MECHANISMS OF FAMILIAL PULMONARY FIBROSIS
S Timothy, Professor
Vanderbilt Universitycity: Nashville country: United States (us)
Grant 5P01HL092870-02 from National Heart, Lung, And Blood Institute
Abstract: This program has arisen from our long-standing interest in familial lung diseases, including familial interstitial pneumonia (FIP). We intend to use this unique cohort of families with FIP to investigate the underlying mechanisms that lead to progressive lung fibrosis. This information will be highly relevant to individuals with sporadic idiopathic pulmonary fibrosis (IPF) and other forms of idiopathic interstitial pneumonia (IIP). There are important advantages to investigating lung fibrosis in families. First, it is possible to study genetic causes of disease in this group. Three genes linked to disease, surfactant protein C and components of the telomerase complex, have already been identified using these FIP families, and this identification has led to new insights into disease pathogenesis. Second, this population allows us the unprecedented opportunity to identify pre-symptomatic patients that can be studied at the earliest stages of disease. This gives us the ability to study the primary manifestations of disease as opposed to pathobiological changes that occur as a result of progressive lung remodeling. Project 1 will identify pre-symptomatic individuals at risk for FIP families by high resolution CT scanning that have radiographic abnormalities in the lungs. At risk individuals (with and without radiographic abnormalities) will undergo bronchoscopy to identify phenotypic characteristics of alveolar epithelial cells, such as ER stress, herpesvirus infection, or telomere shortening that contribute to disease progression in FIP. Project 2 will utilize a candidate gene approach to identify and characterize rare genetic vanants in the surfactant and telomerase pathways, as well as genes known to be associated with secondary forms of pulmonary fibrosis, that impact development of FIP. Project 3 will perform a linkage study to discover new genetic loci associated with disease in FIP and sporadic MP patients. Additional studies will identify viruses present in lung tissue of patients with IIP and investigate gene-gene and gene-environment interactions that influence disease manifestation. The integrated approach in this Program will lead to new concepts in pathogenesis of FIP and sporadic IPF (as well as other forms of IIP), and suggest opportunities for novel treatment or prevention strategies. (End of ) INDIVIDUAL PROJECTS AND CORE UNITS PROJECT 1 EPITHELIAL DYSFUNCTION IN EARLY PULMONARY FIBROSIS (Blackwell, Timothy S.) RESUME AND SUMMARY OF DISCUSSION This revised Project 1 by Dr. Blackwell addresses a highly significant disease entity idiopathic pulmonary with a reasonably well thought out series of related hypotheses related to the role of the alveolar epithelium in the genesis of fibrosis. As originally proposed and maintained in the current application this amounts to a significant paradigm shift away from a focus on inflammation as a primary entity and to an ineffective injury repair hypothesis. In this revised application it was felt that Dr. Blackwell responded to the major criticisms of the previous application it an effective manner although some minor concerns about the sampling strategy and possible safety concerns remained. The use of the well characterized cohort of FIP subjects and the use of HRCT to identify areas of disease involvement was judged to be an effective approach although the possibility remains that many samples will not be representative of disease process. The proposal is strengthened by the effective use of power calculations and biostatistical support. A minor concerned raised was how the investigators would proceed if all the hypotheses turned out to not be supported by the data or if these things were determined to play only a minor role. There was also some discussion and concern about whether samples were being shared across the projects or if interactions were limited to information flow. Lack of clarity on this was considered a weakness. It was clearly recognized that this is an exceptional team of investigators with a reasonable set of tools and good supporting data to address the questions posed. Overall it was the consensus that this was an outstanding to exceptional project with high potential impact on the field. The pathogenesis of idiopathic pulmonary fibrosis (IPF) remains poorly defined; however, identification of mutations in the gene encoding surfactant protein C (SP-C) in the familial form of IPF (FIP), along with several other lines of evidence, suggests that alveolar epithelial cells (AECs) play a key role in disease progression. Our data indicate that abnormal processing of pro-SP-C by AECs leads to endoplasmic reticulum (ER) stress, activation of the unfolded protein response, and cell death. We also show that ER stress occurs frequently in AECs in IPF, suggesting that this pathway contributes to disease. In addition, we show that herpes viruses are commonly localized to AECs in IPF and could contribute to ER stress and AEC injury. Finally, we have identified loss-of-function mutations in telomerase genes that segregate with disease in some FIP families, suggesting that defective telomerase leads to telomere shortening and apoptosis of type II AECs. Identification of individuals in FIP families with early fibrotic changes will provide a valuable resource for investigations aimed at defining primary disease mechanisms. In this study, we will utilize CT scanning to identify asymptomatic individuals at risk for FIP who have radiographic changes consistent with early fibrosis. Subjects with early FIP and controls will undergo bronchoscopy for sample collection to test the following hypothesis. Genetic or acquired factors that increase the susceptibility of lung epithelial cells to injury and/or apoptosis underlie the pathogenesis of IPF. Exposure of vulnerable epithelial cells to common injurious/toxic environmental stimuli results in extensive injury with limited capacity for alveolar repair, leading to fibrotic remodeling. The following specific aims will investigate the role of AECs in early FIP 1) to evaluate epithelial cell injury/apoptosis, markers of ER stress, and surfactant protein production in the lungs of patients with early FIP, 2) to investigate whether herpes virus infection occurs in early FIP, contributes to ER stress, and is associated with alveolar epithelial cell injury and, 3) to determine whether differential telomere length occurs in epithelial cells from patients with early FIP and correlates with epithelial cell injury. By elucidating critical components of early stage disease, our study will define novel therapeutic targets. (End of )
Project start date: 2010-01-19
Project end date: 2014-12-31
Budget start date: 1-JAN-2011
Budget end date: 31-DEC-2011
5P01HL092870-02 (2011): $2251871
Sponsored Links Excellgen http://Excellgen.com
EPITHELIAL DYSFUNCTION IN EARLY PULMONARY FIBROSIS
S Timothy, Professor
Vanderbilt Universitycity: Nashville country: United States (us)
Abstract: PROJECT 1 The pathogenesis of idiopathic pulmonary fibrosis (IPF) remains poorly defined; however, identificafion of mutafions in the gene encoding surfactant protein C (SP-C) in the familial form of IPF (FIP), along with several other lines of evidence, suggests that alveolar epithelial cells (AECs) play a key role in disease progression. Our data indicate that abnormal processing of pro-SP-C by AECs leads to endoplasmic reficulum (ER) stress, acfivafion ofthe unfolded protein response, and cell death. We also show that ER stress occurs frequently in AECs in IPF, suggesfing that this pathway contributes to disease. In addifion, we show that herpesviruses are commonly localized to AECs in IPF and could contribute to ER stress and AEC injury. Finally, we have identified loss-of-funcfion mutations in telomerase genes that segregate with disease in some FIP families, suggesting that defective telomerase leads to telomere shortening and apoptosis of type II AECs. Identificafion of individuals in FIP families with early fibrotic changes will provide a valuable resource for invesfigafions aimed at defining primary disease mechanisms. In this study, we will ufilize CT scanning to identify asymptomafic individuals at risk for FIP who have radiographic changes consistent with early fibrosis. Subjects with eariy FIP and controls will undergo bronchoscopy for sample collecfion to test the following hypothesis. Genefic or acquired factors that increase the suscepfibility of lung epithelial cells to injury and/or apoptosis underiie the pathogenesis of IPF. Exposure of vulnerable epithelial cells to common injurious/toxic environmental sfimuli results in extensive injury with limited capacity for alveolar repair, leading to fibrofic remodeling. The following specific aims will invesfigate the role of AECs in early FIP 1) to evaluate epithelial cell injury/apoptosis, markers of ER stress, and surfactant protein producfion in the lungs of pafients with eariy FIP, 2) to invesfigate whether herpesvirus infection occurs in eariy FIP, contributes to ER stress, and is associated with alveolar epithelial cell injury and, 3) to determine whether differenfial telomere length occurs in epithelial cells from patients with eariy FIP and correlates with epithelial cell injury. By elucidafing crifical components of eariy stage disease, our study will define novel therapeufic targets
Keywords: 21+ years old; aberrant protein folding; abnormal protein folding; Accelerated interstitial pneumonitis; acquired factor; Acute Interstitial Pneumonitis; Adult; adult human (21+); Affect; Alveolar; alveolar epithelium; Apoptosis; Apoptosis Pathway; Biological; Bronchoscopy; CAT scan; CAT Scan, X-Ray; catscan; Cell Cycle Arrest; cell damage; Cell Death; Cell Death, Programmed; cell injury; Cells; Cellular injury; Clinical; cohort; computed axial tomography; Computed Tomography; computerized axial tomography; Computerized Axial Tomography (Computerized Tomography); computerized tomography; Computerized Tomography, X-Ray; CT scan; CT X Ray; Cultured Cells; Data; Desquamative interstitial pneumonitis; Diagnosis; diffuse interstitial pulmonary fibrosis; Disease; disease phenotype; Disease Progression; disease/disorder; Disorder; Dysfunction; effective therapy; EMI scan; Epithelial; Epithelial Cells; Event; Family; Family member; Fibrosing Alveolitis; Fibrosis; Functional disorder; Future; gene product; Genes; Genetic; Genetic Alteration; Genetic Change; Genetic defect; genome mutation; Goals; Hamman-Rich syndrome; heavy metal lead; heavy metal Pb; herpes virus; Herpesviridae; Herpesviridae disease; Herpesviridae Infections; Herpesvirus Infections; Herpesviruses; Human, Adult; Idiopathic ARDS; Idiopathic Interstitial Pneumonia; idiopathic pulmonary fibrosis; ILD; Individual; Injury; Interstitial Lung Diseases; Interstitial Lung Inflammation; Interstitial Pneumonia; Interstitial Pneumonitis; Interstitial Pulmonary Inflammation; intervention therapy; Lead; Length; Low Prevalence; Lung; Lung Alveolar Epithelia; Lung diseases; Lung Diseases, Interstitial; lung disorder; Methods and Techniques; Methods, Other; Morbidity; Morbidity - disease rate; Mortality; Mortality Vital Statistics; mutant; Mutation; necrocytosis; Non-specific interstitial neumonia/fibrosis; novel; Pathogenesis; pathologic protein folding; Pathology; pathophysiology; pathway; Pathway interactions; Patients; Pattern; Pb element; Persons; Phenotype; Physiopathology; Play; Pneumonia; Pneumonia, Interstitial; Pneumonitis; pre-clinical; preclinical; Predisposition; prevent; preventing; Process; Production; programs; Programs (PT); Programs [Publication Type]; protein mis-folding; protein misfolding; Proteins; pulmonary; Pulmonary Diseases; Pulmonary Disorder; Pulmonary Fibrosis; Pulmonary Inflammation; Pulmonary Surfactant Protein C; Pulmonary Surfactant-Associated Protein C; Pulmonary Surfactant-Associated Protein SP-C; repair; repaired; Research; Research Resources; Research Specimen; Resolution; Resources; Respiratory Disease; Respiratory Disorder; Respiratory System Disease; Respiratory System Disorder; Respiratory System, Lung; response; Risk; Role; Sampling; screening; Screening procedure; screenings; social role; SP-C peptide; SP-C protein; Specimen; Staging; Stimulus; Stress; Structure of alveolar epithelium; surfactant; Surfactant Polypeptide SP-C; Susceptibility; Symptoms; Techniques; Telomerase; telomere; Telomere Shortening; Testing; Therapeutic Intervention; Tomodensitometry; Tomography, Xray Computed; treatment strategy; Usual Interstitial Pneumonia; Usual Interstitial Pneumonitis; X-Ray Computed Tomography
Relevance: Interstifial lung diseases, including the idiopathic interstifial pneumonias, are a substanfial cause of morbidity and mortality for which there are no effective treatments. In this program, we will study the genefics and underlying biological mechanisms that lead to progressive fibrosis in the lungs. Our integrated approach will lead to new concepts in disease pathogenesis and identificafion of novel treatment strategies
Budget start date: 1-JAN-2011
Budget end date: 31-DEC-2011
5P01HL092870-02_6707 (2011): $576944
S Timothy, Professor
Vanderbilt Universitycity: Nashville country: United States (us)
Abstract: CORE A Given the size, complexity, duration, and scope involved, Core A will be fundamental In enabling the successful establishment and operation ofthe entire program. All costs for administrative support for the Cores and Projects are included here. Dr. Timothy Blackwell will assume ultimate administrative responsibility for the management of this Core. Administrative and clerical support for the entire program will be shared by all Project Leaders, Core Leaders and investigators. This Core will provide and maintain all of the administrative space and functions for the entire program, including but not limited to, offices for all investigators, an administrative area with secretarial and administrative support, a copy/work room, and a conference room with up to date audiovisual amenities. Administrative functions will include ordering of supplies and equipment, maintenance of all records, keeping and monitoring of budgets, maintenance ofthe personnel database for grant effort, interactions with University administrative offices and the NIH regarding budgetary and other administrative matters, and scheduling and organizing meetings and presentations. This Core will support all of the computer hardware and software resources for the administrative and clerical functions ofthe program. This Core will coordinate and support the activities ofthe Internal Advisory Board and the External Scientific Advisory Board. The basic functions and objectives ofthe Core include quality management of program resources, integration ofthe program, oversight of deliverables within the timeframe, assistance with data management, and leading the vision. To achieve these objectives, detailed plans are presented for administrative structure and leadership, project management, external Scientific Advisory Board, and communication/meetings
Keywords: ing; Accelerated interstitial pneumonitis; Acute Interstitial Pneumonitis; Apparatus and Instruments; Area; Back; balance; balance function; base; Biological; Budgets; Characteristics; Charge; clinical data repository; clinical data warehouse; Communication; Communication Board; Computer Hardware; computer program/software; Computer Programs; Computer Security; Computer software; computer system hardware; conference; Consensus; cost; Cost Savings; Data Banks; Data Bases; data management; data repository; Databank, Electronic; Databanks; Database, Electronic; Databases; Desquamative interstitial pneumonitis; diffuse interstitial pulmonary fibrosis; Disasters; Discipline; Disease; disease/disorder; Disorder; Dorsum; effective therapy; Environment; Equilibrium; Equipment and Supplies; Ethics Committees, Research; Fibrosing Alveolitis; Fibrosis; flexibility; follower of religion Jewish; Funding; Generations; Genetic; Goals; Grant; Hamman-Rich syndrome; heavy metal lead; heavy metal Pb; Housing; Human Resources; Idiopathic ARDS; Idiopathic Interstitial Pneumonia; idiopathic pulmonary fibrosis; ILD; Institutional Review Boards; Interdisciplinary Research; Interdisciplinary Study; Interstitial Lung Diseases; Investigators; IRBs; Jewish; Jewish, follower of religion; Lead; Leadership; Light; Lung; Lung Diseases, Interstitial; Maintenance; Maintenances; Manpower; Manuscripts; Medical Research; meetings; Mission; Monitor; Morbidity; Morbidity - disease rate; Mortality; Mortality Vital Statistics; Multidisciplinary Collaboration; Multidisciplinary Research; National Institutes of Health; National Institutes of Health (U.S.); NIH; Non-specific interstitial neumonia/fibrosis; novel; operation; Pathogenesis; Pb element; personnel; Photoradiation; Preparation; Privacy; Procedures; Productivity; programs; Programs (PT); Programs [Publication Type]; pulmonary; Pulmonary Fibrosis; Records; Recovery; relational database; Reporting; Research; Research Ethics Committees; Research Personnel; Research Resources; Researchers; Resources; Respiratory System, Lung; Saving, Cost; SCHED; Schedule; Scientist; Sight; skills; Software; Structure; Study, Interdisciplinary; symposium; system security; treatment strategy; United States National Institutes of Health; Universities; Usual Interstitial Pneumonia; Usual Interstitial Pneumonitis; Vision; willingness; Work
Relevance: Interstitial lung diseases, including the idiopathic Interstitial pneumonias, are a substantial cause of morbidity and mortality for which there are no effective treatments. In this program, we will study the genetics and underlying biological mechanisms that lead to progressive fibrosis In the lungs. Our Integrated approach will lead to new concepts in disease pathogenesis and Identification of novel treatment strategies
Budget start date: 1-JAN-2011
Budget end date: 31-DEC-2011
5P01HL092870-02_6710 (2011): $187789
Grants awarded to S Timothy
MICROSCOPY & IMAGE ANALYSIS OF UNSTAINED MACROMOLECULES
S Timothy, Professor
University Of California San Diegocity: La Jolla country: United States (us)
Grant 5R37GM033050-29 from National Institute Of General Medical Sciences
Abstract: Viruses are among the best-known and studied pathogens and infect virtually every living organism from bacteria to man. As viruses are parasites of their hosts, the life cycle of any virus is inextricably tied to that of the host cell. Despite this dependence, all viruses share a number of essential tasks, which they must accomplish for survival. A virus must find and recognize a cell in which it can replicate, release its genome into the cell, generate new viral components and assemble these components into precursors that mature into a stable progeny virion which is released from the host cell and transmitted to encounter a new host. Viruses accomplish these tasks in different ways as a result of adaptation to different cellular environments. Each task involves interactions between components within the context of the whole virion and hence requires the visualization of the entire structure at which the techniques of cryo-transmissionelectron microscopy and three-dimensional image reconstruction (´cryo-reconstruction´) excel. We are exploiting these techniques to study a diverse range of viruses, including those that infect humans and other mammals, insects, bacteria, algae, and fungi. Numerous projects funded by the current grant have illustrated the structural response of different viruses to the common tasks of the viral life cycle. This proposal involves continued and new studies that focus on structural investigations of viruses and virus complexes and dynamic events that lie beyond the current realm of crystallographic technology. The large number and extent of our studies are made possible through several fruitful collaborations, which provide important correlative information from biochemical, genetic, and X-ray crystallographic experiments. Our analyses often gain important insights by combining cryo-reconstruction data with available atomic models. Modeling experiments can provide ´pseudo-atomic´ resolution details about the orientation and binding sites of antibody and receptor molecules on viral capsid surfaces that can be tested and refined by means of targeted molecular genetics experiments. Icosahedral as well as non-icosahedral viruses will be studied. These include representatives of several different virus families, all of which are excellent model systems for studying form and function Parvoviridae, Partitiviridae, Phycodnaviridae, Podoviridae, Reoviridae, and Iridoviridae
Keywords: Algae; Animal Viruses; Arts; Bacteria; Binding Sites; Biochemical; Biochemical Genetics; Biological Models; Capsid; Capsid Proteins; CD3D gene; Cells; Collaborations; Complex; Data; Dependence; Double Stranded RNA Virus; electron tomography; Environment; Event; Family; Fc Receptor; Funding; fungus; Genome; Goals; Grant; Human; Image Analysis; Imagery; Insecta; insight; instrument; Investigation; Iridoviridae; Kentucky; Life; Life Cycle Stages; macromolecule; Mammals; man; Manuscripts; Maps; Mediating; member; Membrane; Methods; Microscopy; Modeling; Molecular; Molecular Genetics; mutant; nanometer; Organism; Orthoreoviruses; Parasites; particle; Partitiviridae; Parvoviridae; pathogen; Penetration; Peptide Hydrolases; Peptides; Phycodnaviridae; Podoviridae; Preparation; Process; Publications; reconstruction; Reoviridae; Reoviridae Infections; Reovirus sp.; Reporting; Research Design; research study; Resolution; response; RNA-Directed RNA Polymerase; Roentgen Rays; Role; Serotyping; software development; Staging; Structure; success; Surface; Techniques; Technology; Testing; Three-Dimensional Image; Viral; Viral Proteins; Virion; Virus; Work
Project start date: 1983-06-01
Project end date: 2014-03-31
Budget start date: 1-APR-2011
Budget end date: 31-MAR-2012
5R37GM033050-29 (2011): $633822
5R37GM033050-28 (2010): $690913
SPECIALIZED ANIMAL RESOURCE MODULE
S Timothy, Professor
Case Western Reserve Universitycity: Cleveland country: United States (us)
Abstract: The Specialized Animal Resource (SAR) Module has been heavily used since the first funding cycle in 1997. Due to increased demand from current investigators and new investigators, we recently added a second full-time technician, Kathryn Zongolowicz, supported by Case Medical Center (CMC), to assist Heather Butler in breeding the large number of mouse strains utilized by VSRC investigators. We also added a part time (20%) veterinary surgeon, Eugenia Diaconu, also supported by CMC, to teach and assist investigators with difficult ocular injection procedures (e.g., corneal, intravitreal and subretinal). Mutant strains of mice and rats are of considerable value in modern research on the eye. Yet, within the current limitations on budgets, the costs of managing mutant strains is often too high to be added to individual investigator grants, especially for pilot projects. Proper handling of mutant strains represents a major problem in that special expertise is needed to establish the strain from valuable founders, overcome possible breeding problems caused by the mutant phenotype, manage the colonies efficiently in order to produce enough but not too many progeny of the right ages at the right time, and to obtain tissue samples (usually toe or tail snips under anesthesia) for determination of genotypes. With valuable strains, it is also necessary to use isolation cages to avoid infections that reduce the number of surviving progeny and can endanger the entire colony. In collaboration with the genotyping arm of the MB Module, the SAR Module provides a service of breeding, management, characterization and weaning of transgenic animals. In addition to animal management, Eugenia Diaconu, D.V.M. assists in the Module on a part-time basis. The SAR Module provides the critical support that allows our group of vision researchers to take greater advantage of genetically engineered mice and mutant rat lines. The Module was established, in a dedicated space in the Animal Resource Center (ARC) in the basement of the School of Medicine. However, due to the current extensive renovation of the animal care facility at the Case School of Medicine that should be completed by late 2007, the SAR Module breeding room is in the nearby Wolstein Research Building. This facility is a new state-of-the-art barrier animal facility dedicated only to virus-free mice. The facility is controlled for air flow, temperature and humidity, and these parameters are monitored continuously by remote sensors. The breeding facility is located within 200 yards of all investigators in the VSRC, and animals are transferred by the Animal Care Unit staff from the VSRC breeding center (and other breeding centers now located at Wolstein) to each investigator´s colony daily or as needed. If more than a cage or two need to be transferred, animal transfer is accomplished in heated/air-conditioned vehicles specifically for this purpose. The ARC is fully accredited and employs a highly skilled staff, including three full-time veterinarians. Space within the facility is assigned on a need basis, and only our full-time veterinary technicians, participating investigators, and ARC staff have access to this room (Appendix A). The investigators of the VSRC have made a dramatic transition over the past five years to studying a large variety of mouse transgenic, knockout and knock-in lines. Having a module with extensive expertise in breeding challenging mouse strains and analyzing and documenting ocular defects meets a critical need for our investigators
Keywords: Age; Air; Air Conditioning; Anesthesia procedures; Animal Barrier Facility; animal breeding; animal care; animal colony; Animal Model; animal resource; Animal Technicians; Animals; arm; base; Breeding; Budgets; Cancer Center Support Grant; Caring; Charge; Collaborations; Communication; Cornea; cost; Defect; Doctor of Veterinary Medicine; Educational process of instructing; Environment; Eugenia; Eye; Funding; Genetically Engineered Mouse; Genotype; Grant; Growth; Health Status; Heating; Hour; Human Resources; Humidity; Individual; Infection; Injection of therapeutic agent; Knock-in Mouse; Knock-out; knockout gene; Maintenance; Medical center; medical schools; meetings; Monitor; Mouse Strains; Mus; mutant; Mutant Strains Mice; Nature; offspring; Phenotype; Pilot Projects; Procedures; Production; programs; pup; ranpirnase; Rattus; Records; remote sensor; Research; Research Personnel; Running; Schedule; Services; Surgeon; Tail; Temperature; Time; Tissue Sample; Toes; Training; Transgenic Animals; Transgenic Mice; Universities; Veterinarians; Virus; Vision; Vision research; Visit; Weaning
Budget start date: 1-MAY-2011
Budget end date: 30-APR-2012
5P30EY011373-15_9003 (2011): $88850
GLOBAL CHARACTERIZATION OF LYSINE ACETYLATION IN DIABETIC RETINA
S Timothy
Case Western Reserve Universitycity: Cleveland country: United States (us)
Grant 1R21EY021595-01A1 from National Eye Institute
Abstract: The goal of this proposed study is to quantitatively characterize the lysine N5-acetylation (LysAc) events (acetylome) in the retinal nuclear fraction from diabetic animals, and determine how the individual LysAc sites are modulated by histone acetyltransferase, p300. Diabetic retinopathy is the most common diabetic eye disease and a leading cause of blindness in American adults. It is caused by changes in the blood vessels of the retina, which eventually leads to the progressive loss of ganglion cells, horizontal cells, amacrine cells and photoreceptors. Unfortunately, currently no effective therapies to prevent or slow the progress of the disease are available. Therefore, we must continue searching new therapeutic targets. Recent proteomic studies on LysAc have revealed that LysAc is a common and dynamic post-translational modification present in both histone and non-histone proteins, suggesting that LysAc is used as a regulatory modification similar to that of protein phosphorylation. Interestingly, modulation of lysine acetylation events in diabetic animals by either inhibiting histone acetyltransferase (p300) or activating NAD+dependent protein deacetylase (Sirt1) has been shown to have beneficial effect on the development of retinopathy. Thus, we hypothesize that dysregulation of lysine acetylation events are involved in the development of diabetic retinopathy. In this R21 proposal, we will quantitatively characterize the acetylome in the retinal nuclear fraction. We will employ cutting-edge mass spectrometry based quantitative proteomic techniques, in conjunction with an affinity enrichment technique for acetyl lysine containing peptides. The obtained acetylome data set will be analyzed using bioinformatics-based tools to map the biological processes that are involved in the development of diabetic retinopathy. We will also verify the identified LysAc sites and biological processes by mass spectrometry as well as using biochemical approaches. The identifications of differentially acetylated sites will provide significant impact in the field, because such knowledge is essential to understand the pathophysiology of the disease and provide a foundation for the development of novel therapeutic strategies for diabetic retinopathy. The following specific aims are proposed. Specific Aim 1 To identify LysAc sites and biological processes/pathways that is involved in the development of diabetic retinopathy and Specific Aim 2 To verify the LysAc sites and biological processes/pathways identified in Aim 1. Diabetes is the leading cause of new cases of blindness among adults aged 20-74 years. However, no effective therapies to prevent or slow the progress of the disease are available. The identifications of differentially acetylated sites will provide a foundation for the development of novel therapeutic strategies for diabetic retinopathy
Keywords: Acetylation; Adult; Affinity; aged; Amacrine Cells; American; Animals; base; Biochemical; Bioinformatics; Biological Process; Blindness; Blood Vessels; Cell Nucleus; Data; Data Set; Deacetylase; Development; Diabetes Mellitus; diabetic; diabetic rat; Diabetic Retinopathy; Disease; effective therapy; EP300 gene; Event; Eye diseases; Foundations; Functional disorder; ganglion cell; Genetic Transcription; Goals; histone acetyltransferase; histone modification; Histones; horizontal cell; Individual; Knowledge; Label; Lead; Lysine; Maps; Mass Spectrum Analysis; Mediating; Methods; Modification; new therapeutic target; non-diabetic; novel therapeutics; Nuclear; Pathway Analysis; Pathway interactions; Peptides; Phosphorylation; Photoreceptors; Post-Translational Protein Processing; prevent; Proteins; Proteomics; Rattus; Retina; retina blood vessel structure; Retinal; Retinal Diseases; Role; Site; Small Interfering RNA; Techniques; Therapeutic Agents; tool
Relevance: Diabetes is the leading cause of new cases of blindness among adults aged 20-74 years. However, no effective therapies to prevent or slow the progress of the disease are available. The identifications of differentially acetylated sites will provide a foundation for the development of novel therapeutic strategies for diabetic retinopathy
Project start date: 2012-01-01
Project end date: 2013-12-31
Budget start date: 1-JAN-2012
Budget end date: 31-DEC-2012
1R21EY021595-01A1 (2012): $235500
PARALLEL SOFTWARE FOR FAST, AUTOMATED DETERMINATION OF VIRUS STRUCTURES
S Timothy, Professor
University Of California San Diegocity: La Jolla country: United States (us)
Grant 5R01GM087708-02 from National Institute Of General Medical Sciences
Abstract: Project Summary Electron cryo-microscopy and computer-based, 3D image reconstruction techniques are revolutionizing the way structures of large, complex biomacromolecular machines are studied. These methods provide keys to understanding how these machines function. Our goal is to determine virus structures reliably at the highest possible resolutions in the shortest amount of time. This will enhance our ability to understand how viruses cause a variety of dieases and may yield important clues about how best to develop anti-viral agents. Microscopy and image reconstruction each pose their own set of significant challenges, and development of novel software clearly represents a key to the success of our research efforts as well as that of others. We aim to substantially enhance the computational capabilities that comprise the heart of the 3D structure determination part of our cryo-reconstruction work. The resolutions achieved and structures solved in cryo-reconstruction are limited by many factors, not the least of which includes the numerical techniques employed. Hence, we will focus on algorithmic improvements that expand the range of problems that can be studied, make it possible to reach higher resolutions, and reduce time to solution. For example, we will extend the capabilities of our software to examine the non-icosahedral components of nominally icosahedral viruses as well as particles with lower symmetries. In addition to the improvements noted above, our software development interests include automation, parallel computing, and enhancements to usability. For many virus structures, especially large and/or asymmetric ones, image reconstruction is the rate limiting step. To reduce the time between image acquisition and structure determination and also narrow the gap between the resolutions that can be reached by novice and expert users, we will enhance AUTO3DEM, our automation system for intelligently integrating the multiple applications required in image reconstruction. This system also provides a test bed for evaluating new ideas and makes the software easily accessible to a wider range of users. The proposed work should take us closer to our goal of real-time image reconstruction at the microscope, or at least tightly coupled to data acquisition, to provide rapid feedback and quickly screen sample properties. Our studies are also heavily leveraged by close ties to colleagues in cryo-microscopy, image processing, and data visualization and interpretation, the acquisition of modern microscopes with support from NIH, UCSD, and the Agouron foundation, and interaction with the San Diego Supercomputer Center. All of our software will be made readily accessible to the electron microscopy community. Relevance Viruses infect nearly every living organism on Earth, causing serious diseases and death in humans and other animals, and also greatly impact food sources often leading to famine as well as weakened economies. Knowledge of virus structure provides an important link to better understand how viruses replicate themselves in susceptible hosts. Computer-based 3D models generated from virus images recorded in transmission electron microscopes, provide an important, first glimpse about the molecular structures of viruses. As the time required to obtain these models often requires weeks or months of dedicated effort, an important goal of this proposal is to reduce this down to hours or minutes
Keywords: Algorithms; Animals; Automation; Bacteriophages; base; Beds; Boxing; Cessation of life; Communities; Complex; Computer software; Computers; Coupled; Cryoelectron Microscopy; Data; data acquisition; Development; Disease; Double Stranded RNA Virus; Electron Microscope; Electron Microscopy; Famines; Feedback; Food; Foundations; Goals; Heart; Hour; Human; Image; image processing; Image Reconstructions; Imagery; interest; Knowledge; Life; Link; Lipid Bilayers; Methods; Microscope; Microscopy; Modeling; Molecular Structure; novel; Organism; parallel computing; particle; Preparation; Property; public health relevance; reconstruction; Research; Resolution; RNA-Directed RNA Polymerase; Sampling; Screening procedure; software development; Solutions; Source; Structure; success; supercomputer; System; Techniques; Testing; three dimensional structure; Three-Dimensional Image; three-dimensional modeling; Time; Totivirus; transmission process; United States National Institutes of Health; usability; Viral; Viral Genome; viral RNA; Virus; Work
Relevance: Relevance Viruses infect nearly every living organism on Earth, causing serious diseases and death in humans and other animals, and also greatly impact food sources often leading to famine as well as weakened economies. Knowledge of virus structure provides an important link to better understand how viruses replicate themselves in susceptible hosts. Computer-based 3D models generated from virus images recorded in transmission electron microscopes, provide an important, first glimpse about the molecular structures of viruses. As the time required to obtain these models often requires weeks or months of dedicated effort, an important goal of this proposal is to reduce this down to hours or minutes
Project start date: 2010-06-01
Project end date: 2014-05-31
Budget start date: 1-JUN-2011
Budget end date: 31-MAY-2012
PFA/PA: PA-07-070
5R01GM087708-02 (2011): $305910
1R01GM087708-01A2 (2010): $309000
EPITHELIAL-FIBROBLAST INTERACTIONS IN LUNG FIBROSIS
S Timothy, Professor
Vanderbilt Universitycity: Nashville country: United States (us)
Grant 5R01HL085317-05 from National Heart, Lung, And Blood Institute
Abstract: Fibrotic lung diseases, including idiopathic pulmonary fibrosis, represent a heterogeneous group of diseases in which progressive parenchymal fibrosis disrupts the structure and gas exchanging functions of the lungs. In the microenvironment of the distal lung, this pathobiology is characterized by an increased population of fibroblasts and augmented collagen and matrix synthesis. In addition to fibroblasts, recent evidence points to a critical role for lung epithelial cells in determining the extent and progression of lung fibrosis. The importance of epithelial cells in fibrogenesis is highlighted by the identification of mutations in the epithelial-restricted gene encoding surfactant protein C (SP-C), that are associated with familial pulmonary fibrosis. While a variety of pathways have been implicated in cross-talk between epithelium and fibroblasts, the critical mechanisms by which epithelial cells influence the effector fibroblast population are not well established. We hypothesize that interactions between lung epithelial cells and fibroblasts determine the severity and progression of lung fibrosis. Specifically, we propose that 1) epithelial cells, predominantly alveolar type II epithelium, contribute to fibrosis by undergoing epithelial-mesenchymal transition in vivo, 2) epithelial cells regulate proliferation and survival of resident fibroblasts, and 3) phenotypic changes in alveolar type II epithelial cells that cause endoplasmic reticulum stress lead to lung fibrosis by impairing re-epithelialization and inducing an increased population of effector fibroblasts following injury. Three specific aims are proposed to investigate this hypothesis 1) to define the extent of epithelial-mesenchymal transition as a source of fibroblasts in experimental pulmonary fibrosis, 2) to determine the pro-fibrotic effects of mutant SP-C expression in alveolar type II epithelial cells, and 3) to investigate the effects of epithelial dysfunction induced by expression of mutant SP-C on fibroblast recruitment/activation and development of lung fibrosis. These studies will use transgenic mice and cell culture systems to define essential roles for epithelial cells in the generation of lung fibrosis. Identifying the impact of alveolar type II epithelial cell phenotype on induction and maintenance of specific populations of effector fibroblasts will provide new insights into the pathogenesis of lung fibrosis and identify new treatment approaches. Relevance to public health Progressive fibrotic lung diseases frequently lead to respiratory failure and death. These studies will provide a better understanding of the basic mechanisms of disease pathogenesis and suggest new treatment approaches for these devastating diseases
Keywords: Affect; AGTR2 gene; Alveolar; Amino Acids; Architecture; Basement membrane; Bleomycin; Bone Marrow; C-terminal; Cell Culture System; Cell Death; Cell Survival; Cells; Cessation of life; Characteristics; Chemicals; clinically relevant; Coculture Techniques; Collagen; Data; Development; Disease; Distal; Doctor of Medicine; Endoplasmic Reticulum; endoplasmic reticulum stress; Epithelial; Epithelial Cell Proliferation; Epithelial Cells; epithelial to mesenchymal transition; Epithelium; Exons; Fibroblasts; fibrogenesis; Fibrosis; Functional disorder; Gases; Generations; Genes; Glutamine; Hamman-Rich syndrome; Healed; healing; Human; In Vitro; in vivo; injured; Injury; insight; Lead; Leucine; Lung; lung development; Lung diseases; Maintenance; Mediator of activation protein; member; Mesenchymal; Modeling; Molecular Chaperones; mouse model; mutant; Mutation; Organ; Pathogenesis; Pathway interactions; Patients; Phenotype; Population; Positioning Attribute; Process; Proliferating; protein folding; Protein Precursors; Proteins; public health medicine (field); Pulmonary Fibrosis; Pulmonary Surfactant-Associated Protein C; Respiratory Failure; Respiratory physiology; response; restoration; Role; S100 Proteins; Severities; Source; Stimulus; Structure; Tetracyclines; Transforming Growth Factors; Transgenic Mice
Project start date: 2007-04-10
Project end date: 2012-03-31
Budget start date: 1-APR-2011
Budget end date: 31-MAR-2012
5R01HL085317-05 (2011): $383750
5R01HL085317-04 (2010): $383750
S Timothy, Professor
Case Western Reserve Universitycity: Cleveland country: United States (us)
Grant 5R01EY000300-43 from National Eye Institute
Abstract: Evidence generated by us and others in animals provides evidence that inflammatory processes contribute to the development of at least the early stages of diabetic retinopathy, and especially the degeneration of retinal capillaries. How this capillary degeneration in diabetes is not clear, but most studies to date have focused on metabolic defects within cells of the retina and its vasculature. The present grant proposal is based on novel observations we have made over the previous several years suggesting that proinflammatory pathways within cells derived from bone marrow are critical in the development of the retinopathy. We have found that the vascular lesions of diabetic retinopathy can be prevented merely by eliminating either iNOS or PARP-1 from the marrow-derived cells only. Which myeloid cells are responsible for development of lesions of diabetic retinopathy, how they cause the diabetes-induced abnormalities in structure and function, and how to inhibit these leukocyte-driven abnormalities are not known, and are the subjects of the proposed research. In this proposal, we will (1) characterize which marrow-derived cell types participate in the development of early stages of diabetic retinopathy, (2) determine the role of chemoattractants in recruitment of marrow-derived cells to the retina in diabetes, and if the chemoattraction is critical to diabetes-induced degeneration of retinal capillaries, and (3) investigate the mechanism(s) by which marrow-derived cells kill retinal cells in diabetes, focusing initially on the role of reactive oxygen species generated by NADPH oxidase in the development of diabetes-induced inflammation. We also will determine if the abnormalities within the marrow-derived cells cause degeneration of neuroglial cells of the retina. We postulate that these diabetes-induced degenerative changes in the retina can be inhibited by blocking the abnormal metabolism within the marrow-derived cells, or by blocking the adhesion between those blood cells and retinal vascular cells. This hypothesis offers a novel approach to inhibiting the retinopathy (cause circulating leukocytes to release a factor that inhibits binding of white blood cells to the endothelium) which we will test in this proposal. This data is novel, and indicates that views of diabetic retinopathy need to expand beyond the traditional retina- or vascular-specific view of pathogenesis to include also bone marrow-derived cells. Diabetic retinopathy is a leading cause of vision loss in working-age adults in industrialized nations. In addition to abnormalities that disruption of light hitting the retina (fibrovascular scars, hemorrhage, retinal thickening), there are also alterations in function of the neural cells of the retina. There is very little understanding how this pathology develops. Our preliminary studies demonstrate for the first time that white blood cells play a critical role in the early stages of diabetic retinopathy. This proposal explores which white blood cells are responsible for this abnormality, how they are attracted to the retina, and how they kill retinal cells. We also test a possible therapeutic option to stop white blood cells from interacting with endothelial cells as an option to inhibit the retinopathy
Keywords: Abbreviations; Abnormal Cell; Adhesions; Adult; Age; Animals; Applications Grants; base; Basophils; Binding (Molecular Function); Blindness; Blood capillaries; Blood Cells; Blood Vessels; Bone Marrow; Bone Marrow Cells; capillary; CCL2 gene; Cell Adhesion Molecules; cell killing; cell type; Cells; Cessation of life; Chemotactic Factors; Chimera organism; Cicatrix; cytokine; Data; Defect; Developed Countries; Development; Diabetes Mellitus; Diabetic mouse; diabetic patient; Diabetic Retinopathy; Emigrations; Endothelial Cells; Endothelium; eosinophil; Functional disorder; Genetic; Green Fluorescent Proteins; Histopathology; Hyperglycemia; Immune system; Inflammation; Inflammatory; inflammatory marker; irradiation; Killings; Lesion; Leukocytes; Light; Lymphocyte; Marrow; Mediating; Metabolic; Metabolism; Microglia; Modification; monocyte; Monocyte Chemoattractant Protein-1; Mus; Myelogenous; Myeloid Cells; NADPH Oxidase; NADPH Oxidase 1; Neural Retina; Neuroglia; Neurons; neutrophil; Nitric Oxide Synthase; Nomenclature; novel; novel strategies; Oxidants; Pathogenesis; Pathology; Pathway interactions; Play; Polymerase; prevent; Procedures; Process; Production; Protein Isoforms; public health relevance; Reactive Oxygen Species; Recruitment Activity; Research; Retina; Retinal; Retinal Degeneration; Retinal Diseases; Retinal Hemorrhage; Role; Staging; Stem cells; Structure; Testing; Therapeutic; Time; vascular inflammation; Work
Relevance: Diabetic retinopathy is a leading cause of vision loss in working-age adults in industrialized nations. In addition to abnormalities that disruption of light hitting the retina (fibrovascular scars, hemorrhage, retinal thickening), there are also alterations in function of the neural cells of the retina. There is very little understanding how this pathology develops. Our preliminary studies demonstrate for the first time that white blood cells play a critical role in the early stages of diabetic retinopathy. This proposal explores which white blood cells are responsible for this abnormality, how they are attracted to the retina, and how they kill retinal cells. We also test a possible therapeutic option to stop white blood cells from interacting with endothelial cells as an option to inhibit the retinopathy
Project start date: 1977-12-01
Project end date: 2014-08-31
Budget start date: 30-SEP-2011
Budget end date: 31-AUG-2012
PFA/PA: PA-07-070
5R01EY000300-43 (2011): $408852
ROLE OF FETAL LUNG MACROPHAGES IN BRONCHOPULMONARY DYSPLASIA
S Timothy, Asst Professor
Vanderbilt Universitycity: Nashville country: United States (us)
Grant 5R01HL097195-03 from National Heart, Lung, And Blood Institute
Abstract: Bronchopulmonary Dysplasia (BPD) is a frequent complication of preterm birth that results from arrested lung development in the saccular and alveolar stages of lung morphogenesis. While innate immune signaling has been implicated in the disruption of epithelial-mesenchymal interactions that regulate normal branching morphogenesis, the mechanisms linking innate immunity and abnormal lung development in BPD remain unclear. This proposal will specifically test the role of macrophages in BPD pathogenesis through regulation of innate immune signaling in the fetal lung. Our preliminary data indicate that exposing the fetal lung to inflammatory stimuli induces expression of NF-kB dependent inflammatory cytokine by fetal lung macrophages. Depletion of macrophages or targeted disruption of the NF-kB signaling pathway in macrophages prevents alterations in lung morphogenesis induced by Gram negative bacterial lipopolysaccharide (LPS). While prenatal exposure to inflammatory stimuli increases the risk of BPD in preterm infants, a second episode of significant inflammation may be key for progression to clinical disease, suggesting that exposure to prenatal inflammatory stimuli primes the innate immunity for a heightened response to subsequent noxious stimuli. Consistent with this idea, we found that in utero exposure to LPS results in a marked increase in neutrophilic lung inflammation following subsequent intratracheal LPS treatment of pups after weaning. Therefore, structural abnormalities in the lungs resulting from prenatal inflammation may define a vulnerable host in which additional insults, such as oxygen toxicity, mechanical ventilation, and recurrent infection, result in excessive lung inflammation, injury, and remodeling that is clinically manifest as BPD. In this proposal, we will test the hypothesis that infections and inflammatory stimuli lead to BPD through activation of innate immune signaling in fetal lung macrophages. Activation of NF-kB signaling in macrophages results in production of cytokines, including TNF1 and IL-12, that inhibit epithelial-mesenchymal cell interactions required for branching morphogenesis in the lungs. Activation of NF-kB in fetal lung macrophages also primes the lung for excessive immune responses later in life by altering the lung macrophage phenotype. Three specific aims are proposed to investigate this hypothesis 1) to determine whether fetal lung macrophages are required for inhibition of lung development by Toll-Like Receptor (TLR) agonists, 2) to identify the role of the NF-kB pathway in inhibition of lung development following innate immune activation, 3) to determine whether activation of fetal lung macrophages alters the macrophage phenotype as lungs mature. Determining the cell types and pathways that lead to arrested lung development and altered innate immunity in the setting of perinatal inflammation could lead to targeted therapies that prevent BPD or improve outcomes. This proposal will test novel mechanisms of disease explaining how infection and inflammation disrupt normal lung development and immune function in preterm infants. These processes contribute to the pathogenesis of bronchopulmonary dysplasia, a chronic lung disease that affects up to 10,000 former preterm children each year in the United States. (End of )
Keywords: ing; Affect; Agonist; Alveolar; Alveolitis; Birth; Bronchopulmonary Dysplasia; Cell Communication; cell type; Cells; Child; Chronic lung disease; Clinical; Clinical Data; Complication; cytokine; Data; Dichloromethylene Diphosphonate; Disease; Effector Cell; Epithelial; Exposure to; fetal; Fetal Lung; fibroblast growth factor 10; Genetic; Health; human disease; Immune; immune activation; immune function; Immune response; Immune system; Impairment; improved; in utero; In Vitro; Individual; Infant; Infection; Infection of amniotic sac and membranes; Inflammation; Inflammatory; Injection of therapeutic agent; Injury; Interleukin-1; Interleukin-12; Knockout Mice; Lead; Life; Link; Lipopolysaccharides; Lung; lung development; Lung Inflammation; macrophage; Measures; Mechanical ventilation; Mediating; Mesenchymal; Mesenchyme; Morphogenesis; mouse model; Mus; Natural Immunity; Newborn Infant; NF-kappa B; NFKB Signaling Pathway; novel; Outcome; oxygen toxicity; Pathogenesis; Pathway interactions; Patients; Perinatal; Perinatal Exposure; Phenotype; Population; Premature Birth; Premature Infant; prenatal; prenatal exposure; prevent; Process; Production; pup; Recurrence; Regulation; response; Risk; Role; Signal Pathway; Signal Transduction; Staging; Stimulus; Testing; TLR4 gene; TNF gene; Toll-like receptors; Transgenic Mice; Transgenic Model; Tumor Necrosis Factor Receptor; United States; Weaning; Wild Type Mouse
Relevance: Narrative This proposal will test novel mechanisms of disease explaining how infection and inflammation disrupt normal lung development and immune function in preterm infants. These processes contribute to the pathogenesis of bronchopulmonary dysplasia, a chronic lung disease that affects up to 10,000 former preterm children each year in the United States
Project start date: 2009-09-14
Project end date: 2014-07-31
Budget start date: 1-AUG-2011
Budget end date: 31-JUL-2012
PFA/PA: RFA-HL-08-009
5R01HL097195-03 (2011): $528278
Sponsored Links Excellgen http://Excellgen.com
INTERDISCIPLINARY TRAINING PROGRAM IN LUNG RESEARCH
S Timothy, Professor
Vanderbilt Universitycity: Nashville country: United States (us)
Grant 5T32HL094296-04 from National Heart, Lung, And Blood Institute
Abstract: Acute and chronic lung diseases are major causes of morbidity and mortality throughout the world. For many of these diseases, the fundamental pathobiology is not well understood and effective, disease-modifying treatments are not available. Therefore, there is a pressing need to train researchers focused on elucidating disease mechanisms. The Division of Allergy, Pulmonary, and Critical Care Medicine at Vanderbilt University and the Center for Lung Research have a long, successful history in developing well trained researchers who have the vision and the skills with which to embark on successful careers in academic research. This new program will train young investigators to study basic mechanisms of lung inflammation, repair, and remodeling. The Vanderbilt Center for Lung Research (VCLR), which was developed to coordinate and enhance collaborative interdisciplinary research and training related to the lungs, will house this program, and Dr. Timothy Blackwell (director of the VCLR) will serve as program director. This training program will support postdoctoral trainees who have completed M.D. and/or Ph.D. programs and show exceptional aptitude for successfully pursuing an academic research career. Trainees will concentrate on one of several disease focused areas of existing expertise in the VCLR acute lung inflammation/injury, asthma, pulmonary fibrosis, pulmonary hypertension, or lung carcinogenesis. A customized mentoring team will be formed for each trainee, consisting of a mentor with nationally recognized expertise in the area and a research advisory committee to provide additional guidance, mentoring, and feedback. The trainee´s experience will be enhanced by interactions with other investigators and trainees in existing lung disease-focused research programs in the VCLR, an extensive program of seminars and conferences, and coursework tailored to meet the needs of each trainee. The unique environment of the VCLR - its rich collaborative interactions between basic scientists and clinical researchers, wide range of supporting Cores and Centers, and exposure to state-of-the-art clinical care - provides an outstanding opportunity to train successful scientists whose discoveries regarding fundamental aspects of lung diseases can be translated into improved patient care
Keywords: Lung; Research; Training Programs
Project start date: 2008-07-01
Project end date: 2013-06-30
Budget start date: 1-JUL-2011
Budget end date: 30-JUN-2012
PFA/PA: PA-06-468
5T32HL094296-04 (2011): $341452
S Timothy
University Of North Carolina Chapel Hillcity: Chapel Hill country: United States (us)
Keywords: Address; Award; Communities; Ethnicity aspects; experience; Faculty; Foundations; Funding; Future; Goals; Grant; Health; health disparity; Individual; innovation; Institution; Intervention; Intervention Studies; Lead; National Center on Minority Health and Health Disparities; Peer Review; Pilot Projects; Plant Roots; Publications; Race; Research; research and development; Research Infrastructure; Research Project Grants; United States National Institutes of Health; Universities
Budget start date: 1-JUN-2011
Budget end date: 31-MAY-2012
5P60MD000244-10_9010 (2011): $155962