Jorge Piedrahita

As stated in PAR-17-341, the goal of this NIGMS initiative is to .���������������encourage changes in biomedical graduate training to keep pace with the rapid evolution of the research enterprise that is increasingly complex, interdisciplinary, and collaborative������������������������������. The objective of this Comparative Molecular Medicine Training Program (CMMTP) is to provide a diverse pool of graduate students with rigorous training in biomedical research with special emphasis in team science. Support is requested for three/six students, to be supported by the T32 for a two-year period. The program emphasizes team interdisciplinary research training and provides extensive hands-on experience in challenging research projects focused on comparative molecular medicine, and extensive professional development designed to prepare trainees for a successful career in the biomedical sciences. To achieve our objectives, we have incorporated a set of novel components including: 1) Two Team Science courses, the first led by an Associate Professor with a PhD degree in Communications, focused on developing the communication skills required to successfully participate in team-based collaborative interdisciplinary research, and the second led by the Senior Associate Director for Operations and Academic Programs, Shelton Leadership Center, focused on team leadership skills related to biomedical sciences. 2) A team science mentoring program, the Young Scholar Program (YSP), which provides an opportunity for CMMTP trainees to apply and further refine competencies in project management, mentoring, and effective group communication, including teaching in an undergraduate course in Team Sciences in the Biomedical Sciences. 3) The requirement for the development of a collaborative aim (a coaim) in the trainee������������������s thesis proposal. 4) A graduate level minor in ����������������Team Leadership and Communication in the Biomedical Sciences". A minor that will help prepare the future leaders in interdisciplinary biomedical research. 5) Close incorporation of science of education expertise within the proposal not only to assess the program, but more importantly, to develop new educational approaches to train PhD students to carry out complex interdisciplinary research in the biomedical sciences. This includes an education/communication PhD student as part of the institutional commitment. This PhD student will use the CMMTP program as the basis for their research on improving education methods in team science. They will ensure that each of our training activities is assessed using evidence-based approaches and that this information is used to further improve methods for team interdisciplinary research training. Other components include: 1) Basing the training grant in the Comparative Medicine Institute (CMI). The CMI������������������s well established cross-departmental organizational infrastructure facilitates the management and implementation of this training grant, and 2)Extensive professional development activities that include career and academic advising, seminar series, preparation to present orally and in poster format, workshops on scientific communication, among others.

LGR5, a marker of adult and fetal stem cells in various tissues and organs, has been studied extensively for the past decades. Cells expressing LGR5 play key roles in organ/tissue development, homeostasis, regeneration, and disease, including cancer. Therefore, having the ability to identify, track, and manipulate these cells in vitro and in vivo, will allow their detailed role in normal and pathological organ function. To date, most of the research on LGR5 cells has been performed using mice, which have significant anatomical, physiological, and molecular differences from the human. Utilizing a newly developed LGR5-H2B-GFP pig line we have demonstrated its wide utility for studying the role of these unique cells and have now made seminal observations in a variety of tissues/organs. However, we have also been unable to address some key aspects due to the inability of the existing model to analyze the differentiated progeny (lineage tracking) of LGR5 cells during development, after transplantation, or during the process of injury and repair. In addition, while we have been able to inactivate both alleles of the LGR5 gene, we do not have the ability to delete the cell itself. Being able to delete the LGR5 expressing cell will increase our understanding of the importance of this unique cell (versus the LGR5 gene itself) in the phenotypes of interest. Thus, to overcome these two deficiencies, and further increase the value and impact of the original LGR5-H2B-GFP animal model, we propose to develop a highly improved LGR5 pig line that will allow labeling of the LGR5 cell, tracking of its progeny after injury, and delete it when desired. The high concordance between human and pig results to date with respect to LGR5 expression and function, not only increases the value of this line for basic research, but also for rapidly translating clinical findings to humans, further increasing the impact of this unique animal model. Successful completion of the proposed aims will generate a powerful animal model for study of translational aspects of LGR5 stem cells of the gut, lung, skin, liver, cochlea, and kidney among others.

The overall goal of this project is to design, construct and outfit a swine biomedical research facility on the campus of the fourth ranked College of Veterinary Medicine in the United States with ready access to trained veterinary specialists and state of the art biomedical (e.g. MRI, CT and nuclear medicine) facilities. The facility will provide additional high-quality space for biomedical research by NIH funded faculty from NC State University, Duke University and the University of North Carolina. The design and construction of the swine biomedical research facility will feature a free-standing masonry and steel building that will house the production and care of gnotobiotic and gene-edited swine, as well as state-of-the-art procedures (surgical, telemetry, arthroscopy, endoscopy). In addition, the unit includes flexible space that can accommodate pregnant and non-pregnant sows, and farrowing facilities to generate needed gene edited progeny from our own lines as well as those obtained from the NIH-supported NSRRC. Procedural space will provide a sterile surgery suite (two tables) to accommodate an increasing bioengineering need for endoscopic and arthroscopic procedures. The building will be placed immediately adjacent to space (referred to as the G20 facility) previously created for the use of severe combined immunodeficiency and other gene edited miniature or juvenile pigs (G20 OD020279) to allow for shared use of the space when possible. The proposed facility has been designed to maximize synergy and minimize overlap with the G20 space. Combined they will give us a high degree of flexibility and will allow us to conduct a broad range of research thus having a broad impact across multiple NIH Centers/Institutes. This project team is uniquely situated to drive the design and development of this facility and the expansion of this program. By serving in leadership roles within the College of Veterinary Medicine and NC State University we have the ability to provide access to the veterinary college biomedical campus, the research animal facilities and the state-of-the-art equipment in the tertiary care veterinary hospital. Our team of investigators has comparative medicine expertise and an extensive collaborative network with biomedical researchers at Duke and the University of North Carolina. As a team, we have successfully managed infrastructure grants, such as the expansion of facilities for housing and studying transgenic and non-transgenic miniature pigs (G20 OD020279) and building projects including a 20,000 square foot wet lab and GMP lab space.

Although an invaluable workhorse for research and training, the current 3D bioprinters available at NC State, such as the 3D Bioplotter (EnvisionTec), BioAssemblyBot (Advanced Solutions), BioX (Cellink), and Allevi 3 (3D Systems) are primarily based on the extrusion printing mechanisms. These systems are well suited for macro-geometric structures, but their micro-scale and cellular level control and precision are limited. Furthermore, these systems lack in-process monitoring abilities. This severely impedes fundamental research about cellular-level functional interactions in bioprinting and the potential to develop new manufacturing strategies and applications that can benefit from single cell-level control across layers of bulk constructs. The proposed multi-modal, high-resolution Next-Generation Bioprinter-Research (NGB-R) system [1] will address this gap and make a huge impact on on-going and future research and training at NC State. The comprehensive standalone BSL-2 system is equipped with micro-extrusion and inkjet bioprinting modalities along with the one-of-its-kind laser induced forward transfer (LIFT) mechanism. The uniqueness of the NGB-R system is further enhanced by the embedded microscope driven by machine learning algorithms, which enables high-throughput, in-line, real-time quality monitoring of bioprinted constructs.

In the US one out of eight people suffers from hearing loss. The common causes for hearing loss are age, frequent exposure to loud noise, genetic disorders and more. Hearing loss is often accompanied with other medical conditions such as a higher rate of depression, social isolation, and cognitive decline (e.g., dementia). Given that hearing loss is a non-lethal condition, most of our understanding about hearing loss in humans is provided by either postmortem examination, or animal models. Rodent models are commonly used to study normal and impaired hearing, and they vastly expanded our understanding of how sound is perceived at a molecular level. However, the rodent inner ear physiology, genetics and anatomy is different from the human, therefore, in some situations, this fact limits our understanding how mutations in deafness genes affect hearing. Moreover, the small size of the mice creates difficulties to assess and evaluate the translational value of emerging technologies to deliver and restore hearing via regeneration of hair cells or spiral ganglion neurons. To address these gaps, we would like to advocate for the use of the pig as a translational animal model to study the inner ear. Pigs are large animal models, and their size, anatomy, intelligence, and genetics is by far closer to humans than rodents. Consequently, the pig is becoming a popular animal model and it is commonly used in cardiovascular research, wound healing, organ transplantation, nutritional studies and more. In general, pigs are readily available for research as they are a popular form of livestock, and only in the US, over 100 million pigs are slaughtered annually. Here, to establish the pig as a large animal model, we will: (i) Characterize and compare the gene expression pattern of major deafness genes to those of mice and marmosets. (ii) Establish an image guided method to deliver therapeutics (e.g., viruses) to the middle and inner ear. (iii) Quantify the baseline expression of cochlear progenitors and hair cells across ages. Technically, we will utilize tissue clearing and labeling techniques together with advanced microscopy to image the whole porcine inner ear with subcellular resolution. This methodology facilitates the detection of subtle biochemical and morphological changes in the tissue. Overall, the success of these goals will open future avenues for testing new regenerative medicine approaches for hearing restoration, characterizing cellular and physiological changes during development, and studying hearing loss progression driven by genetic mutations that do not produce a deafness phenotype in mice.

We are proposing an innovative undergraduate research program at the Chemistry:Life Science interface across NC State, building on the success of a unique team-based undergraduate research program being developed in the Comparative Medicine Institute, referred to as the SIRI program (Summer Interdisciplinary Research Initiative). This program has already led to a NIH T34 grant in an orthogonal translational research area, and is primed to serve as the foundation for a Beckman Scholars Program. Although building on this successful framework, this program will be distinct and tailored for this call. NC State������������������s Beckman Scholars Program will set itself apart nationally by leveraging team science (faculty co mentors), a mentoring of mentors program, and communication and leadership programs to train students across traditional barriers while having them participate in innovative research projects at the Chemistry:Life Science interface. The focus on faculty co-mentors, engaged graduate/postdoc mentors and cutting edge, vetted research projects positions us well to compete successfully to bring the Beckman Scholars Program to NC State.

NC State is the largest university in North Carolina, with nearly 34,000 students, including 8,500 graduate students. Approximately 56% of all students are men, and 44% are women; approximately 16% are members of underrepresented groups. Yet, the number of programs focusing on preparing highly qualified UR students for PhD or MD/PhD is low (1). There is, therefore, a strong need for this program at NC State. In addition, team or interdisciplinary science has become an integral component of biomedical research. Yet most training programs still rely on the traditional single discipline training. This U-STAR program combines the strengths of a university-wide biomedical institute, the Comparative Medicine Institute (CMI), and the University Honors Program at NC State in order to train the next generation of UR biomedical scientists in a rigorous inter/trans disciplinary environment. Major program components include: 1) A pre-U-STAR feeder program designed to introduce students to the program and to the unique skills required to carry team science; 2) A ����������������Mentor-the-Mentor��������������� program for Graduate students, Postdoctoral Fellows and Junior Faculty mentoring U-STAR Scholars; 3) A U-STAR program that provides rigorous hands-on research mentoring in inter/transdisciplinary sciences; and 4) Professional development activities that include career and academic advising, seminar series, preparation to present orally and in poster format, workshops on scientific communication, introduction to existing T32 programs at NC State and at our partner institution, UNC Chapel Hill, and assistance with application to graduate school. Our overall goal is to enroll 10 new U-STAR scholars each year, 70% of which will matriculate in a biomedical sciences PhD or an MD/PhD program.

Recent data indicates that the fastest rising rates of anterior cruciate ligament (ACL) injuries of the knee are reported in children and adolescents with significant growth remaining. In the skeletally immature patient population, surgical reconstruction is increasingly suggested for complete ACL tears. However, the choice of non-surgical treatment or immediate surgical reconstruction of ACL tears remains a subject of debate in young patients with significant growth remaining or in the case of partial tears involving one ACL bundle. Sex appears to be a major risk factor for ACL injury during adolescence, but not in childhood, adding another layer of complexity. For both complete and partial ACL injuries, treatment algorithms have been developed without considering the potential sex- and age-dependent function of the ACL, due to the paucity of available data. Thus, the objective of this proposal is to determine how age and sex impact ACL maturation and joint function during skeletal growth and to assess if this knowledge can be applied to improve treatment after ACL injury. Aim 1 will determine how sex impacts the maturation of the ACL as well as that of its individual AM and PL bundles during skeletal growth. Aim 2 will determine how age and sex impact the immediate loading of secondary tissues in-vitro and the remodeling response of the joint in-vivo following loss of function of the AM bundle, PL bundle, or the entire ACL. Aim 3 will determine if a replacement graft positioned to more accurately reflect age- and sex-specific ACL function improves graft viability and function and reduces the risk of secondary injury and long-term degenerative changes. Successful completion of these aims will provide a basic science foundation for the development of age- and sex-specific algorithms for the treatment of ACL injuries.

In the United States, colorectal cancer (CRC) is the 3rd leading cause of cancer death among women and men combined, with of 1 in 20 people being diagnosed with CRC each year. Yet, understanding factors controlling disease progression and metastasis has been hampered by the lack of a suitable animal model of CRC. At present, there is considerable information regarding candidate genes that are mutated in CRC and contribute to tumor locations, tumor phenotype and patient response to treatment1. While candidate genes can be studied in mice by use of transgenic lines carrying gain-of-function and loss-of function genetic modifications, mutations in candidate genes such as Apc, result in lesions in mice that differ from those seem in humans. As a result, modifications of the traditional transgenic model have been required to advance our understanding of CRC. In contrast, an analogous APC-/- pig model demonstrates predominantly colon tumors, which are remarkably similar to the human disease. We propose to combine the strengths of an existing gene edited LGR5-H2B-GFP pig line with the power of a Cre-inducible Cas9 system to generate a novel line that will allow a careful dissection both in vivo and in vitro of the molecular pathways associated with the behavior of LGR5 cells in colon cancer. This proposal will allow for the generation and the initial in vitro and in vivo validation of the model. This highly relevant large animal model will greatly improve our understanding of how LGR5 cells behavior is controlled at the molecular level during oncogenic transformation and will be of great use to develop novel strategies to treat human colorectal cancer, as well as other cancers.

The National Research Council has documented a dire national need for veterinary specialists trained in biomedical research. Furthermore, veterinary researchers play a key role in comparative and translational research activities since they naturally bridge basic and clinical research. To address this training need, we request continued NIH funding for 7 fellows per year for 3 years. NC State University will provide funding for up to 2 fellows per year and 2 pre-T32 positions. Trainees will be degree-seeking fellows in the Comparative Medicine and Translational Research training program established by the faculty in the College of Veterinary Medicine (CVM) and the Comparative Medicine Institute (CMI) at North Carolina State University. This training program specifically targets individuals with the DVM degree who have completed specialty training and is designed to prepare trainees to compete for an early career development award and a rapid transition to independence as a principal investigator or in another research-intensive career. Trainees complete requirements leading to the PhD degree in Comparative Biomedical Sciences (CBS) in one of 7 areas of oncentration: 1) Immunology 2) CellBiology, 3) Pharmacology, 4) Neurosciences, 5) Infectious Diseases, 6) Population Medicine, and 7) Pathology. Training faculty are well-funded productive scientists that have a strong training track-record and diverse research expertise. Training faculty are all members of the CBS graduate program and the CMI and represent 4 departments from 2 Colleges. Research projects emphasize comparative and translational themes fostered by the CVM and CMI in tissue engineering, pharmacology and physiology, genetics and genomics, and emerging infectious diseases. Program requirements include: (1) a capstone comparative medicine and translational research seminar course; (2) professional development courses and workshops; (3) a course in research ethics; (4) a grant writing course and a pilot grant program that provide a pathway to a K award; and (5) annual research symposia. These requirements are in addition to those associated with the graduate program. Sixteen fellows have completed training. Thirteen hold faculty positions in academia, one is a research pathologist, and two are research fellows at other institutions. Fellows were awarded 7 NIH or other career development grants and 15 extramural research grants and published 67 papers (48 first author) arising from their research while in training.