1989 — 1991 |
Pittler, Steven J |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Functional Studies of Retinal Cgmp Phosphodiesterase (S) @ Baylor College of Medicine |
1 |
1994 — 2002 |
Pittler, Steven J [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Structure and Regulation of Retinal Cgmp Pde Alpha Gene @ University of South Alabama
The long term objectives of the research program are to define the structure, expression, and spatial and temporal regulation of genes which encode the molecules essential to flow of information through the visual transduction pathway. The focus of this current proposal is on the gene encoding a large subunit, alpha, of rod photoreceptor cGMP phosphodiesterase (PDE), an essential component of mammalian phototransduction. The sequence of the human PDE alpha-subunit gene will be determined by polymerase chain reaction (PCR) of genomic DNA and direct sequencing, and by isolation and sequence analysis of genomic clones. Primer extension in conjunction with the ribonuclease protection assay or S1 nuclease mapping will be used to identify the transcriptional start point and putative core promoter region of the mouse alpha gene. Direct demonstration of promoter activity and specificity will be obtained in transgenic mice by directed expression of the reporter gene, Beta-galactosidase. As a first step towards promoter characterization, the sequences of mouse and human PDE alpha- and Beta-subunit genes upstream regulatory regions will be determined. Comparison of the upstream sequences may identify potentially important cis regulatory elements that will be confirmed for the mouse alpha-subunit gene by DNA footprinting, and gel retardation analysis. Studies will then be initiated to identify and characterize specific transcription factors in mouse retina that interact with the mouse PDE-alpha-subunit promoter. The proposed studies will provide the foundation necessary to understand the regulation of genes whose functions are essential for rod cell phototransduction and cyclic nucleotide metabolism. The information gained can be used to initiate candidate gene studies to assess the potential for a defective PDE alpha gene to be causal of any form of hereditary retinal degeneration. Additionally, new photoreceptor specific genes encoding transcriptional regulators may be identified. Thus, these studies should have direct impact on understanding the molecular basis of certain forms of human hereditary retinopathies.
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0.907 |
2004 — 2008 |
Pittler, Steven J [⬀] |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Core--Molecular Biology @ University of Alabama At Birmingham |
0.904 |
2008 — 2011 |
Pittler, Steven J [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Analysis of Retina Rod Photoreceptor Garp and Cgmp-Gated Cation Channel @ University of Alabama At Birmingham
[unreadable] DESCRIPTION (provided by applicant): The long term objectives of the research program are to understand protein structure/function relationships in retinal photoreceptors, and to translate this understanding to the treatment of patients with retinitis pigmentosa and related disorders. In humans, CNGB1 gene defects cause hereditary retinal degeneration. We have therefore focused on the murine Cngbl locus that expresses, exclusively in the rod outer segment (ROS), two relatively abundant proteins: plasma membrane localized 2-subunit of the cGMP-gated cation channel and cytoplasmic glutamic acid-proline rich polypeptide (GARP-2). The rod cGMP-gated cation channel plays a critical role in vision by regulating the flow of calcium and sodium ions into the rod cell. In addition, both the 2-subunit and GARP-2 interact with peripherin-2/rds, a disk membrane protein, and thus may play essential structural roles. To determine the contribution of the proteins in ROS, we generated a homozygous Cngbl photoreceptor null (5' KO), and are analyzing the mice using light and EM level analysis, ERG and single cell recordings, and Western analysis. Homozygous 5' KO mice show a significant reduction (>30 fold) in light sensitivity and attenuated b-wave response, but are functional prior to the onset of retinal degeneration. Surprisingly, in the 59 KO not only is the 2-subunit absent but the channel 1-subunit is also greatly reduced in abundance in ROS. We hypothesize that the GARP region has an important structural role in the photoreceptor and that GARP-2, like the 2-subunit has also an important functional role. We will directly examine the structural and functional roles of the GARP region of the beta subunit and of GARP-2 by targeted deletion of GARP-2 expression and introduction of transgenes into 59 KO and 2-subunit knockout mice (39 KO). The proposed studies will help to establish the function of the Cngbl encoded GARP region and of GARP-2 in the photoreceptor, and hence provide important insights into the mechanisms whereby CNGB1 gene defects cause hereditary retinal degeneration. The studies may also yield new targets for intervention as possible treatments for certain forms of hereditary retina degeneration. [unreadable] [unreadable] [unreadable]
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0.904 |
2010 |
Pittler, Steven J [⬀] |
S10Activity Code Description: To make available to institutions with a high concentration of NIH extramural research awards, research instruments which will be used on a shared basis. |
Noninvasive Visualization and Analysis of Organ Systems Using Oct and Digital Fun @ University of Alabama At Birmingham
DESCRIPTION (provided by applicant): We propose the acquisition of Bioptigen, Inc. 840 nm SDOCT Optical Coherence Tomography (OCT) bundled with Phoenix Research Laboratories, Inc. Micron II Retinal Imaging Microscope package (total cost $274,940) for use with both humans and other animals. These instruments, which are currently not available at UAB will make an essential addition to our existing NIH-funded (P30 EY03039) Molecular Biology/Cellular Analysis Core grant module. The availability of the instruments will complement existing ERG capability and provide a powerful non-invasive analytical suite of instruments to analyze and correlate structure and function of the eye in vivo in the living animal. The proposed OCT/fundus imaging equipment bundle is planned to be housed in newly renovated space created to bring together researchers in Ophthalmology and Optometry and establish a strong cohesive group of retina researchers. The instruments are critical and essential new tools in five current NIH-funded projects (RO1 EY018143, Analysis of rod photoreceptor GARP and cGMP-gated cation channel in retina;RO1 EY019311, Rhodopsin Trafficking and Retinal Degenerations;RO1 EY006109, Quantitative Analysis of Aging Retina;RO1 EY05922, Mechanisms of Ocular Development;and RO1 EY006400, Proteinase Inhibitors and Crystallin Fragments in Cataract). The OCT instrument will also be utilized by a group of minor users that are studying retina, optic nerve, heart and brain. Relevance: OCT is an emerging diagnostic imaging method enabling in vivo cross-sectional tomography of internal microstructures in living biological systems. While the instrumentation was first invented in the early 1990's, only recently has the technology evolved to allow ultra high resolution imaging that is approaching the quality and informational content of conventional invasive histologic analyses. No organ system is more amenable to OCT than the eye. OCT is now in routine use by Ophthalmologists and is becoming increasingly used in research in vision science. OCT is also being increasingly used in medical diagnostics in cardiology, gastroenterology, and other fields. While, routine histologic analysis is an end point experiment, OCT will allow the investigator to follow changes over time in the same animals. The proposed OCT and Retina Digital Imaging Equipment will enhance the already strong Vision Science Research Center Core Grant and adds a new dimension of analytical prowess to advance studies in many aspects of vision science and the study of other organ systems. Public Health Relevance: The instrumentation requested in this application will allow tissues to be visualized in living animals noninvasively. This is a monumental breakthrough in diagnostic capability that has been in use in the clinics for several years and is now available for large and small animals. The use of this new technology will dramatically advance our ability to correlate structure and function in living animals and in isolated tissues and will ultimately lead to new ways to treat diseases in humans.
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0.904 |
2010 — 2015 |
Pittler, Steven J [⬀] |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Vision Science Research Center @ University of Alabama At Birmingham
DESCRIPTION (provided by applicant): In 1979, a NEI Core Grant was awarded to the University of Alabama at Birmingham (UAB). Over the next 30 years, the institution's continued commitment to growth resulted in a remarkable expansion in vision research at UAB. Participants in the Core Grant have increased from thirteen in two departments to twenty-nine representing eight departments, and there has been a concomitant increase in extramural funding. Annually, NEI funding to Core Grant participants exceeds $3 million, not including institutional awards. Total extramural funding for vision-related research at UAB has grown from just over $6 million in 1998 to $15 million in 2008. A major research focus of the group is visual neuroscience, with seventeen of the participants actively involved in anatomical, biochemical, physiological, and psychophysical studies of the retina and central visual pathways. Other areas of research include myopia, biochemistry of the lens and cornea, and vitreous biochemistry. This application requests funds to continue to provide services and facilities to UAB Core Grant participants through the Electronics, Machine Shop, Computer and Molecular and Cellular Analysis modules. These modules provide easily accessible technical facilities and the expertise to use them in dedicated spaces designed specifically for each purpose. The Electronics Module is staffed by an Electronics Engineer and includes all of the equipment required to design and build new electronic apparatus, and to provide repair and maintenance services. The Computer Module provides access to high-end computers and peripherals, custom programming capability, and assistance with office and lab computers and software. A computer programmer is available for systems administration, and networking. The Machine Shop is fully equipped with a lathe, milling machine and other machine tools, and is staffed by a Tool and Die Maker who designs and fabricates new mechanical devices and can repair or maintain mechanical equipment. The Molecular and Cellular Analysis Core provides access to major instrumentation for DNA, RNA and protein studies. A PhD level Research Associate provides expertise and training in all of these areas. In addition, equipment and expertise for tissue analyses including routine tissue processing, histological stains, immunohistochemical studies, and in situ hybridization is available. A part time histological technician provides consultation for experimental design, technical expertise, and training of laboratory personnel.
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0.904 |
2013 — 2016 |
Pittler, Steven J [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Analysis of Retina Rod Photoreceptor Garp and Cgmp-Gated Channel @ University of Alabama At Birmingham
7. Project Summary/Abstract The long-term objectives of this research program are to understand cGMP-gated cation channel protein structure/function relationships in rod photoreceptors and to translate this understanding to the treatment of patients with retinitis pigmentosa (RP) and related disorders. We will focus our studies on the channel b- subunit and the associated soluble glutamic acid rich protein (GARP) GARP2, both encoded by the Cngb1 locus, examining the structural and functional roles of the GARP region in three specific aims. We previously generated a homozygous Cngb1 photoreceptor null (X1 KO) mouse resulting not only in partial loss of channel function but also severe structural perturbations establishing in vivo that these proteins are necessary for normal rod outer segment ROS disk morphogenesis and structural integrity. We hypothesized that the GARP2 sequence on the b-subunit is required for plasma membrane/disc membrane interaction. To test this we created transgenic mice expressing an N-terminally truncated b-subunit (Tb) devoid of all GARP2 sequence on the X1 KO background. Despite the absence of soluble GARP2, the entire b-subunit GARP2 region and only one glutamate rich segment remaining on Tb, there is significant but not complete structural and functional rescue. To determine the basis for the observed rescue (A.) we will analyze these mice structurally using histology, immunocytochemistry and ultrastructure analysis by transmission and Cryo-EM and functionally using ERG, single cell physiology and a now established retina punch preparation. In the X1 KO and X26 mice that do not express the b-subunit and in X1 KO also missing soluble GARPs the photoresponse is attenuated. In WT mice overexpressing GARP2 a significant increase in phototransduction gain was observed demonstrating a previously unknown role for GARP2 in modulating phototransduction. (B.) We hypothesize that GARP2 regulates rod dark noise and also contributes to novel slow Burnsian adaptation. To test this we will use our established physiologic tools to compare phototransduction parameters in WT, GARP2 overexpressing, Tb transgenic mice and mice expressing full length b-subunit during activation, recovery and adaptation to further define the role of GARP2 and the b-subunit in modulating the photoresponse. Using novel zinc finger nuclease technology we have established GARP2-specific knockout mice deleted for the last unique GARP2 exon and a potential hypomorph that is missing the GARP2 3'-UT region. (C.) We will use these mice to test the hypothesis that GARP2 is required for structure and function in the rods. We will perform structural analysis to determine effects on disk morphogenesis and overall outer segment structure and functional analysis to complement studies proposed in Aims A. and B. and to directly examine the role of GARP2 in rod function. The proposed studies document a comprehensive plan to define the structural importance of the GARP region and to define the role of GARP2 in modulating the rod photoresponse. The studies may also yield new targets for intervention treatments for certain forms of hereditary retinal degeneration.
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0.904 |
2016 — 2020 |
Pittler, Steven J [⬀] |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Administrative Core @ University of Alabama At Birmingham
Administrative Core 7. Project/Summary Abstract The Administrative Core of the UAB Vision Science Research Center P30 grant performs an essential function in overseeing the overall activity of the scientific Cores, and making sure that funds are appropriately utilized to support the highest quality science in a diverse range of vision disciplines. Another major role of this Core in conjunction with the efforts of the Vision Science Research Center is to promote and actively foster new collaboration, and to provide support and guidance to new and less established investigators to develop and strengthen their research programs. Both the Core Director/PI, Dr. Pittler and Co-Director/Co-PI, Dr. Gamlin are NEI R01 supported and maintain active and productive labs. Their combined expertise in diverse areas of vision research, and now 16 year established strong working relationship, makes them an excellent team to lead the P30 grant. They will receive at least bi-annual input from an advisory committee comprised of both UAB internal and external NEI R01 grant holders and additional input from UAB P30 participants and others in a monthly open forum of all UAB vision scientists that precedes Vision Research at UAB seminars. Additionally, the quality of Core provided resources and services is measured by annual surveys and through interaction with faculty at faculty meetings and retreats. Support to continue two staff personnel with several years of administrative experience is requested to facilitate day-to-day operations such as ordering supplies, maintaining compliance and budgets, coordinating repairs if needed and assisting with document preparation and record keeping. Due to strong institutional support, secretarial/business officer support costs represent only 9 % of the total budget each year. The Administrative Core has an essential role in maintaining and coordinating P30 activities and keeping the Cores running smoothly and effectively.
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0.904 |
2016 — 2021 |
Pittler, Steven J [⬀] |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Molecular & Cellular Analysis Core @ University of Alabama At Birmingham
Molecular & Cellular Analysis Core 7. Project/Summary Abstract The Molecular & Cellular Analysis Core was approved as a molecular biology/histology core in the last competitive renewal and has been an active and critical core for many Core participants. Core participants since the last grant renewal have demonstrated outstanding productivity publishing 159 papers. For the proposed funding cycle, of the 11 moderate or extensive Core participants, seven are NEI R01-funded (64%) and the other four are actively seeking new or continued NEI R01-funding. The two limited Core participants proposed have NIH new investigator status and are actively seeking NEI R01 support. User needs have progressed to require more advanced molecular analysis tools and expertise warranting several core enhancements that have been made to meet these needs. Major institutional support has allowed the purchase of new equipment or the upgrade of some of the key Core equipment to help meet user needs by providing the most up to date equipment. The Core encompasses protein and nucleic analysis, histology, and advanced microscopy imaging and analysis. Dr. Pittler, the Core Director is an expert in cell and molecular biology, and biochemistry and will continue as the Core Director. Dr. Marina Gorbatyuk has been added as an Associate Director to assist with Core governance and metrics analysis. She brings additional cell and molecular and biochemical expertise including extensive experience with AAV production and use. In addition to having advanced equipment for analysis, the Core has a highly educated, fully trained MD, PhD scientist, Dr. Vincenzo Guarcello as the Laboratory Director. His broad knowledge and experience base and overall scientific skill is a major asset. The Core equipment is primarily housed dedicated space in close proximity to most users and is accessible 24/7. Conflicts in usage scheduling is minimized through a comprehensive online scheduler for all major equipment. Outstanding institutional support has been critical in many ways: (1) to provide the facilities that house Core equipment and an office for the laboratory director, (2) to maintain the equipment, (3) to obtain new equipment to increase core capability and (4) to partially support the Laboratory Director. Core usage is carefully monitored to ensure priority of use among Core participants as follows: NEI R01-funded participants have first priority for use (1), followed by participants with NIH new investigator status that are actively seeking an NEI R01 (2), then by participants that are collaborating as key personnel on an NEI R01 and actively seeking lead PI NEI R01 support (3), and lastly by participants that have recently in the last three years been NEI R01-funded and are actively seeking new NEI R01 support (4).
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0.904 |
2016 — 2018 |
Pittler, Steven J [⬀] |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Uab Vision Science Research Center @ University of Alabama At Birmingham
UAB Vision Science Research Center - Overall Core 7. Project Summary/Abstract In 1979, an NEI Core grant was first awarded to UAB. Over the next 35 years, the institution's continued commitment to growth resulted in a remarkable expansion in vision research. Participants have now increased from 13 in two departments to 23 in five departments, and there has been a concomitant increase in extramural funding. Eleven of the Core participants currently hold 14 eligble grants, another 7 have NEI new investigator status and are seeking NEI R01 support (four of these have submitted NEI R01s). Of the reminaing five participants, two are Co-Investigators an an active NEI R01 and three have submitted NEI R01s. Annually, NEI funding exceeds $3M million, not including internal UAB awards. Total annual extramural funding for vision-related research at UAB has grown to nearly $13 million. A major research focus of the participants is visual neuroscience, with 16 actively involved in anatomical, biochemical, physiological, or psychophysical studies of the retina and central visual pathways. Additionally, UAB vision research groups are focused on the study of glaucoma, AMD, myopia, keratoconus, dry eye and cataractogenesis. This application requests funds to continue to provide services and facilities to support the research activity of the participants through four professionally staffed cores. These cores provide state-of-the-art facilities and expertise in dedicated spaces designed specifically for each purpose. The Instrumentation Core is staffed by an electronics engineer who designs and builds complex novel electronic apparati and an expert machinist/tool and die maker who designs and fabricates unique mechanical devices. Both also repair and maintain current instruments greatly reducing repair costs. The Research Programming & Computational Analysis Core provides access to high-end analytical tools and is staffed by an experienced programmer/analyst with an MS in computer sciences who offers custom software development and software/hardware interfacing, and by a part-time experienced biostatistician who will assist in experimental design and statistical analysis. The Molecular & Cellular Analysis Core is staffed by an MD/PhD trained scientist who provides expertise for study design, training and access to major instrumentation for DNA, RNA and protein studies and morphologic analysis of tissues and cells. The new Ocular Phenotyping Core will be part-time staffed by a PhD who has several years of expertise in the use of all instrumentation for analysis of the structure and function of small animal models of human ocular disease. Since 1979, more than $17M has been provided in institutional support of vision research to date and more than $800,000 is committed over the next 5 years of requested NIH support. UAB vision science is in an exciting growth phase that is focused on the use of advanced technology and the recruitment of the best scientists to study and devise treatments for ocular disorders. The continuation of the P30 core grant for vision research is an essential element at the core of new discovery in vision science at UAB.
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0.904 |
2019 — 2021 |
Fliesler, Steven J. Pittler, Steven J [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Development and Characterization of Mouse Models of Rp59 Dhdds Deficiency @ University of Alabama At Birmingham
7. Project Summary/Abstract Within the family of Congenital Disorders of Glycosylation (CDGs), defects in the dehydrodolichyl diphosphate synthase (DHDDS) gene cause a recessive form of retinitis pigmentosa (RP59; OMIM #613861). Patients with a K42E mutation in DHDDS exhibit progressive loss of both rod and cone function, as well as macular changes, suggestive of RPE involvement. The DHDDS enzyme is ubiquitously required in all cells for protein N- glycosylation. We wish to understand the basis for selective ocular pathology associated with ubiquitous DHDDS mutation and the contribution of specific ocular cell types to the pathology of mutant Dhdds-mediated retinal degeneration. As a first step, we devised a selective knockout scheme to study the importance of the enzyme in specific retinal cell types. To circumvent known embryonic lethality associated with DHDDS knockout, we generated a Cre-dependent knockout allele of murine Dhdds (Dhddsflx/flx). Additionally, we used CRISPR/Cas technology to generate a knock-in K42E mouse model of RP59. We propose to use these novel mouse lines to examine the mechanism of disease induced by DHDDS enzyme deficiency and to identify the primary site(s) of ocular pathology. In Aim 1, using rod-specific Cre expression, we will address the prevailing hypothesis that defective rhodopsin glycosylation is the major cause of Dhdds mutation-mediated pathology. In Aim 2, using retinal pigment epithelium (RPE)-specific Cre expression, we will examine the effects of perturbation of DHDDS activity on RPE structure and function. In Aim 3, we will selectively perturb DHDDS activity in Müller glia, and examine their contribution to Dhdds-dependent retinal degeneration. In Aim 4, we will characterize a newly generated K42E knock-in RP mouse model and compare the resulting structural and functional changes with those observed in the three knockout models to assess the pathological importance of each cell type. These four Aims will utilize a combination of state-of-the-art methodologies, including Cre-lox technology, CRISPR-Cas9- mediated genome editing, electroretinography (ERG), ultrahigh resolution spectral domain-optical coherence tomography (UHR SD-OCT), optokinetic reflex (OKR), light and electron microscopy, immunohistochemistry and lectin histochemistry, Western blot analysis, HPLC, and MALDI imaging mass spectrometry (IMS). Each stand- alone aim will provide significant new insights into the importance of DHDDS activity in the neural retina and RPE, and collectively will provide information that can guide rational treatment design for specific intervention in DHDDS-mediated RP. The overarching hypothesis is that, contrary to conclusions from initial reports, RP59 is not a simple disorder of altered rhodopsin glycosylation; rather, it is a complex disorder involving primary pathology in more than one cell type that may require a more global approach for effective therapeutic intervention. Upon completion of the proposed studies, we will be well positioned to pursue future proof-of- principle preclinical gene therapy studies to correct the genetic defect that otherwise would result in blindness.
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0.904 |