Lei Wei, Ph.D. - US grants

0216381
Wei

The phenomenal growth of wireless systems in thc last 15 years represents a major paradigm change for society. Today, wireless, personal, and mobile communications are essential for our modern economy and the general well being of the country. Wireless technologies have not only brought in USA billions of dollars in technology export, but also significantly increased the productivity of the whole nation.

The instrumentation the PIs are requesting in this proposal is a wireless communication systems emulator. In short, it can transmit and receive not only standard wireless signals and display them in detail, but it also allows them to deviate from these standards. It contains a universal wireless signal generator/transmitter, a universal wireless signal receiver, a high resolution network analyzer and a multi-input network analyzer.

Wireless communications has been a major research focus area in the School of Electrical Engineering and Computer Science (SEECS), UCF, over the past few years. A major mission of SEECS is to advance the future of wireless communications through education and research. The PIs involved in this effort have been conducting research in wireless communications for a number of years now. Some examples of the state-of-the art that they have been engaged are included below:

(a) Invented new decoding algorithms that are not only best known for block lengths from 100-250 bits, but also specifically geared towards practical implementation and compatibility with existing wireless systems. With the proposed emulator, they will be able to test these algorithms for applications in the real world.
(b) Introduced a new intelligent packet network architecture, called a cognitive packet network (CPN), in which the packets have intelligent capabilities for routing and flow control themselves. The PIs currently have a network test-bed, with fixed wire-line linkage, and their plan, with the help of the emulator, is to extend it to incorporate wireless linkages. With the proposed instrument, they can also increase the intelligence of the packets using the information gained from link-quality, channel state information, power levels. etc.
(c) Teleoperated robots have wide application from search-rescue operation to factory operation. The robotics and controls lab has several robots, real-time control systems, and other equipments. The proposed instrument will enable the PIs to extend their capability to mobile robots with advanced wireless communication capabilities.

The faculty members on this team have published several hundreds of papers in leading technical journals and conferences. In fact, all PIs and senior personnel have served or are serving as Editors / Associate Editors for prestigious international journals in the areas of their expertise. This state-of-the-art instrumentation will help them verify their research results, extend their capabilities, and justify their future research directions.

This equipment will also enhance their educational capabilities. The PIs currently offer a Wireless Communications option for their electrical engineering undergraduate students and have several graduate students working on projects in the field of wireless communications. This emulator will not only help demonstrate to the students the theory presented in the classroom, but also provide a hands-on platform for thesis and dissertation works.

DESCRIPTION (provided by applicant): The initiation of cardiac differentiation has been a topic of vigorous investigation, and many transcription factors have been described as regulators of the genesis of cardiomyocytes from mesodermal stem cells and the subsequent activation of genes responsible for cardiac contractility and morphogenesis. However, the mechanisms regulating transcriptional activity of cardiac transcription factors, especially at the post-translational level, are largely unknown in early cardiogenesis. Our preliminary results point to a role for Rho kinase in inhibiting cardiac cell differentiation and in regulating cardiac morphogenesis. We observed that both p160ROCK and ROKalpha, two members of the Rho kinase family, are expressed in early mouse embryos before the onset of cardiac differentiation, and p160ROCK is highly enriched in the developing heart. Treatment of early stage chick embryos with a specific pharmacological Rho kinase inhibitor (Y27632) induced precocious expression of cardiac alpha-actin (an early marker of cardiomyocyte differentiation), cardia bifida, an open neural tube and abnormal left-right asymmetry. In cell culture, we observed that Rho kinase phosphorylates SRF, a critical transcription factor in mesoderm specification and cardiac differentiation, and selectively inhibits SRF transcriptional activity on the cardiac alpha-actin promoter. It is thus important to investigate the role of Rho kinase in mammalian cardiac development by a direct genetic approach. We have successfully generated p160ROCK deficient mice for loss-of-function studies. The Specific Aims of this proposal are: 1) to determine the spatial-temporal regulation of Rho kinase expression and activity during cardiomyocyte differentiation; 2) to demonstrate the role of Rho kinase in mammalian cardiac development through Rho kinase knockout and conditional knockout mouse models; 3) to determine if Rho kinase selectively represses SRF-dependent cardiac gene expression in undifferentiated cardiac cells through direct phosphorylation of SRF. The proposed study will elucidate fundamental roles of Rho kinase in mammalian cardiogenesis and embryogenesis, and gain insight into the mechanisms by which Rho kinase regulates cardiomyocyte differentiation. Understanding the mechanisms of cardiomyocyte differentiation not only has fundamental importance for understanding heart development, but also has important implications for the possibility of cardiac repair through genetic manipulation of embryonic stem cells.

Core B is the Genetically Modified Mouse Resources Core. All three projects will make extensive use of genetically modified mice for their proposed experiments. Centralized localization of the animals, rational control and sharing of the breeding stocks, and centralization of genotype analyses will maximize efficiency and minimize costs associated with the production of the mice needed for the propose experiments. Specific activities of Core B include (1) Assistance in the generation of additional genetically modified mouse models, (2) Establishment and maintenance of breeding stocks, (3) Generation and genotype analysis of transgenic and/or knock-out mice, (4) Generation and collection of timed pregnancies, (5) Fostering, weaning and storage of the experimental animals, (6) Distribution of genetically modified mouse models, and (7) Delivery of experimental animals to the appropriate laboratory for use. Core personnel are already well-trained in all aspects of the services to be provided. Given the heavy reliance on genetically modified animals, the importance of Core B to the success of the Program Project Grant application cannot be overstated.

Heart Failure (HF) is a common event in childhood with significant morbidity and mortality. Current research indicates that cardiomyocyte apoptosis may contribute significantly to the development of HF. We have recently demonstrated that ROCK1 (Rho-associated, coiled-coil containing protein kinase 1) is a key mediator which links pro-apoptotic stimuli to apoptosis in neonatal cardiomyocytes. Our results suggest a model wherein low levels of activated caspase 3 directly cleave and activate ROCK1;activated ROCK1 in turn amplifies caspase 3 activation, resulting in a marked amplification of cardiac apoptosis. Importantly, this mechanistic relationship between ROCK1 activation and caspase 3 activation occurs in failing human hearts, suggesting that this pathway is a valid therapeutic target. The experiments proposed in Project 2 will further validate the importance of, as well as establish the mechanistic underpinnings of, ROCK1-mediated cardiomyocyte apoptosis. Specific Aim 1 will characterize the role of ROCK1 activation in cardiomyocyte apoptosis. Initial experiments will establish the importance of caspase 3-dependent ROCK1 activation on cardiomyocyte survival and HF progression following treatment with cardiotoxic drugs which induce childhood HF. Other studies will test the hypothesis that ROCK1 activation is sufficient to amplify caspase 3 activation and induce cardiomyocyte apoptosis in vivo. Experiments proposed in Specific Aim 2 will establish the molecular mechanism by which activated ROCK1 induces cardiomyocyte apoptosis. Initial studies will test the hypothesis that activated ROCK1 amplifies caspase 3 activation via post-mitochondrial regulation. Other studies will determine if TAT-based delivery of anti-apoptotic proteins can attenuate activated ROCK1-induced cardiomyocyteapoptosis in vitro, and if warranted, in vivo. Collectively, the experiments proposed in Project 2 will test the hypothesis that ROCK1-mediated amplification of caspase 3 activation plays a critical role in cardiomyocyte apoptosis, and furthermore will establish the role of ROCK1 signaling in response to acquired myocardial injuries which lead to childhood HF. This project will also determine if manipulation of ROCK1 signaling can be exploited to therapeutically inhibit cardiomyocyte apoptosis in a mouse model of acquired postnatal HF.

This award provides funding for a new collaborative Research Experiences for Teachers (RET) Site focused on Signal and Image Processing at the University of Central Florida and Florida Institute of Technology. Each year 13 high school teachers from school districts in four Central Florida counties will participate in research projects at the universities. The teachers will also develop modules related to their research which they will implement in their classes in the following school year. The teachers will organize annual science events to share their work with the communities around their schools and they will participate in the Florida Science Olympiad. Through participation in the RET Site the teachers will have an enhanced knowledge base in engineering and computer science and the skills to translate this into their classroom practices, thus impacting their students and motivating them towards science, technology, computing, and engineering disciplines.

The intellectual merit of this project revolves around the expertise of the research team and outstanding research environment in which the teachers will work. The research projects are compelling and are in areas that are of current interest.

The broader impacts of this project include substantial impact on the area schools and dissemination to a broad community. Teachers will incorporate new computing and engineering topics into their classes and develop hands-on ways to impart these topics to secondary students. The teachers will also engage in public outreach to convey the concepts and appreciation of computer science and engineering to the public. Through this project, a long-term relationship between the university and the schools will be forged and cemented. The partners will work together to build a foundation of outstanding computing and engineering education in the region.

DESCRIPTION (provided by applicant): Heart failure remains a leading cause of human morbidity and mortality. Rho kinase (also named ROCK) has recently emerged as a potential therapeutic target for the treatment of cardiac diseases with the overall promising studies showing beneficial effects of ROCK inhibitors in experimental and clinical studies. However, one important question needing to be addressed is whether ROCK truly represents a viable target for the treatment of human disease as currently available ROCK inhibitors have broad specificity. In addition, the two members of the ROCK family, ROCK1 and ROCK2, are inhibited by ROCK inhibitors with equal potency, and little is known about ROCK isoform functions in vivo. We recent discovered that systemic ROCK1 deficiency is protective against cardiac decompensation and the anti-apoptotic effect of ROCK1 deficiency is a critical contributor. In contrast to the beneficial effects of ROCK1 deletion, we observed that cardiac-specific ROCK2 deficiency results in spontaneous cardiac hypertrophy and dysfunction, suggesting a novel role for ROCK2 in cardiac protection. Our in vitro studies using ROCK1 or ROCK2 deficient embryo-derived fibroblasts support a novel mechanistic concept that ROCK1 preferentially mediates stress-induced acto-myosin contraction via the ROCK1/MYPT/MLC pathway leading to increased cell death, while ROCK2 preferentially contributes to actin polymerization via the ROCK2/LIMK/cofilin pathway leading to improved cell survival under stress conditions. The goal of this application is to dissect isoform functions of ROCK in hypertrophic cardiac remodeling and to test a novel central hypothesis that ROCK1 and ROCK2 are functionally different in regulating cardiomyocyte death and cardiac remodeling in response to cardiac stress. Specific Aim 1 will test the hypothesis that ROCK2 promotes cardiomyocyte survival and cardiac protection. The studies will further characterize the onset and progression of spontaneous cardiac hypertrophy in cardiac-specific ROCK2 knockout mice, and will determine if conditional ROCK2 deletion in cardiomyocytes accelerates heart failure progression. Specific Aim 2 will determine the ultimate role of ROCK1 in cardiac decompensation. The studies will determine if conditional ROCK1 deletion in cardiomyocytes can limit the progression of heart failure when cardiac hypertrophy or dilated cardiomyopathy has already occurred through chronic pressure overload. Specific Aim 3 will test the hypothesis that ROCK1 and ROCK2 play opposite roles in mediating stress-induced cardiomyocyte death and characterize the underlying mechanisms. Results of these studies will significantly advance our knowledge in ROCK isoform pathophysiology and inform clinical trials testing ROCK pan- inhibitors, and eventually isoform selective inhibitors, with the ultimate goal of developing therapeutic interventions to prevent cardiomyocyte death and reduce heart failure progression.

ABSTRACT Core C is the Mouse Resources Core. All three Projects will make extensive use of genetically modified mice for their proposed experiments. The purpose of Core C is to provide a centralized localization of the animals, sharing of common breeding stocks, and uniform performance of genotype analyses, which will maximize efficiency and minimize costs associated with the production and maintenance of the mice needed for the proposed experiments. Specific activities of the Core C include (1) assistance in the generation of new genetically modified mouse models; (2) establishment and maintenance of breeding stocks; (3) generation of timed pregnancies; (4) generation of postnatal experimental animals; (5) delivery of experimental animals to the appropriate laboratory for use; (6) distribution of genetically modified mouse models. Core C personnel are well-trained in all aspects of the services to be provided. Given the heavy reliance on genetically modified animals and the proven performance of the Core personnel, the importance of Core C to the success of the Program Project Grant application cannot be overstated. RELEVANCE The major benefit of this Mouse Resources Core to these three Projects is to facilitate development of new animal models and efficient usage of existing animal models of human disease thereby accelerating basic discovery. The benefit of this Core to society is to maximize efficiency and minimize the costs associated with usage of animal models of human disease.

1 Squamous cell skin cancer (SCC) is the second most common cancer in the US. There are methods available 2 to prevent SCC but are not appropriately used because we lack methods of evaluating their effectiveness in a 3 timely manner. Ultraviolet light (UV) from the sun induces genomic damage which is the most important cause 4 of skin cancer. Early in the process of cancer formation UV causes mutations in cells which result in small 5 clones, clusters of mutated cells. The early mutations that result in the growth of these clones are called 6 clonogenic mutations (CM). CMs are early changes during SCC formation, which appear decades before 7 clinically detectable cancer. Based on previous evidence CMs may signal skin cancer risk and evaluate the 8 efficacy of preventative treatment strategies and sun protection. CM are in low abundance in the skin which 9 make them challenging to detect. However, recent advances in genomic sequencing technology and 10 computational tools allow accurate identification and quantitation of CMs in the skin. Preliminary data has shown 11 that CMS can be accurately detected and used to evaluate sun damaged skin areas. Many of the CMs found in 12 normal sun exposed skin are also common in SCC. The central hypothesis for this application is that CMs are 13 biomarkers of sun induced skin damaged and that CMs can measure how well strategies for skin cancer 14 prevention and preventative treatment work. In the first set of studies we will refine the previously developed 15 panel of sun induced CMs by identifying the most common CMs in sun exposed versus non-sun exposed skin. 16 Subsequent studies will examine the impact of UV exposure on changes in the CM panel and development of 17 skin cancer. These studies will evaluate patterns of CMs and the risk of developing skin cancer. Next, the 18 refined panel of CMs will be used to examine how well treatments designed to prevent skin cancer in heavily 19 sun damaged skin areas reduce CMs and skin cancer formation. In the final set of studies, CMs will be used to 20 evaluate the efficacy of sun protection strategies, such as sunscreens. Sun protection factor (SPF) is widely 21 used to evaluate sunscreens. However, SPF measures reduction in redness of the skin instead of the actual 22 DNA damage. Genomic DNA damage contributes to skin cancer, not ?redness? in the skin. Genomic damage 23 can be caused by long term sun damage that does not cause a sunburn. In the final set of studies, CMs are used 24 to evaluate the effectiveness of sunscreens to protect against genomic damage and skin cancer. These studies 25 will change how we evaluate a patient?s risk of developing skin cancer and how we determine the effect of skin 26 cancer prevention. These studies have the potential to shift the focus from treating cancer to preventing the 27 occurrence of skin cancer. This would result in an improvement in cancer care outcomes, improve treatment 28 strategies and ultimately improve the life of individual with a history of sun damage and pre-cancerous lesions. 29 This work focuses on skin cancer but as CMs play a crucial first step in cancer growth in most human cancers 30 our findings and the framework of this study will have implications for the wider field of preventative oncology.

PROJECT ABSTRACT The prevention and treatment of heart disease remain challenging. Rho kinase (also named ROCK) has recently emerged as a potential therapeutic target for various cardiovascular diseases. A long-term goal of our past twenty years of research on ROCK pathophysiology is to define the roles and underlying mechanisms of ROCK-mediated signal pathway in regulating cardiac remodeling. The two members of the ROCK family, ROCK1 and ROCK2, have both shared and distinct cellular functions and can compensate each other in numerous single isoform knockout conditions. The majority of our knowledge on the cellular and molecular function of ROCKs comes from research on proliferative cell types in which ROCKs modulate actin cytoskeleton organization through promoting actomyocin contraction and F-actin stabilization. Cardiomyocytes stand apart from other cell types because they contain both sarcomeric and non-sarcomeric cytoskeleton. There is a gap in our knowledge on how ROCKs regulate sarcomeric and non-sarcomeric F-actin in cardiomyocytes and how these processes contribute to overall heart function. Recently, for the first time to use inducible approach to delete both ROCK isoforms in cardiomyocytes, we have discovered that although ROCKs are not required for maintaining sarcomeric cytoskeleton in adult hearts, they do participate in the regulation of non-sarcomeric F-actin organization, inhibit autophagy by promoting mammalian target of rapamycin (mTOR) activity and contribute to age-related cardiac fibrosis. In contrast, the non-sarcomeric F- actin dynamics are able to be maintained with the presence of either isoform in the cardiomyocytes where the other isoform has been deleted; this might be attributed to compensatory over-activation of the remaining isoform in cardiomyocytes having single ROCK isoform deletion. The proposed research aims to further elucidate the pathophysiological roles and downstream pathways of ROCK-mediated actin cytoskeleton changes in cardiomyocytes and fibroblasts under pathological stress sceneries. Aim 1 will determine if deletion of both ROCK1 and ROCK2 from adult cardiomyocytes limits the progression of heart failure in pathological hypertrophy and myocardial ischemic injury through activating autophagy and facilitating autophagic flux by inhibiting mTOR signaling. Aim 2 will determine if deletion of both ROCK1 and ROCK2 from adult fibroblasts limits the activation of myofibroblasts and fibrotic response through inhibition of F-actin regulated transcription factor activation including the serum response factor (SRF) and myocardin-related transcription factors (MRTFs); the direct contribution of ROCKs/F-actin/MRTFs/SRF axis in fibroblasts to cardiac fibrosis has never been demonstrated in vivo, and our preliminary results indicate that the inducible approach is required for double ROCK knockout in fibroblasts. The biomedical significance of this work is to provide the cutting-edge concepts for understanding pathophysiological roles of ROCKs in heart failure. The ultimate goal is to develop new therapeutic intervention to ameliorate compromised cardiac function.