides alone and in combination will be used to treat experimental liver disease. Relaxin receptors-null mice will be used in models of liver disease to directly determine the role of the relaxin receptors in the progression of and recovery from of liver disease. The relaxin signaling through the cAMP/protein kinase A and peroxisome proliferator-activated receptor gamma (PPARy) pathways will be defined, and the combined effect of relaxin peptides and PPARy agonists on treatment of experimental hepatic fibrosis will be determined. The proposed research is significant, because understanding the response to treatment with relaxin receptor ligands, the role of the relaxin receptors in liver disease progression and resolution, and the signaling pathways regulated by relaxin receptors, will allow the development of new strategies to treat hepatic fibrosis. Cirrhosis affects 900,000 people and causes 36,000 deaths in the US annually. Treatment is currently limited to removal of the cause of the injury, which is not always possible or effective, or transplantation. The research proposed in this application may lead to targeted therapeutic strategies for the treatment of liver disease, a major health concern in the United States
Keywords: 3`5`-cyclic ester of AMP; Abscission; adenosine 3`5` monophosphate; Adenosine Cyclic 3`, 5`-Monophosphate; Adenosine Cyclic Monophosphate; Adenosine Cyclic Monophosphate-Dependent Protein Kinases; Adenosine, cyclic 3`, 5`-(hydrogen phosphate); Affect; Agonist; Alcohol abuse; alcohol induced hepatic injury; alcohol induced liver disorder; alcohol induced liver injury; alcohol problem; alcohol research; alcohol withdrawal; Alcohol withdrawal syndrome; alcohol-induced hepatic dysfunction; alcohol-induced liver disease; alcohol-induced liver dysfunction; alcohol-mediated liver dysfunction; alcohol-mediated liver injury; Alcoholic Liver Diseases; Antiviral Agents; Antiviral Drugs; Antivirals; base; Biochemical; biological signal transduction; body system, hepatic; Body Tissues; cAMP; cAMP-Dependent Protein Kinases; Cell Communication and Signaling; Cell Signaling; Cell-Extracellular Matrix; Cessation of life; Chemotherapy-Hormones/Steroids; Chimp; Chimpanzee; Chronic; Cirrhosis; Collagen; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Death; defined contribution; Deposit; Deposition; design; designing; Development; Disease; Disease Progression; disease/disorder; Disorder; DNA Molecular Biology; ECM; effective therapy; Effectiveness; Endocrine; Endocrine Gland Secretion; Environment; ethanol abuse; ethanol induced hepatic injury; ethanol induced liver disorder; ethanol induced liver injury; ethanol research; ethanol withdrawal; ethanol-induced hepatic dysfunction; ethanol-induced liver disease; ethanol-induced liver dysfunction; ethanol-mediated liver dysfunction; ethanol-mediated liver injury; Excision; expectation; experience; Extirpation; Extracellular Matrix; Family; Family member; Fibrillar Collagen; Fibrosis; Goals; hazardous alcohol use; Health; heavy metal lead; heavy metal Pb; Hepatic Cells; Hepatic Disorder; Hepatic Failure; hepatic fibrosis; Hepatic Parenchymal Cell; Hepatic Stellate Cell; Hepatitis; Hepatocyte; hepatopathy; Hormones; Human; Human, General; in vivo Model; Injury; innovate; innovation; innovative; Intracellular Communication and Signaling; Investigation; Investigators; Ito Cell; Knockout Mice; Knowledge; Lead; Ligands; Liver; Liver Cells; Liver diseases; liver disorder; Liver Dysfunction; Liver Failure; Liver Fibrosis; Man (Taxonomy); Man, Modern; Mice, Knock-out; Mice, Knockout; Modeling; Molecular; Molecular Biology; Null Mouse; organ system, hepatic; Outcome; Pan; Pan Genus; Pan Species; Pathogenesis; Pathologic; pathway; Pathway interactions; Pb element; peptide hormone; Peptides; Peroxisome Proliferative Activated Receptor Gamma; Peroxisome Proliferator-Activated Receptor gamma; Peroxisome Proliferators; Phenotype; PKA; PPAR gamma; PPARG; PPARG1; PPARG2; PPARgamma; prevent; preventing; problem drinking; programs; Programs (PT); Programs [Publication Type]; Protein Kinase A; receptor; Receptor Activation; Receptor Protein; Receptor Signaling; Recovery; Regulation; Relative; Relative (related person); Relaxin; relaxin receptor; Removal; Research; Research Personnel; Research Resources; Researchers; resection; Resolution; Resources; response; Role; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; social role; Source; Surgical Removal; System; System, LOINC Axis 4; Testing; Therapeutic; Therapeutic Agents; therapeutic effectiveness; therapeutic efficacy; Therapeutic Hormone; therapeutic target; therapeutically effective; Thiazolidinedione Receptor; Time; Tissues; transplant; Transplantation; Treatment Efficacy; treatment strategy; United States; withdrawal from alcohol; Work
Project start date: 2007-07-05
Project end date: 2011-06-30
Budget start date: 1-JUL-2009
Budget end date: 30-JUN-2011
PFA/PA: PA-05-074
5R01AA015509-03 (2009): $226800
MOLECULAR CHIMERISM THERAPY FOR HEMOPHILIA A
G Robert, Professor And Chair
George Washington Universitycity: Washington country: United States (us)
Grant 5R01HL065519-08 from National Heart, Lung, And Blood Institute
Abstract: Hemophilia A is an X-linked recessive genetic bleeding disorder caused by a deficiency or functional defect in coagulation factor VIII (FVIII). There is currently no cure for hemophilia A and patients receive infusion of FVIII concentrates or recombinant proteins at the time of bleeding. Although this treatment regimen has increased the life expectancy of hemophiliacs significantly, it is inconvenient and has potentially serious complications such as the development of inhibitory antibodies to FVIII, which occurs in approximately 25% of patients, rendering them refractory to further treatment. The objective of this research is to evaluate the curative efficacy of retroviral vectors encoding modified human FVIII transgenes targeted to hematopoietic stem cells (HSCs) in a murine hemophilia A model. HSCs are an attractive target cell population for hemophilia A gene therapy because they are readily accessible for ex vivo genetic modification and allow for the possibility of sustained expression of a FVIII transgene in circulating peripheral blood cells for the recipient´s lifetime. Moreover, a potential benefit of targeting HSCs is the possibility of inducing immunological tolerance to the FVIII transgene product. For almost two decades, our laboratory has been designing and optimizing retroviral vectors for gene transfer studies of HSC biology and gene therapy modeling. In particular, our MSCV (murine stem cell virus) retroviral vector is in use in several HSC gene therapy trials currently underway in the United States. However, the emergence of adverse events in a French clinical trial for X- linked severe combined immunodeficiency disease demands a reevaluation of the risks of retroviral-induced mutagenesis. Therefore, building upon our recent success at achieving clinically-relevant FVIII plasma levels in hemophilia A mice by MSCV-based HSC-directed gene delivery, our Specific Aims are (1) To further optimize FVIII transgene sequences for more efficient secretion in hematopoietic cells and decreased immunogenicity of the protein; (2) To develop nonmyeloablative HSC transplant conditioning regimens that allow sufficient levels of transgene molecular chimerism for long-term therapeutic FVIII production and tolerance induction; and (3) To create biologically safer FVIII retroviral vectors - devoid of transcriptional regulatory elements within their long terminal repeats and flanked by enhancer/promoter-blocking elements - displaying reduced HSC genotoxicity
Keywords: A Mouse; Adverse event; Adverse Experience; Animal Model; Animal Models and Related Studies; Antibodies; Antihemophilic Factor; antihemophilic factor A; arthropathic; arthropathies; Arthropathies NOS; Articulation; B-domain-deleted factor VIII; backbone; base; Biosynthetic Proteins; Bleeding; Bleeding Time; Bleeding time procedure; Blood; Blood (Leukemia); Blood Cells; Blood Circulation; Blood Coagulation Factor VIII; blood loss; Blood Plasma; Blood Precursor Cell; Blood-coagulation factor VIII, complex; Bloodstream; Body Tissues; Bruise; Candidate Disease Gene; Candidate Gene; canine; Canine Species; Canis familiaris; Cells; Chimerism; Chronic; Circulation; clinical investigation; clinical relevance; Clinical Trials; Clinical Trials, Unspecified; clinically relevant; Coagulation Factor VIII; coagulation factor VIII, procoagulant component (hemophilia A) protein, human; Coagulation Factor VIIIc; Complication; conditioning; Contusions; Defect; design; designing; Development; Disease; disease/disorder; Disorder; Dogs; domestic dog; Elements; Engineering; Engineerings; Enhancers; enroll; Enrollment; enzyme replacement therapy; F8 protein, human; F8B protein, human; Factor VIII; Factor VIII Deficiency; Factor VIII F8B; factor VIII, human; FVIII protein, human; GC-rAHF; Gene Delivery; gene product; gene therapy; Gene Transfer; Gene Transfer Clinical; Gene Transfer Procedure; Gene-Tx; Genes, Regulator; Genetic; Genetic Intervention; genetic recessive; genetic therapy; Genetics-Mutagenesis; genotoxicity; Hematoma; Hematopoietic; Hematopoietic stem cells; Hemophilia; Hemophilia A; Hemophilia As; Hemorrhage; Human; human F8 protein; Human, General; Immune; Immunocompetent; immunogenicity; improved; Infusion; Infusion procedures; inhibitor; inhibitor/antagonist; Intervention, Genetic; Intracranial Hemorrhages; Investigators; Joint Diseases; joint disorder; Joints; Knock-out; Knockout; Laboratories; Lentivirinae; Lentivirus; leukemia; Leukemias, General; Life Expectancy; Link; long terminal repeat, nucleic acid; Long Terminal Repeats; Mammals, Dogs; Mammals, Mice; Man (Taxonomy); Man, Modern; Mice; model organism; Modeling; Modification; Molecular; Molecular Biology, Gene Therapy; Molecular Biology, Mutagenesis; Mother Cells; Murine; Mus; Muscle; Muscle Tissue; Mutagenesis; Operation; Operative Procedures; Operative Surgical Procedures; Patients; Peripheral Blood Cell; Plasma; platelet cofactor I; Population; pre-clinical; preclinical; Prion Proteins; Prions; Procoagulant Component; Production; Progenitor Cell Transplantation; Progenitor Cells; Progenitor Cells, Hematopoietic; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); prophylactic; Protein Replacement Therapy; Proteins; Protocols, Treatment; PrP Proteins; recessive genetic trait; recessive trait; recombinant antihemophilic factor VIII; recombinant factor VIII; Recombinant Proteins; Refractory; Regimen; Regulation; Regulator Genes; regulatory gene; Replacement Therapy; Research; Research Personnel; Researchers; Reticuloendothelial System, Blood; Reticuloendothelial System, Serum, Plasma; Retroperitoneal; Retroperitoneal Cavity; Retroperitoneal Space; Retroperitoneum; Retroviral Vector; retroviral-mediated; Retrovirus Vector; rFVIII; rFVIII (B-domain-deleted); RGM; Risk; RNA Splicing; Serum, Plasma; Spinal Column; Spine; Splicing; stem cell biology; Stem cell transplant; Stem Cell Transplantation; Stem cells; Subfamily lentivirinae; success; surgery; Surgical; Surgical Interventions; Surgical Procedure; Terminal Repeats, Long; Therapeutic; Therapy, DNA; Thromboplastinogen; thromboplastinogen A; Tissues; trans acting element; Transcriptional Regulatory Elements; transfer of a gene; Transgenes; Transmission; transmission process; transplant; Transplantation; Trauma; Treatment Protocols; Treatment Regimen; Treatment Schedule; United States; vector; Vertebral column; Virus; Virus-Lenti; Viruses, General; X-Linked Severe Combined Immunodeficiency
Project start date: 2000-07-01
Project end date: 2011-06-30
Budget start date: 1-JUL-2009
Budget end date: 30-JUN-2011
5R01HL065519-08 (2009): $371408
TRAINING PROGRAM IN ORAL SCIENCE
G Robert, Professor
University Of Rochestercity: Rochester country: United States (us)
Grant 1T90DE021985-01 from National Institute Of Dental & Craniofacial Research
Abstract: Funds are requested in this application to support the Rochester Training Program in Oral Science. Additional clinician-scientists must be generated for society to take advantage of the rapid and dramatic advances being made in the biomedical sciences. The Center for Oral Biology´s training programs have been highly successful in recruiting, training and retaining Oral Science researchers over the past 20+ years. The University of Rochester Medical Center has committed substantial resources to recruit new faculty, to improve our physical plant, and to acquire new technologies. This enhanced commitment to our research environment expands the depth and breadth of training necessary to excel in the biomedical sciences. The University of Rochester has a long history and commitment to training graduate dentists. The overall goal of the Training Program in Oral Science (TPOS) is to provide improved training opportunities for dentist-scientists and oral biologists that will enable them to pursue research careers in dentistry and medicine at academic, federal and industrial organizations. Cross-disciplinary training will focus on the general fields of oral infectious diseases, the cellular and developmental biology of craniofacial structures, with training in clinical/translational approaches to the manifestations of oral disease. TPOS targets the recruitment of dentists who wish to pursue a PhD and dentist-PhDs who want to engage in post-doctoral level training. We will also recruit PhDs and baccalaureate degree-holders pursuing a PhD. In this manner, PhD level scientists will be recruited to the field of Oral Science. Our program features a DST Program for dental students who wish to coordinate clinical studies with PhD training. This novel program partners the excellent clinical programs at Marquette University, the University of Puerto Rico and the University of Kentucky with the PhD programs at the research-intensive University of Rochester Medical Center. Finally, our program will include R90 research education for internationally trained DDS, PhD dual degree holding individuals. The various components of TPOS are integrated through educational options of the Rochester Graduate Program, the clinical training programs of the Eastman Institute for Oral Health, and the clinical/translational educational resources of the Rochester Clinical and Translational Sciences Institute. TPOS trainees are expected to be highly prepared for careers in Oral Science research. The proposed Training Program aims to prepare scientists and dentists to become leaders in the research of oral disease and oral/facial birth defects. The program will integrate classroom work, research in laboratories and hospitals, and in dental clinics in a multidisciplinary fashion. Our long-term goal is to accelerate the pace at which effective, new treatments for diseases can be brought to the public to lessen suffering and improve lives
Keywords: Bachelor`s Degree; career; Cellular biology; Clinical; Clinical Research; Clinical Sciences; Commit; Communicable Diseases; craniofacial; Dental Students; Dentistry; Dentists; Developmental Biology; Doctor of Philosophy; Educational aspects; Educational Status; Environment; Faculty; Funding Applicant; Goals; improved; Individual; Institutes; Kentucky; Medical center; Medicine; Mouth Diseases; new technology; novel; Oral; oral biology; Oral health; oral infection; Plants; Postdoctoral Fellow; programs; Puerto Rico; Recording of previous events; Recruitment Activity; Research; Research Personnel; Resources; Science; Scientist; Societies; Structure; Training; Training Programs; translational approach; Translational Research; Universities
Relevance: The proposed Training Program aims to prepare scientists and dentists to become leaders in the research of oral disease and oral/facial birth defects. The program will integrate classroom work, research in laboratories and hospitals, and in dental clinics in a multidisciplinary fashion. Our long-term goal is to accelerate the pace at which effective, new treatments for diseases can be brought to the public to lessen suffering and improve lives
Project start date: 2011-08-01
Project end date: 2016-07-31
Budget start date: 1-AUG-2011
Budget end date: 31-JUL-2012
PFA/PA: PAR-10-170
1T90DE021985-01 (2011): $723854
FITNESS PROFILING OF THE STREPTOCOCCUS MUTANS GENOME
G Robert, Professor
University Of Rochestercity: Rochester country: United States (us)
Grant 5R01DE017425-05 from National Institute Of Dental & Craniofacial Research
Abstract: Fitness profiling of the S. mutans genome. Caries formation by S. mutans requires the organism to form a biofilm, become a prominent member of the microbial community of dental plaque and to survive stresses within plaque (Marsh, 2003). The initiation of caries involves demineralization of tooth enamel by organic acids produced by S. mutans fermentation of sugars. S. mutans survives acid environments by the up- regulation of a number of proteins, or, fatty acids in a process referred to as acid-adaptation. Evidence from our laboratory, and others, shows that acid-adaptation is a complex, multi-gene process that is only partially understood at present. With the availability of the S.mutans genome sequence, the annotation of 1,963 open reading frames, and the development of methods precisely to delete individual open reading frames, it is possible to analyze every ssential gene in S. mutans. In this project, we will examine the effect of each gene on the ability of S. mutans to grow in acidic conditions. The contribution of each gene in the organism to events implicated in caries formation will be determined. Specific Aim 1a. High throughput methods will be tailored to reproducibly delete individual genes in the entire S. mutans genome. Each deletion will be marked by the presence of two unique DMA sequences (18-20 bp) such that abundance of each particular mutant strain in a population of mutants and wild type can be scored using these "bar codes." Specific Aim 1b. A genomic set of marked deletions will be constructed, resulting in a library of S. mutans strains bearing knockouts of all non-essential genes. Methods to analyze gene function by fitness profiling will be used to determine a function for each gene in S. mutans. Specific Aim 2. To find genes that may affect growth in the oral cavity, we will test mutant strains for their ability to grow, in vitro, in acidic conditions. Then, we will use the knockout library in fitness profiling experiments under defined conditions, which will include the following 1) co-culture of the library of mutant strains growing in acidic environments, including planktonic and biofilm cultures; and, 2) a mouse infection model, in which the mice are fed a highly cariogenic diet. These experiments will be done as screens for candidate strains to be examined individually in the mouse caries model. Specific Aim 3. We will also examine the ability of the library of mutant strains to grow in the presence of additional species of oral microbes, with the aim of identifying those genes in S. mutans that contribute to its ability to out-compete other oral organisms. From these studies, we expect to identify genes in S. mutans that participate in acid-resistance. The relevance to public health is that the information derived from these studies will lead to new insights, and potentially new targets for therapeutic intervention, into specific mechanisms that S. mutans uses to cause disease in humans
Keywords: Acids; Actinomyces naeslundii; Affect; Bar Codes; base; Cariogenic Diet; Coculture Techniques; Communities; Complex; Defect; demineralization; Dental caries; Dental Enamel; Dental Plaque; Diet; Disease; Environment; Essential Genes; Event; Exhibits; Fatty Acids; feeding; Fermentation; fitness; Fusobacterium nucleatum; Future; gene function; Gene Mutation; Gene Targeting; Genes; genetic analysis; Genome; genome sequencing; Genomics; Growth; Homologous Gene; Human; In Vitro; indexing; Individual; Infection; insight; Knock-out; Laboratories; Lead; Learning; Libraries; member; method development; Methods; Microbe; Microbial Biofilms; microbial community; Modeling; mouse model; Mus; Mutagenesis; mutant; Open Reading Frames; Oral; oral bacteria; Oral cavity; organic acid; Organism; Population; Process; Processed Genes; Production; programs; Proteins; Protocols documentation; public health medicine (field); Publishing; Research Personnel; research study; Resistance; Resources; response; Streptococcus; Streptococcus mutans; Streptococcus sanguis; Stress; success; sugar; Testing; Therapeutic Intervention; trait; Up-Regulation (Physiology); Yeasts
Project start date: 2007-06-01
Project end date: 2012-05-31
Budget start date: 1-JUN-2011
Budget end date: 31-MAY-2012
PFA/PA: PA-04-093
5R01DE017425-05 (2011): $681030
GAP JUNCTIONAL PATTERNING IN ARRHYTHMIC HEART
G Robert, Professor
Medical University Of South Carolinacity: Charleston country: United States (us)
Grant 5R01HL056728-11 from National Heart, Lung, And Blood Institute
Abstract: Direct cytosolic communication between cells is mediated by an aggregate of intercellular channels in the plasma membrane called the gap junction (GJ). Remodeling of connexin43 (Cx43) GJs has been linked to cardiac arrhythmias. Work accomplished during the previous period of this RO1 identified a biological function of interaction between Cx43 and the actin-binding protein Zonula Occludens (ZO)-1 in GJ remodeling. In recent work, we have determined that a peptide known to inhibit Cx43/ZO-1 interaction, 1CT1, reduces the frequency of inducible arrhythmias following injury to the left ventricle. Preliminary data from work performed with 1CT1 in vitro, indicates that the transition from free connexon channels in the membrane to paired connexons in intercellular channel aggregates underlies GJ remodeling. We hypothesize that ZO-1 regulates the rate at which free connexons in the membrane accrete to GJs-the "connexon switch". The aims in this renewal will test this hypothesis and its implications for GJ intercellular communication, membrane excitability, and a novel role for Cx43 in differential adhesion between myocytes and fibroblasts following myocardial infarction - all processes likely to impact susceptibility to re-entrant arrhythmia. The proposed experiments will quantitatively determine the role of Cx43/ZO-1 interaction in the "connexon switch" using methods including live cell imaging, fluorescent fusion proteins, loss-of-function mutants, whole cell patch clamp, single channel electrophysiology and biochemical assays. Implications of Cx43/ZO-1 interaction within the physiological framework of cardiac injury in vivo will be assessed by echocardiography, EKG telemetry, optical mapping of electrical activation and arrhythmia induction protocols. The data generated will broaden our understanding of fundamental mechanisms of Cx43 function and may translate to new therapies for the prevention and treatment of cardiac arrhythmia. Injury and scarring of the heart following myocardial infarction (heart attack) is one the most frequent causes of sudden death in the USA. This project will deliver new insights on the function of a protein essential to a stable heartbeat called connexin43. The project aims to provide novel approaches to the prevention and treatment of heart attacks
Keywords: Actin-Binding Protein; Actins; Action Potentials; Adhesions; Arrhythmia; Binding (Molecular Function); Biochemical; Biological Assay; Biological Process; Biotinylation; Cardiac; Cardiomyopathies; Cell Culture Techniques; Cell Line; Cell membrane; Cell physiology; Cells; cellular imaging; Chimeric Proteins; Cicatrix; Clinical; Coculture Techniques; Communication; Competence; Connexin 43; Connexins; Connexon; Coronary artery; Coupling; CTF1 gene; Data; Dyes; Echocardiography; Electrocardiogram; Electrophysiology (science); Fibroblasts; Frequencies (time pattern); Gap Junctions; Goals; Heart; Hela Cells; Heterogeneity; Human; Imagery; Implant; In Vitro; in vivo; Infarction; Injury; insight; intercellular communication; Left ventricular structure; Life; Ligation; Link; loss of function; Manuscripts; Maps; Mediating; Membrane; Methods; Molecular; Morbidity - disease rate; Mortality Vital Statistics; Mus; Muscle Cells; mutant; Myocardial; Myocardial Infarction; Myofibroblast; Neonatal; novel; novel strategies; Optics; patch clamp; Pattern; Peer Review; Peptides; Physiological; Predisposition; Prevention approach; Prevention therapy; Process; Proteins; Protocols documentation; public health relevance; Publishing; research study; response; Role; Silicones; Skin; Sudden Death; Surface; Telemetry; Testing; Tight Junctions; Time; Tissues; Transgenic Mice; Translating; uptake; Ventricular Remodeling; Work
Relevance: Injury and scarring of the heart following myocardial infarction (heart attack) is one the most frequent causes of sudden death in the USA. This project will deliver new insights on the function of a protein essential to a stable heartbeart called connexin43. The project aims to provide novel approaches to the prevention and treatment of heart attacks
Project start date: 2010-04-01
Project end date: 2014-02-28
Budget start date: 1-MAR-2011
Budget end date: 29-FEB-2012
PFA/PA: PA-07-070
5R01HL056728-11 (2011): $331875
2R01HL056728-10A2 (2010): $331875
HTS FOR CYTOCHROME C SYNTHESIS PATHWAYS
G Robert
Washington Universitycity: Saint Louis country: United States (us)
Grant 3R21NS061663-01S1 from Office Of The Director, National Institutes Of Health
Abstract: Cytochromes are heme proteins essential for the aerobic and anaerobic growth of most organisms, including humans and human pathogens. The assembly of c-type cytochromes occurs by one of three pathways, called systems I, II, and III. Defects in these pathways are the basis for certain human genetic diseases (in system III) and because only prokaryotes, plants, and protozoa use systems I or II, these pathways are ideal extracytoplasmic targets for antimicrobial agents (against pathogens). Many organisms that can not be cultured and/or genetically manipulated use these pathways. A specific inhibitor molecule would facilitate an understanding of how they grow and survive. Since cytochrome c is directly involved in apoptosis, and implicated (by mitochondrial bioenergetic functions) in aging and neurological manifestations, inhibitors of cytochrome c synthesis would provide valuable tools to study these processes. A major breakthrough in the field has come from the PIs lab in engineering the complete pathways of systems I and II to function in Escherichia coli. Moreover, the PIs lab has developed recombinant, inducible cytochrome c reporters, as well as very sensitive chemiluminescent (ECL) methods that detect subpicomolar levels of the cytochrome c product. Here it is proposed to advance these engineered E.coli for ultimately the discovery of specific small molecule inhibitors of the pathways. Modifications of the strains and the culture conditions will optimize and validate 96 and 384 well microtiter plate technologies for cytochrome c product detection. Two different assays will be optimized i) the ECL-based high-throughput detection of cytochrome c in whole recombinant E.coli cells and ii) the immunological detection of holo cytochrome c (his-tagged and as a PhoA fusion) with antisera already generated. These screens will then be used for the discovery of small molecule inhibitors towards one or all of the three pathways. Strains and methods already developed will establish inhibitory specificity towards the pathways and will dissect the step(s) inhibited in the pathways. The PIs group has recently shown that large molecule, heme analogs inhibit specific steps, but these analogs require specific porins for access. The cell-based assays will also be validated by detection of this inhibition and by the use of known heme biosynthesis inhibitors. Thus, the HTS will also discover new inhibitors of heme biosynthesis, and the PIs group has developed the tools to quickly determine which enzyme (of nine) is inhibited in heme synthesis
Keywords: Aerobic; Aging; Alkaline Phosphatase; alkaline phosphomonoesterase; analog; anti-microbial agent; anti-microbial drug; antimicrobial agent; antimicrobial drug; Antisera; Apoptosis; Apoptosis Pathway; Assay; base; Bioassay; Bioenergetic; Bioenergetics; Biologic Assays; Biological Assay; Cell Death, Programmed; Cells; Chimera Protein; Chimeric Proteins; cofactor; Complex; CYP; cytochrome c; Cytochrome c Group; Cytochromes; Cytochromes Type c; Data; Defect; Detection; E coli; Engineering; Engineerings; Enzymes; Escherichia coli; Eukaryota; Eukaryote; eukaryotida; Ferrate(2-), (7, 12-diethenyl-3, 8, 13, 17-tetramethyl-21H, 23H-porphine-2, 18-dipropanoato(4-)-N21, N22, N23, N24)-, dihydrogen, (SP-4-2)-; Ferricytochrome c; Ferrocytochrome c; ferroheme; Ferroprotoporphyrin; Figs; Figs - dietary; Fusion Protein; Generalized Growth; Genes; Genetic Condition; Genetic Diseases; genetic disorder; glycerophosphatase; Growth; Helicobacter; Heme; Heme b; heme biosynthesis; Heme Proteins; Hemeproteins; hemoprotein; Hereditary Disease; hereditary disorder; his6 tag; Human; Human Genetics; Human, General; Immune Sera; inhibitor; inhibitor/antagonist; Ligase; living system; Man (Taxonomy); Man, Modern; Methods; milliliter; Mitochondria; mitochondrial; Modeling; Modification; Molecular Disease; Nature; neural manifestation; Neurologic Manifestations; Neurologic Signs and Symptoms; Neurological Manifestations; ontogeny; Organism; Orthophosphoric-monoester phosphohydrolase (alkaline optimum); pathogen; pathway; Pathway interactions; Phosphatases; Phosphohydrolases; Phosphomonoesterases; Phosphoric Monoester Hydrolases; Plants; Plants, General; Plasmids; pore forming protein; porin; Process; Prokaryotae; prokaryote; Prokaryotic Cells; Protoheme; Protoheme IX; Protozoa; Protozoal; Reagent; Recombinants; Reporter; Senescence; senescent; small molecule; Sn protoporphyrin; SnPP protoporphyrin; Specificity; success; Synthetases; System; System, LOINC Axis 4; Technology; Therapeutic; thioether; tin-protoporphyrin; Tissue Growth; tool
Project start date: 2007-09-30
Project end date: 2011-08-31
Budget start date: 30-SEP-2007
Budget end date: 31-AUG-2011
PFA/PA: RFA-RM-07-008
3R21NS061663-01S1 (2011): $38000
Sponsored Links Excellgen http://Excellgen.com
PROBING LIGHT RESPONSES OF ON BIPOLAR AND AII AMACRINE CELLS WITH CALCIUM IMAGING
G Robert
University Of Pennsylvaniacity: Philadelphia country: United States (us)
Grant 5R21EY021308-02 from National Eye Institute
Keywords: ing; Address; Amacrine Cells; Calcium; Calcium Channel; calcium indicator; Calcium Signaling; cell type; Cells; Color; computerized data processing; Coupled; Coupling; Dendrites; design; Dyes; Electrical Synapse; Electrophysiology (science); Fluorescent Dyes; Frequencies (time pattern); ganglion cell; Gap Junctions; gene therapy; Goals; Heparin; Image; Inner Plexiform Layer; Knowledge; Light; light intensity; Link; Maps; Measures; Meclofenamic Acid; Mediating; Methods; Microelectrodes; Microscopy; Morphology; Neuromodulator; Neurons; Noise; novel; Ocular Physiology; Output; parallel processing; Photons; Photoreceptors; Physiological; Presynaptic Terminals; Process; Promotor (Genetics); Property; Proteins; receptor; research study; response; Retina; Retinal; Retinal Cone; Rods (Retina); Running; Ryanodine; Ryanodine Receptor Calcium Release Channel; Signal Transduction; Source; stem; Stimulus; Stratification; Synapses; Testing; Thapsigargin; tool; two-photon; Vision; Visual; visual information; visual process; visual processing; voltage
Relevance: Our goal of imaging light-evoked calcium responses in the ON bipolar cells and the AII amacrine cells will have a substantial impact on the field because these recordings are still novel and they promise to pave the way for efficient recordings from specific cell compartments in the retina. These will yield important new information relatively fast, and will gain greater understanding of the principle of visual processing in night and day vision. This understanding in turn will help design more optimal approaches for the ever developing tools of genetic therapy
Project start date: 2011-01-01
Project end date: 2012-12-31
Budget start date: 1-JAN-2012
Budget end date: 31-DEC-2012
5R21EY021308-02 (2012): $180000
TRAINING PROGRAM IN ORAL SCIENCE
G Robert, Professor
University Of Rochestercity: Rochester country: United States (us)
Grant 1R90DE022529-01 from National Institute Of Dental & Craniofacial Research
Keywords: Bachelor`s Degree; career; Cellular biology; Clinical; Clinical Research; Clinical Sciences; Commit; Communicable Diseases; craniofacial; Dental Students; Dentistry; Dentists; Developmental Biology; Doctor of Philosophy; Educational aspects; Educational Status; Environment; Faculty; Funding; Goals; improved; Individual; Institutes; Kentucky; Medical center; Medicine; Mouth Diseases; new technology; novel; Oral; oral biology; Oral health; oral infection; Plants; Postdoctoral Fellow; programs; Puerto Rico; Recording of previous events; Recruitment Activity; Research; Research Personnel; Resources; Science; Scientist; Societies; Structure; Training; Training Programs; translational approach; Translational Research; Universities
Relevance: Narrative The proposed Training Program aims to prepare scientists and dentists to become leaders in the research of oral disease and oral/facial birth defects. The program will integrate classroom work, research in laboratories and hospitals, and in dental clinics in a multidisciplinary fashion. Our long-term goal is to accelerate the pace at which effective, new treatments for diseases can be brought to the public to lessen suffering and improve lives
Project start date: 2011-08-01
Project end date: 2016-07-31
Budget start date: 1-AUG-2011
Budget end date: 31-JUL-2012
PFA/PA: PAR-10-170
1R90DE022529-01 (2011): $87069