Daniel Peterson - US grants

Scanning laser confocal microscopy provides powerful analytic capabilities for biomedical research. Among these is the capability to simultaneously identify the location of several tagged particles and to determine their spatial relationship with one another in tissue slices or in cell cultures. The ability to combine fluorescent probes indicating the physiological state of the tissue with other optical and electrophysiological measurements further enhances the usefulness of this instrument. The Chicago Medical School does not have a confocal microscope. This application requests funding for a basic confocal microscope and accessory equipment for live cell studies and post- acquisition digital analysis to be placed in a dedicated core facility for the shared use of the investigators on this application. To ensure the successful installation and use of this instrument, The Chicago Medical School has committed to a three year support plan equal in value to half the purchase amount of the instrument. The users on this application represent four basic science departments at the University, the majority of whom have research interests in neuroscience. The Principal Investigator is in the Department of Neuroscience, which will serve as the core facility's home department. Users in the group have interests in the mechanisms of neurodegenerative diseases, including Alzheimer's and Parkinson's diseases, and the development of therapeutic intervention strategies, in the interaction of dopamine receptors and excitatory amino acid receptors in the mediation of drug addiction, in the regulation and function of neuropeptides and their receptors, in the hormonal regulation of neuronal transcription and neurite extension, in the regulation of the blood-brain-barrier, and in the regulation of neurogenesis in the adult CNS. Other research interests include the functional properties of gap junction mutations and in the synthesis of extracellular matrix. All of these research interests are relevant to a number human disease states or to the problem of drug addiction. The availability of a confocal facility at the Chicago Medical School would provide new analytic capabilities and greatly enhance research into each of these areas.

This application is responsive to Research Topics: 7. Sensory and Motor Processing and 13. Genetic, cellular and biochemical basis of functional senescence (including phenotypic characterization of age-related changes) The neurons of the adult brain are post-mitotic and are not replaced following injury or disease, contributing to functional decline in aging and with neurodegenerative diseases. However, the continued generation of neurons from neural stem cells in certain regions of the adult brain suggests that signals for neuronal proliferation, migration and differentiation may persist beyond development. Understanding these signals and how to regulate their expression may provide new therapeutic strategies for brain repair. Adult neurogenesis has been intensively investigated in the olfactory bulb (one of two neurogenic sites in the adult brain) of young animals. Reports investigating the dentate gyrus (the other neurogenic site) suggest that proliferation there is reduced with aging. The olfactory bulb provides a useful model for studying adult neurogenesis in that the sites of proliferation, migration, and differentiation are spatially distinct and there are three different neuronal phenotypes produced. By comparing these processes in young and aged olfactory bulb, it may be possible to detect differences that would suggest which signals are important for maintaining and regulating neurogenesis. Unfortunately, little is known about the proliferation, migration, and differentiation of neural stem cells in the aged olfactory bulb. This proposal will use multiple immunofluorescence confocal microscopy in conjunction with design-based stereology to quantify labeled progenitor cells in total and by phenotype in the aged olfactory bulb. These studies will provide a foundation for subsequent investigations into the mechanisms regulating neurogenesis in the aged olfactory bulb.

DESCRIPTION (provided by applicant): The goal of this study is to recruit endogenous neural progenitor cells (NPCs) within the neocortex and direct their cell fate so that they can contribute to brain repair. The brain and spinal cord are vulnerable to cell death or loss of neuronal function as a result of neurodegenerative disease, stroke, or injury. There is little spontaneous generation of replacement cells, resulting in limited neurological recovery and sustained impairment in those afflicted, while placing a burden on the public health system. The identification of neural stem cells that can produce new neurons has generated hope for their use as a therapy to treat such neurological conditions. Neurogenesis, the generation of new neurons from stem/progenitor cells, does persist in the adult brain, but is spatially restricted to two, small regions, leaving most of the brain, where therapeutic intervention is critically needed, without new neurons. However, NPCs have been isolated and cultured from widespread brain regions, despite their lack of differentiation in vivo. Furthermore, limited spontaneous migration of NPCs from germinal centers to sites of injury in some experimental models has been reported with limited and transient neuronal differentiation. The prevalent view is that most of the brain lacks a suitable environment, or niche, to support neurogenesis, suggesting the creation of a suitable niche may permit recruitment of local, endogenous quiescent NPCs. Thus one of the critical questions facing the field at this time is how neuronal differentiation can be directed and the resulting new neurons integrated to support brain repair outside of the small neurogenic centers. This study will investigate recruitment of local, quiescent endogenous NPCs in the entorhinal cortex by using gene delivery approaches to create a neurogenic niche where candidate cell autonomous factors can be expressed. The entorhinal cortex was chosen as a target, non- neurogenic region for its distance from migratory subventricular zone neuroblasts, its participation in the processing of spatial memory, and its relevance to the early development of Alzheimer's disease pathology. This study will address recruitment of endogenous NPCs with three specific aims. Aim One will characterize in vivo the properties of endogenous NPCs in regard to cell cycle frequency, self renewal, and multipotentiality using an innovative birthdating approach in combination with selective killing of proliferating cells and lineage analysis. To assess the recruitment of expanded NPCs to the desired cell lineage, Aim Two will use in vivo gene delivery to express proneuronal environmental factors to create a stem cell niche, while Aim Three will express transcription factors for neuronal differentiation in proliferating NPCs to assess the role of cell autonomous fate determinants. Neurological disease and injury are increased with advancing age, yet recent reports indicate that changes in the aging brain may limit its ability to support neurogenesis. This study will include both young and aged animals to address this important, clinically relevant factor.

0208311
Paterson

BAC libraries for key branches of the flowering plant family tree will greatly accelerate progress in unraveling the 200-million year history of angiosperm evolution. Extensive prior investments in the Poales and Brassicales make them important nucleation points for the monocots and dicots, respectively, from which to reach out across plant diversity to further investigate evolution of genome size, gene repertoire and function, and plant morphology. We will make 5 libraries totaling 423,936 BACs from carefully-selected angiosperm genomes (see below) to empower new investigations of well-delineated questions in developmental, evolutionary, and comparative biology. Among these, two (Ananas, Opuntia) will be the first BAC libraries known in their taxonomic orders, and two will be the first for important genome types (A, F) within a species complex (Gossypium) that is especially facile for asking fundamental questions about morphological transformations, polyploid evolution, and genome organization

These libraries will empower new investigations in monocots and dicots respectively, of general mechanisms associated with plant diversification, and the specific events that may account for major morphological-developmental transformations, by virtue of making possible important new comparisons to many taxa already well studied. These libraries will also offer opportunities to shed new light on the genomic consequences of polyploidy, and new insights into the developmental genetics of other scientifically important features that are inadequately studied at the molecular level. All of the taxa we will study are closely-related to economically-significant crops that exhibit features 'amplified' by human selection, and as such comprise botanical models in their own right for various aspects of plant growth and development.

This work will empower many scientific synergies to be realized by links with prior investments by the Arabidopsis and Rice Genome Initiatives, NSF Plant Genome Program (in the Poales and Malvales), USDA-IFAFS (in the Brassicales), and USDA-NRI genome programs (Malvales). Community involvement in decisions relating to this project has been substantial. Our research is closely-tied to strong training and outreach programs with a successful history of engaging undergraduates and groups under-represented in the sciences.

Binomial Common name Family 1C value (Mbp) # of BACs Genome coverage
Ananas comosus pineapple Bromeliaceae 531 55,296 10x
Gossypium herbaceum A-genome cotton Malvaceae 1785 92,160 5x
Gossypium longicalyx F-genome cotton Malvaceae 1592 92,160 6x
Gossypioides kirkii Malvaceae 579 73,728 12x
Opuntia cochellinifera prickly-pear cactus Cactaceae 869 110,592 10x
TOTAL 423,936

DESCRIPTION (provided by applicant): Stem cells hold great promise for treating many age-related disorders through cell replacement therapy, yet little is known about the ability of aged tissue to support critical events in the survival, spatial targeting, or differentiation of grafted stem cells or primitive progenitor cells. In the central nervous system, an age-related decline in neurogenesis in the dentate gyrus has been reported. We have recently found a massive decline in neurogenesis in the aged olfactory bulb, the other region supporting neurogenesis in the adult brain. Therefore, the environment of the aging brain may be impaired for supporting differentiation and integration of either endogenous or grafted stem cells. In this regard, comparison of aging brain with young brain may provide a useful model to discriminate between critical environmental factors regulating neurogenesis and supporting neuronal differentiation. Identifying such age-related deficits and restoring their expression in the aged brain would be necessary to extend the possible use of cell replacement strategies for treating age-related neurodegeneration, stroke, or cognitive decline. Alternatively, the environment of the aging brain may retain adequate neuronal differentiation signals in these neurogenic regions, but the endogenous stem cells may exhibit age-related impairment in their ability to differentiate. This project will test these two possibilities by 1) evaluating differences in expression of identified stem cell proliferation and differentiation signals between young and aged adult brain and 2) restoring expression of deficient signals by gene delivery to enhance neurogenesis in the aged brain. The subsequent grafting of rodent stem cells derived from embryonic, young adult and aged adult tissue sources will reveal the extent to which environmental modification of aged tissue can facilitate effective cell replacement therapy.

DESCRIPTION (provided by applicant): The continuation of neurogenesis in certain regions of the adult mammalian brain suggests that these regions may express signals to maintain continued proliferation of neural stem cells with subsequent differentiation of their progeny. Understanding these mechanisms may be useful for the development of therapeutic strategies for structural brain repair. However, neurogenesis declines in the aging hippocampus, suggesting that the signals maintaining neurogenesis may also decline with aging. We have recently determined that neurogenesis is also significantly reduced in the olfactory bulb with aging, despite previous reports that proliferation in the subventricular zone is unaffected by age. As proliferation also occurs in the rostral migratory stream, this project will quantify age-related changes in the rostral migratory stream by confocal stereology using our recently described thymidine analog markers to reveal the proliferative history of cells in subregions of the rostral migratory stream. Equivalent regions will also be sampled by laser microdissection for subsequent quantitative RT-PCR analysis of age-related changes in gene expression for candidate neurogenic signals. The use of stereology will reveal age-related changes in cell number in the regions analyzed for gene expression. This relationship will be used to normalize the gene expression data to actual cell number for greater biological relevance. Data resulting from these studies will advance our knowledge of neural stem cell regulation and may lead to restorative strategies for the age-impaired brain.

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

Support from the National Science Foundation Major Research Instrumentation Program has allowed Mississippi State University to purchase a Thermo Scientific LTQ Velos Orbitrap mass spectrometer for quantifying and describing proteins and their modifications. Being able to do such work is a fundamental component for understanding the functional complexity and emergent properties of life from genome sequences upwards. This equipment advances our "omic" biology research and education in 13 departments in five colleges and promotes greater trans-disciplinary research and teaching collaborations. It is used in many areas of bioscience in Mississippi including: bacteriology, cell physiology, computer science, bioenergy, forestry, immunology, informatics, neuroscience, parasitology, plant science, reproductive biology, toxicology and virology. Specific example projects include: genome-wide mapping of histone state and epigenetic regulatory networks in Arabidopsis to understand transcription control; enabling proteomics mass spectrometrists to derive more value from their mass spectral data by combining quantum mechanics computation with experimental- and computational-proteomics using new algorithms for peptide/protein identification; developing novel computational methods to improve signal processing sensitivity in biological mass spectrometry; understanding interactions between the foodborne pathogen Listeria monocytogenes and it's human and animal hosts; using computational systems biology and experimental validation to understand how complex interconnected functional networks of proteins, metabolites, nucleic acids and other molecules orchestrate cellular functions and responses to external and intrinsic signals. We continue our existing work to leverage methods used in biomedical organisms, like mouse, to solve problems in species that have important economic and environmental impacts. Training workshops will be conducted for teachers and students, and networking will foster collaborative research and teaching interactions between regional institutions. Project outcomes will be disseminated by student and faculty presentations at regional or national meetings, published in peer-reviewed journals, and made available via the AgBase Database (www.agbase.msstate.edu).

This proposal will fund the R&D needed for future lepton collider detector and in particular the International Large Detector (ILD) detector concept. The R&D projects will address critical issues related to the design of the ILD concept and associated beam instrumentation. The future lepton collider is expected to be a frontier scientific facility, exploring the interactions of electrons and positrons at energy scales of up to about 1 TeV. To fully exploit the physics opportunities of such an accelerator, it is essential to design a particle physics detector of unprecedented granularity, robustness and precision, and demonstrate that its feasibility. ILD is based around particle flow, a paradigm which aims for reconstruction of individual particles, using a highly robust tracking system centered on a time projection chamber supplemented with silicon tracking, and high granularity "camera-like" electro-magnetic and hadronic calorimetry suited to separation of individual energy deposits. Particle Flow calorimeters offer an exciting new approach to achieve significant better energy resolution needed by the new physics. If proven to work they could be useful for many future applications in High energy and Nuclear Physics experiments. This proposal will support the development and validation of Particle Flow Algorithms using test beam data. The detector projects address complementary aspects associated with advancing the design of the ILD detector with projects focused on tracking, calorimetry as well as beam instrumentation. This proposal will support the development of Low mass TPC endplates and luminosity monitoring system.

The proposal will have broader impact in that a number of the R&D projects utilize a significant numbers of undergraduate students who are highly integrated into the planned research. The university groups have a strong track record of encouraging the participation of under-represented groups in their research and this will continue to be emphasized. Many of the detector R&D projects have potential to lead to applications in areas such as medical imaging, and will lead to a cadre of scientists well trained in the art of experimentation and detector technologies.

Crocodylia (alligators, gharials, and crocodiles) is one of the major extant reptilian clades. Therefore,a basic grasp of the structure and function of crocodilian genomes is essential to developing a comprehensive understanding of genome evolution, structure, and function in amniotes. Furthermore, because the crocodilians are the closest living relatives of birds, their genomes represent the best group through which we can gain an outside perspective on avian genome evolution and function. To facilitate comparative genomics and the testing of a variety of hypotheses involving Crocodylia and other amniotes including birds and mammals, this research focuses on (1) investigating crocodilian genome diversity and evolution through detailed analysis of mobile elements (MEs), and (2) providing fundamental information about genome organization, gene synteny and regulatory sequences in three crocodilian species. These goals will be accomplished through a combination of molecular and computational tools, including BAC library construction and analysis, Cot library construction and analysis, and structure/function analyses of the resulting data via GBrowse and GO (Gene Ontology) annotation. The research will complement and enhance international efforts to understand the genetic underpinnings of genome structure and function by illuminating the impact of mobile elements on genome structure and function in a critical amniote lineage and by providing information on important developmental genes and conserved non-coding elements in the lineage.

Broader Impacts
The research will serve as a focal point for local high school teachers to experience current research in molecular biology and evolution through integration into a functional research laboratory. The teachers will then be helped in incorporating their new knowledge and experiences into their teaching for the benefit of their students. The research will also be part of ongoing collaborative partnerships between Mississippi State University and student researchers from local historically black colleges and universities. Finally, these efforts will inform general aspects of evolution and genomics and highlight crocodilians, organisms of broad public interest. The use of these charismatic megafauna will enhance the public's interest in evolution and genome biology, along with its understanding of both.

DESCRIPTION (provided by applicant): The goal of this STTR project is to develop a pre-formed, naturally-based hydrogel postoperative adhesion barrier with improved handling characteristics, laparoscopic deliverability, and consistent efficacy. Our technology is based on a novel, patented process that imparts exceptional elasticity and toughness on normally brittle, weak materials. Postoperative adhesions carry a profound public health burden. An annual $3.45 billion (US) is spent in hospitalization costs associated with adhesion-related complications. Despite tremendous efforts to resolve this unwanted scar formation there exists no consistently efficacious and safe solution. To meet the criteria of an ideal adhesion barrier and to overcome current anti-adhesion technology limitations, we propose a pre-formed barrier that exhibits exceptional handling properties and improved anti- adhesive effectiveness. Our membrane consists of hyaluronic acid (HA) and alginate, natural polysaccharides well established for wound healing and anti-adhesion. HA-based anti-adhesion barriers have been FDA-regulated for over 14 years. HA is metabolized following enzymatic degradation, and with non-toxic modification, degradation rate can be tuned. Alginate-based wound dressings have been FDA- regulated for over 20 years. Alginate is quickly hydrolyzed with subsequent renal clearance. Our films utilize a novel, patented processing technology developed in our lab that enables mechanical properties such as elasticity and improved toughness in otherwise weak materials. This simple processing method does not require specialized or expensive equipment, toxic components, and is easily scaled up. In Phase I, we will optimize our anti-adhesive membrane for handling properties and degradation tunability, and perform a pilot safety and efficacy study. The goal of these tests will be to a) understand the basic science supporting the mechanical behavior from pre-implanted membrane to fully-bioabsorbed, and b) ensure feasibility of safe and effective adhesion prevention. In Phase II, we will to prepare our technology for commercialization by addressing product development, regulatory, and other clinically relevant issues. These issues include shelf-life stability, in vivo degradation rate, tisue adherence timing, healing mechanism for prevention, localized anti-adhesive efficacy, and secondary indication of use. Finally, we will outsource specific biocompatibility assessment per FDA guidance and International Organization for Standardization (ISO) 10993 standards. At the end of the Phase II program we will have developed and manufactured a final product and will have accomplished all prerequisites to initiate first in human trials.

Project Summary Central neurons are highly specialized and long-lived cells that form precise circuitry to support normal brain function. The cerebral cortex does not add new neurons to maintain or increase function nor replace neurons lost to injury or disease. Adult hippocampal neurogenesis in the human brain shows that adult neurogenesis is possible. However, for human cerebral cortex, there are two possible strategies for neuronal addition, 1) replace with exogenous neurons generated by cell culture or 2) recruit local endogenous cells by converting them to neurons. The ultimate goal of this project is to investigate the second approach and to establish the capacity for neuronal reprogramming of human glial progenitor cells and to assess their potential for functional integration. For neuronal reprogramming to have therapeutic potential, it will be necessary to precisely engineer neurons with a predictable and sustainable rate of survival. Aim 1 will address issues of survival efficiency and subtype precision, developing an innovative vector toolbox to conduct these studies. But there also remains the question of how authentic these induced neurons become. The forced transcription factor expression may cause expression of certain neuronal phenotypes, including the capacity to fire an action potential, without necessarily resulting in functional maturation. While our preliminary data demonstrate the most advanced neuronal morphology reported to date, true circuit integration still remains to be shown. Aim 2 is designed to utilize current advances in connectivity tracing to investigate the state of integration of newly engineered neurons into local and distant circuits. In particular Aim 2b will ask if newly engineered neurons are capable of actively rewiring in the brain. We see application for this approach in rebuilding any damaged circuit, including ultimately those experiencing dysregulation such as in epilepsy or neuropathic pain. There is no information about the capacity to reprogram non-neuronal cells in the human brain into new neurons, despite the compelling need for such data to advance therapeutic application of this approach. As we cannot conduct these experiments in human brain, Aim 3 will develop a chimeric model, engrafting human cells into rodent brain to allow targeting of human glial progenitor cells for neuronal reprogramming and evaluating the extent of circuit integration with rat neurons. By using human iPSC-derived glial progenitor cells as our starting population, it will be possible to expand these studies to address the impact of disease-specific factors as well as providing a basis for eventual patient-specific therapies.