Stephen Harris - US grants

DESCRIPTION (Adapted from the Applicant's Abstract): The bone morphogenetic proteins (BMPs) are involved in a variety of developmental and differentiation processes in skin and hair, heart, kidney, reproductive tissues, neural tissues and cartilage, bone and tooth development. In this application, the investigators' major goal is to characterize the mechanisms by which transcription of the BMP2 and BMP4 genes are regulated in osteoblasts and other tissues in vivo and to examine the mechanisms by which BMP2 affects osteoblast function. The stated goals are: 1. To extend prior studies on BMP effects on osteoblasts and chondroblasts and in particular to identify homeobox and other mRNA species which are regulated by BMP2 and which may regulate BMP2 and BMP4 expression during differentiation. 2. To characterize transcriptional regulation of the BMP2 and BMP4 genes in vivo, they will use transgenic mice in which BMP2 or BMP4 gene fragments from the 5' flanking region and other regions are linked to bacterial beta-galactosidase (LacZ) and/or luciferase (LUC). BMP2 and 4 gene transcription control will be examined. Using these mice, genetic crosses to candidate mutant mice can be established to evaluate gene networks and interactions with the BMP2 and BMP4 genes. 3. Using specific regions of the BMP4 promoter driving human BMP4 cDNA that directs expression in the normal BMP4 domains of 6.5-8.5 dpc embryos, the investigators will attempt to rescue the BMP4 homozygous null mutant phenotype. New phenotypes may develop that will give insight into the role of BMP4 later in development.

Osteoblasts are the precursors to osteocytes. A specific genetic program and gene expression profile underlies this differentiation process to a mature osteocyte, but the detailed components of that program are unknown. We have begun to define a subset of gene expression patterns that represent the state of the osteoblast as it progresses towards an osteocyte surrounded by osteoid that is actively mineralizing. Using osteoblast and osteocyte models composed of two cell lines, 2T3, an osteoblast-like cell, and MLO-Y4, an osteocyte-like cell, we have defined a subset of genes that are low in osteoblasts and become highly expressed in the osteocytes model. Additionally, a subset of genes in the osteocyte that respond to fluid flow have been identified Additionally, a subset of genes in the osteocyte that respond to fluid flow have been identified. One genes that encodes for Monocyte Chemoattractant Protein, MCP-3, is 13 TO 19 fold higher in osteocytes compared to osteoblast becomes an osteocyte and identify genes important for osteocyte function. The short- term goal is to determine the role of MCP-3 in osteocyte function. Our hypothesis is that MCP-3 is a marker for differentiation of osteoblasts into osteocytes and a critical molecule in recruitment of osteoclast precursors to bone. In this proposal we will address 1) the role of MCP-3 in osteocyte function and 2) the regulation of MCP-3 gene expression under mechanical strain both in vitro and in vivo. To accomplish these goals, the MPO-Y4 cell line, primary osteocytes, primary osteoblasts and the 2T3 cell line that forms mineralized nodules containing osteocyte-like cells in culture will be used. Increasing magnitude and frequency of fluid flow will be tested for effect on MCP-3 production, gene expression patterns and function in these models. Two rodent in vivo models of mechanical strain, the tooth movement model and ulnae-fatigue loading model will be used to access and validate gene expression changes found in the in vitro models. We will also use transgenic mice with a null allele of the MCP receptor, CCR2 mice will have defects in osteoclast precursor recruitment. Results from these studies will dramatically expand our database on genes important for osteocyte function and should also lead to insights into the mechanism of osteoclast recruitment to sties of bone microfracture.

Integrated computational- experimental program for ductility and failure in cast aluminum alloys
Somnath Ghosh, Bhaskar Majumdar and Steve Harris

Today's The automotive industry is faced with major challenge of improvedmustis faced with major challenges to improve performance to and reduce weight ratio in order to meet lofty fuel efficiency and emissions standards at low cost. Improved mAs designs become more complex and the power output requirements increase, the practical limits of their ductility and ultimate strength are being reached.,. which Scrapped parts and downtime can cost a manufacturer millions of dollars in terms of scrapped parts and downtime. Compounding The proposed Industry-University Collaborative GOALI research is aimed at addressing thisese issues for meeting goals of materials with high ductility, ultimate strength and strength at low cost. It will build a collaborative relation between the Ohio State University (PI), New Mexico Institute of Technology and Ford Research Laboratory (industry partner) to launch an integrated experimental-computational research program. The program will augment a major thrust area at FRLord called Virtual Aluminum Casting or VAC that is targeted to (a) reduce product development time (b), improve quality and performance, and reduce scrap and (c) improve performance and lower weight, and (d) reduce costs.
and cycle time.
The proposed program will develop a system of experimentally validated adaptive multiple scale computational models for predicting localization and ductile fracture of cast Al-Si components from microstructural information and process conditions. The models will simulate the evolution of microstructural features such as voids and secondary phases into incipient cracks and determine how the loss of ductilityductile failure depends on alloy properties and on the , distributions and interactions of different phases in the microstructure. The mechanics of particle fracture, interface decohesion, matrix rupture and damage percolation through the dendritic network will be studied. The role of porosity size and distribution on failure will also be investigated. Various dDevelopmental modules will include: (i) Quantitative metallography using SEM, and orientation imaging microscopy (OIM), and microstructural characterization to identify and characterize critical microstructure features that control important material response; (ii) Mechanical tests accompanied bywith in-situ SEM and fracture surface observations, computer imaging and microstructural characterization observation to generate strain fields and to provide understanding of critical mechanisms in the failure process; (iii) Neutron diffraction measurements and Raman microprobe techniques for microstress evolution and probabilistic strength estimation of particles; (iv) Development of an adaptive multi-level model for multiple scale analysis to predict the failure process as a phenomenon of multi-scale incidence and propagation of cracks; (v) Development of image-based microstructural Voronoi Cell finite element model for efficient and accurate analysis of plastic deformation, strain localization and damage evolution in nonuniform heterogeneous microstructures; and (vi) Incorporation of a probabilistic analysis framework to account for the effect of input variabilities on ductility and failure. The major intellectual merit of the proposed research is in its innovative blend of state state-of of-the the-art computational tools andwith experimental methods to advance provide a comprehensive analysis tool and design methodology for advanced metallic materialscast metals to increase their effective utilization. The uniqueness of this approach is in the broad attack on the problem: (a) iIntroduction of adaptive hierarchical and multi-scale computational models, incorporating image-based microstructural models to depict the percolation of damage at different length scales;. T (b) he iIncorporation of detailed microstructures at the critical regions of evolving damage and localization is possible through the efficient and accurate Voronoi Cell finite elementFE model, being developed by the PI.; and (c) Robust validation of the models through rigorous feedback from multi-scale experiments and material characterization by using in-situ SEM, orientation imaging microscopy (OIM), in-situ neutron diffraction and Raman microprobe. To the best knowledge of the investigators, there is a lack of such a necessary comprehensive approach to the understanding of response and failure characteristics of complex cast microstructures. The program, upon completion, is expected towill provide a good understanding of stress and strain evolution ofin the the complex phases in cast Al, their strength levels, and damage initiation and percolation through the network of brittle, ductile and porous phases.

The broader impact of the program will occur on two fronts. front, the It will reach beyond the automotive industry to aid the entire casting industry, where significant gains in alloying and solidification technology is are often stymied by unknowns regardingnot knowing how variability in material and process parameters affect damage tolerance and ductility. The methodology will allow industry be able to leapfrog thepresent technology and use these lightweight allows into in new safety -critical applications, armed with the knowledge that ductility and fracture can now be predicted with a reasonable degree of confidence. The second front will be oGraduate students will intern at FRL every summer and NMT students will have access to equipment at the national labs. As a consequence of the university-industry collaboration collaboration, students in this program will have a strong interaction with and mentorship from industrial researchers . There will also be student interaction with researchers at Sandia National Laboratory. researchers. In addition, the national laboratories will be involved in the experimental component of the work.

Development and validation of an adaptive, scale invariant materials model, based on microstructural
information that will efficiently account for the material and mechanical behaviors inherent in
composites, is proposed. Our approach involves: (a) Introduction of an adaptive hierarchical and multi-scale
computational model, incorporating image analysis to correlate computational predictions; (b)
Incorporation of architectural flexibility based on Unit Cell approach; (c) Utilization of Independent State
Variables that bridge the micro-to-macro scales; and (d) Account of interfacial mechanisms through
cohesive elements. The intellectual merit relies on the creation of an interdisciplinary program to tailor
computational and fundamental material science, that will ultimately provide the underpinnings for
understanding the mechanics of reinforced structures such as woven, bio, and nanocomposites. Broader
Impact: In the area of basic research and education the innovative and comprehensive approach taken by
this work will promote other research on composite materials aimed at developing a unified, scale
invariant model for FRPCs. (B) In the area of industrial research, this program will affect a wide variety
of industries where significant gains in microscopic characterization and macroscopic behavior is often
stymied by lack of knowledge of micro effects and ignorance of how to bridge the scales.

It is now known that small changes in bone adaption to mechanical load can lead to large changes in skeletal resistance to fracture. Osteocytes are believed to be the mechanosensory cells of bone receiving these physiological signals and responding in a manner to regulate their local microenvironment and to globally control bone formation and bone resorption in selective regions of bone. Dentin Matrix Protein 1, DMP1, and Matrix Extracellular Phosphoglycoprotein, MEPE, are highly expressed in osteocytes and respond to mechanical load. Both proteins are highly localized in the canaliculi and lacunae of osteocytes, with DMP1 found predominately on the canalicular walls. Our goal is to use these two genes as representative of osteocyte selective genes responsive to mechanical strain to identify molecular signalling mechanisms responsible for changes in bone properties. Our hypothesis is that specific osteocyte selective and mechanically responsive enhancer regions exist in the promoters of DMP1 and MEPE that are controlled by specific transcription family pathways in response to strain. To test this hypothesis three specific aims are proposed: Specific Aim 1. Determine the relationship between DMP1 and MEPE gene expression patterns with strain field analysis upon mechanical loading in vivo. Specific Aim 2. Determine the relationship of osteocyte deformation in the mouse ulna and femur to different levels of strain and gene activation of the DMP1 and MEPE cis-regulatory regions. Specific Aim 3. Determine the cis-regulatory regions of the DMP1 and MEPE genes that control the response to loading selectively in osteocytes. This project is unique in that DMP1 and MEPE gene expression will be correlated with macroscopic strain in vivo and with local cell deformation ex vivo. These genes and their appropriate cis-regulatory regions linked to reporters will serve as sensitive read-outs of osteocyte responsiveness in different loading conditions in different genetic backgrounds. This project will be devoted to understanding the cis-regulatory systems of both the DMP1 and MEPE genes in terms of their osteocyte selectivity and to identifying transcription factors responsible for this selectivity and responsiveness to mechanical loading. The goals of this project will be accomplished using cell models to identify molecular mechanisms, animal models for in vivo validation, together with engineering principles, combined with a molecular and a systems biology approach. Increased fatigue resistance is a major means to prevent fracture. Mapping osteocyte genes and pathways that are selectively responsive to load will provide information important to prevention or treatment of bone disease such as disuse osteoporosis, post menopausal osteoporosis and other pathological conditions of bone loss.

This project will develop an adaptive, scale invariant material model based on microstructural information that will efficiently account for and predict the material and mechanical behaviors inherent in Random Fiber polymer Composites (RaFC). The proposed framework uniquely incorporates representative volume elements (RVE) and independent state variables (ISV) The successful development and implementation of the proposed model depends on: (i) the fundamental theory of ISV, which provides a carrier and invariant algorithm to bridge the micro phenomena building up to macroscopic behaviors; (ii) a quantitative micro description of the geometry and the structure by a set of generalized RVEs, which compose unified geometric primitives used to construct and characterize RaFCs. These primitives also provide an ideal approach for systematically investigating the relationship between microstructure and damage mechanisms. The major intellectual merit lies on establishing an interdisciplinary program to create and tailor a state-of-the-art computational model that integrates material science and applied mechanics to provide a comprehensive analysis tool for RaFCs. This program will provide the underpinnings for understanding more fiber reinforced material systems such as biomaterials and carbon nanotube composites.
The societal impact lies in that the innovative and comprehensive approach taken by this work creates enabling technologies for better material characterization. Improved material modeling results in creative product design, improved efficiency, increased applicability and decreased environmental burden. Educational impact will be also significant since the students involved will intern at Ford Research Labs. Participation of undergraduate female and minority students will be actively recruited to perform experimental and modeling tasks.

DESCRIPTION (provided by applicant): Mutations in the human BMP4 and BMP2 genes have been associated with osteoporosis and these BMPs signal through a variety of BMP receptors. The role for BMP2 and BMP4 in bone biology is limited since deletions of BMP4 and BMP2 are embryonic lethals. Data from our lab has shown that conditional knock-out of BMP4 in matrix producing osteoblasts, using Cre/loxP, results in postnatal animals with an osteopenic phenotype. BMP4 cKO mice have decreased osteoblast activity and increased osteoclasts in older animals. We have recently shown that the Shh responsive gene, Gli2, directly regulates BMP2 expression and transcription in osteoblast precursors through Gli response elements. Our hypothesis is that BMP4 and BMP2 are required at early mesenchymal precursor cells to drive the precursors to a commitment osteoblast stage before expansion and differentiation to mature osteoblasts. Further elevated BMP levels and signaling through BMP receptor 1A, is then required to drive the development of the mature matrix/mineralizing osteoblasts and osteocytes. We will test this hypothesis by selectively removing BMP2 and BMP4 at two different osteoblast stages, the early preosteoblast stage and at later matrix producing osteoblast stage. We will then attempt to rescue some of these bone phenotypes by selectively activating one of the important BMP receptors in bone, BMPR1A. Specific Aim 1 will be directed at determining the specific and overlapping roles of BMP4 and BMP2 in osteoblast biology, using the Osterix-CreERt2 mouse model to temporally delete BMP4 and/or BMP2 in preosteoblast stage, specifically in animals after birth. Specific Aim 2 will be to determine the role of BMP2 and BMP4 in later stage osteoblasts, using the 3.2 Col1a1-CreERtm model to induce deletions after birth. Specific Aim 3 will be to determine the mechanism of action of BMP2 and BMP4 in vitro, using bone marrow mesenchymal precursor cultures in combination with BMP2 and BMP4 deletions in vitro with Adenovirus Cre. Growth, apoptosis, differentiation, and altered pathways will be determined, as well as gene expression patterns using microarray analysis. Specific Aim 4 will be directed at determining what aspects of the bone specific deletions of BMP2 and BMP4 are due to signaling through the BMP receptor 1A. This hypothesis will be tested using quantitative in situ hybridization, immunocytochemistry. Western analysis of altered signaling pathways will also be determined in a quantitative manner. b-catenin/TCF and BMP signaling reporter mice and lineage marker mice will be used to determine levels of these pathways and alterations in the BMP2 and BMP4 deletion mice. In vitro primary osteoblast cell cultures, before and after specific gene deletion will be used to determine candidate mechanism of action of the single BMP2 or BMP4 and the combined deletion of both BMP2 and BMP4.

The research objective of this grant is to fundamentally understand the thermodynamic driving forces and kinetic mechanisms leading to the formation of lithium metal dendrites in Li-batteries. One of the most significant challenges for Li-ion battery design is the prevention of Li-dendrite growth, which would allow faster charging for current Li-ion battery technology and the use of Li metal anodes for future "beyond Li-ion batteries." A computational model based on the phase-field method will be developed to predict the conditions for dendrite growth and morphological changes with input thermodynamic, mechanical and kinetic parameters from atomistic/first principles calculations and experimental measurements. The proposed model will be based on a nonlinear kinetics in which the dependence of the rate of changes of a phase-field parameter is nonlinear with respect to the thermodynamic driving force, and hence it is applicable to modeling the microstructure evolution under large overpotentials or high charging rates. One of the key parameters is the Li metal/electrolyte interface energy, which will be directly computed by connecting DFT calculations and liquid thermodynamic data. This three-year grant will lead to (1) fundamental understanding of the transport and chemical kinetics of dendrite formation and growth and their relationships to their solid electrolyte interphase (SEI) film properties and (2) the development of a physics-based microstructure evolution model that does not rely on non-transferable fitting parameters to predict the conditions for dendrite formation and growth morphology. The ultimate goal for this work is to eliminate the formation-- or at least to limit the growth-- of dendrites on Li metal electrodes.

Dendrite formation is the primary degradation and failure mechanism and a safety concern in Li batteries, either because dendrite pieces lose electrical contact with the rest of the Li electrode or because growing dendrites penetrate the separator and lead to short circuits. The fundamental understanding achieved from this research program is expected to contribute to the Li ion battery safety improvement, a critical need for the near-term development of hybrid and electric vehicles. The planned research, both the methodology and the actual results, are designed to make significant contributions to new battery technology by providing important fundamental information about electrode materials behavior under various electrochemical conditions. The direct involvement of GM scientists provides an important avenue for disseminating the knowledge generated from this project. The primary research results will be shared with the public on-line to the public at http://lithiumbatteryresearch.com/ in addition to peer-reviewed publication and conference proceedings. The graduate student and postdoc supported by this project will spend extended periods of time in an industrial environment, which will provide an important added dimension to their education. Both of these individuals will thus be very well positioned for future work in battery-related fields. In addition, undergraduate students will be integral to the program via Penn State's MURE (Minority Undergraduate Research Experience) programs and senior thesis projects in the Department of Materials Science and Engineering at Penn State.

? DESCRIPTION (provided by applicant): Fundamental knowledge of the periodontium is required to develop new approaches to understand and treat this major health problem. This project is directed at understanding the role and mechanism of the Bmp2 gene and Sost gene in periodontium function. Our recent published results indicate that the mouse Bmp2 gene in stem-progenitor cells in the periodontium is required for the development of the periodontium and formation of tooth root. Deletion of the Bmp2 gene in mesenchymal stem cells of bone marrow suggests that Bmp2 is a critical factor that activates these stem cells located on the small capillary vascular bed to differentiate toward mature osteoblasts and osteocytes. We now want to test the idea that the Bmp2 gene in stem cells of the periodontium, similar to mesenchymal cells in bone is critical for their lineage progression to the more complex situation of differentiation to cellular and acellular cementum and the tendon-like periodontal ligaments within the periodontium, using state of the art lineage tracing methods in vivo. In this proposal, the Bmp2 gene will be removed from two major classes of stem cells (aSmooth Muscle actin positive or aSMA+ and Osterix+ cells) of the periodontium and determine the role of the Bmp2 gene in specification and differentiation of the PDL and its role in attachment to the bone and teeth. Recently, the function of the Sost gene has been shown to be a negative regulator of cementum and alveolar-basal bone surrounding the teeth. In a model of periodontal degeneration, the Periostin KO deletion of the Sost gene greatly corrects many of the periodontal defects, especially in the alveolar bone, and increases differentiation of PDL progenitors to new alveolar bone. Treatment of mice with Sclerostin antibody (Scl-Ab), in clinical trials for treating osteoporosis, also greatly corrects these periodontal defects in the Periostin KO. We will now cross the Bmp2 cKO model with Sost KO and treat the Bmp2 cKO model with Scl Ab and determine the rescue of the periodontal defects in the Bmp2 cKO model. Using a periodontium stromal cell model, highly enriched in periodontal stem cells, we will delete the Bmp2 gene and determine the gene expression profiles during differentiation using RNA-seq methods and chromatin maps of key markers of promoters and enhancers, and DNase hypersensitive site maps. We will use RNA-seq methods to determine the kinetics of differentiation from stem cell to periodontal-like and mineralized structures. We will isolate primary periodontal cell, from 2 week animals that have no cellular cementum related but acellular cementoblast precursors, and PDL and osteoblast cells, 2 month animals undergoing active cellular cementogenesis, PDL formation and osteogenesis, and 6 month animals in which cementogenesis is complete. We will determine the expression profiles at these 3 stages and determine a set of genes selective for acellular cementoblast and cellular cementoblasts and cementocytes. By validation in vivo, we will determine a set of genes that is selectively expressed during cementogenesis and not osteogenesis. These experiments have high clinical relevance and fundamental basic periodontal biology.

How host-parasite interactions are maintained over ecological time and evolutionary time (i.e. many generations) is a significant biological question, with real-world applications in medicine, public health, and conservation. It is often overlooked, though, that a single host can contain a diverse community (i.e. infracommunity) of parasites.. The proposed research focuses on duck hosts and seeks to understand their parasite infracommunity structure and predictability. Ducks transmit several diseases of human importance, including Avian Influenza Virus (AIV) and Human Cercarial Dermatitis (HCD). As a group, ducks can be divided based on ecological traits (habitat selection, feeding behaviors) into two groupings, dabbling and diving ducks. Prior studies have suggested that dabbling ducks may support a higher rate of transmission of AIV and HCD. This work will characterize infracommunities of four dabbling and four diving duck species within the Eastern USA, to determine if infracommunities are specific to host species and/or ecological group. We will look deeply into the genetics of recovered parasite populations to determine if host species and/or ecological group help explain critical public health parameters, such has higher rates of transmission. Human-induced environmental change has resulted in significant changes to duck populations, such that some species are thriving in altered habitats, and others are in decline. There is thus an urgency to understand the ecology and evolution of duck parasite infracommunities to better model diseases such as AIV and HCD in a changing world. <br/><br/>Within an individual host, a community of symbionts (infracommunity) assembles in response to both ecological and evolutionary processes. Does the shared host environment act in a concerted way to shape the structure, assembly, and microevolution of infracommunities? The proposed research takes a community genetics approach to provide robust insights into the evolutionary processes within and across species of a shared host environment. This work will investigate the helminth (parasitic worms) and viral communities of eight duck species, which can be divided into two distinct ecological groups (dabbling vs. diving species) based on host-traits. The proposed research will use long-read Oxford Nanopore Sequencing to 1) characterize infracommunity structure across hosts and ecological groups and 2) compare population genetic structure and diversity of recovered core taxa (i.e. >70% prevalence). Merging community ecology and population genetics will help uncover the ecological determinants of infracommunity assembly, microevolution and ultimately provide insights into the evolution of the hologenome. Prior work with both helminths (Trematoda: Trichobilharzia) and Avian Influenza Virus (AIV) have shown higher prevalence, genetic diversity, and larger effective sizes are associated with dabblers, suggesting host-traits shape infracommunity assembly and within-host microevolutionary patterns. Understanding the predictability and taxonomic scalability of infracommunity assembly, and identifying what ecological factors support transmission, could improve our ability to model zoonotic waterborne diseases associated with waterfowl.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.