Mark S. Miller - US grants

Fetal tissues are more sensitive to the effects of chemical and physical carcinogens than are adult tissues. This suggests that the embryos and fetuses of pregnant women are at a greater risk of developing cancers from environmental exposures than is the adult population. It is thus important to gain further understanding of the mechanism(s) that help modulate the fetal organism's response to environmental toxicants, and to determine how the genetic background of the individual fetus can modulate its response to environmental carcinogens. The goal of this research will be to determine the molecular mechanisms governing the pathogenesis of lung tumors in mice differing in their susceptibility to polycyclic hydrocarbon-mediated tumor formation. Bioassays will determine the lung tumor yield following transplacental exposure to 3-methylcholanthrene in mice differing in their inducibility for CYPIA1, which will allow the tumor incidence to be correlated with the metabolic phenotype of the fetus. By analysis of oncogene RNA expression levels and examination of selected oncogenic loci for mutations by the highly sensitive polymerase chain reaction technique, particular patterns of oncogene over/under- expression and/or mutation can be established in the tumor models. It will be particularly interesting to determine whether differences in the metabolic capacity of the fetus toward 3-methylcholanthrene result in differences in the mutational spectrum at the molecular level, possibly as a result of damage occurring to different segments of the same gene or mutations occurring at different genetic loci . This project offers an unique opportunity to demonstrate the important role played by both genetic and environmental factors in determining the susceptibility of the individual to the induction of lung tumors. The data will show how the genetic make-up of the individual can influence susceptibility to cancer as a result of early in utero exposure to environmental chemicals.

During the preparation of meat by conventional cooking, highly carcinogenic heterocyclic amines are generated. These compounds require metabolic activation by the cytochrome P-4501A family of enzymes in order to exert their carcinogenic effects. Transplacental exposure to these heterocyclic amines may play an important role int he initiation of tumors, as studies by this and other laboratories have demonstrated the unique qualities of the fetus that render it particularly susceptible to tumor initiation by environmental carcinogens. The goal of this research will be to determine the transplacental carcinogenicity of a typical dietary heterocyclic amine, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). This study will assess the target organ specificity and the potential role of Cyp1a1 in the conversion of iQ to proximate carcinogenic metabolites following in utero exposure to the carcinogen. The pathogenesis of the induced tumors will also be examined at the molecular biological level. Bioassays will determine the tumor yield in various organs following transplacental exposure of mouse fetuses to IQ. The ability of IQ to increase the levels of Cyp1a1 will be assessed by biochemical and northern blot assays. Tissue generated from the bioassay will be embedded in paraffin and used to determine the types of genetic damage mediated by IQ at selected oncogenic loci implicated in human cancer, in particular the ki-ras and p53 genes. By analysis of these oncogenic loci for mutations by the highly sensitive polymerase chain reaction technique, particular patterns of oncogene mutation can be established in the tumor models. The induction of tumors following treatment during the transplacental period with IQ will show how chemicals contained in the mother's diet can lead to cancer initiation as a result of the in utero exposure to these dietary carcinogens.

Interactions between environmental and genetic factors have been implicated in the etiology of breast cancer. In particular, several studies have suggested that the type of genetic damage observed in human breast tumors may be influenced by exposure to chemical toxicants. However, few attempts have been made to compare the ability of breast tissue to metabolize chemical carcinogens with the types of mutations observed at critical oncogenic loci. To better understand the etiology of breast carcinogenesis and determine the role of gene/environmental interactions in determining individual susceptibility to breast cancer formation, a prospective case-case study design will be utilized to compare mutations in the p53 gene with the genotype of affected cancer patients for 4 metabolic enzymes (CYP1A1, GSTM, GSTT, and GSTP) that play key roles in the metabolism of human environmental carcinogens. We hypothesize that those breast cancer patients containing either specific alleles of CYP1A1 that enhance the metabolic activation of environmental toxicants or genotypes of GSTs that would result in less detoxification will be more likely to have accrued genetic damage at the p53 locus, and that combinations of alleles that increase the burden of reactive electrophiles will be more susceptible to tumor initiation. Tumor tissue samples will be analyzed for genetic alterations in p53 by SSCP and gene sequencing analyses. DNA obtained from blood will be genotyped by PCR-RFLP to determine if patients harboring genetic damage to p53 more frequently exhibit metabolic genotypes that increase formation of reactive electrophiles from invironmental toxicants. A prospective study design will allow use of a questionnaire to identify other potential factors (including smoking, diet, occupation, race, and reproductive history) that may modify the association between genotype and mutations to p53. As mutation at p53 has been implicated in poor patient prognosis, these studies should aid in identifying those patients at risk for damage to key regulatory genes that play a role in the pathogenesis of breast cancer, and will further our understanding of the etiology and risk factors for this disease.

DESCRIPTION (provided by applicant): Studies in humans and animal models have shown that mutation of Ki-ras is a critical, early event in lung tumorigenesis. We and others have shown that the type of mutation present in Ki-ras may influence tumor progression and appears to correlate with patient survival. We thus hypothesize that different mutations induced in Ki-ras by environmental carcinogens exhibit different oncogenic potential, and that the type of mutation initially induced in this gene will determine how rapidly tumors progress to adenocarcinomas. It is difficult to definitively determine the role of mutated ras genes in tumor initiation and progression because treatment with chemical carcinogens may cause alterations at other genetic loci. We thus propose to develop 3 strains of transgenic mice that contain either the wild type, VAL12, or CYS12 mutant alleles of the human Ki-ras gene linked to a tet-inducible promoter that specifies lung specific expression of the transgene. The biochemical characteristics and activity of the 3 alleles will be compared and correlated with the oncogenicity of each allele. Carcinogenicity bioassays will be employed to determine the effects of different ras mutations on tumor development and progression. The effect of each of the 3 alleles on subsequent damage to tumor suppressor loci implicated in both human and murine lung tumor pathogenesis, in particular the pl6Ink4a, Rb, and cyclin D1 genes, will be assessed by PCR-SSCP, methylation specific PCR, and determination of the levels of expression of the gene products by reverse transcription PCR. The development of these "humanized" Ki-ras transgenic mice will represent a critical advance in this field and provide an important new research tool that will allow us to determine the role of Ki-ras mutations in the pathogenesis of lung cancer. We anticipate that the proposed studies will elucidate the effect of different mutant ras alleles on lung tumor pathogenesis and will have important long term implications for future research on the etiology, genetics, prevention, and development of novel therapies for lung cancer.

This is a planning grant for department level reform at the University of Utah, Department of Electrical and Computer Engineering. In this project, the already strong laboratory component within the ECE program at the University of Utah will be enhanced by developing system-level design projects to be integrated within individual courses and also spanning multiple courses. This builds on a particular strength of the department, which already has a top-notch industrially-sponsored senior design sequence a few courses with strong system design projects. Through this change, it is expected to (1) increase student recruitment and retention (including diverse students who might otherwise not have chosen or stayed in engineering), (2) increase student motivation (system design projects are FUN), (3) increase knowledge acquisition and retention (because you can't forget something that you need to integrate at the end of the semester and perhaps next semester too), and (4) develop system-level design understanding (the formal training of which is very limited within ours and other traditional engineering curricula).

The novel aspects of this proposal include the projects themselves, the methods and concepts of integrating multiple classes within the curriculum, and the methods to assess and implement these projects. Undergraduate students, graduate students, and faculty will work together to build this new program within our department.

The objective of this research is the development and characterization of new silicon devices that should exhibit a variety of magnetic behaviors at will - even though consisting of non-magnetic silicon. Recent theoretical results demonstrated that on a finely-patterned two-dimensional lattice, electrons are confined to intersection sites; at higher concentrations, additional electrons also occupy interstitial sites. Many-body quantum theory predicts that at these differing concentrations, Ferromagnetic, Antiferromagnetic, and several Other Phases will arise, triggered by electron-electron interactions. It should be possible to "switch" between these thermodynamically stable phases merely by changing a control voltage. Such controllable "Spin Lattices" should prove useful in spintronic devices. The approach is to etch lattices into the gate electrodes of silicon metal-oxide-semiconductor transistors. Theoretical and numerical efforts will evaluate their novel electrical and magnetic properties. Importantly, the phenomena should scale up to room temperature as lattice features scale down below 10 nm.

A broader intellectual contribution of this project is the connection of the most basic quantum physics with advanced silicon microelectronics. Successful devices may play an important economic role by replacing transistors when further transistor miniaturization according to "Moore's Law" becomes impractical, potentially finding uses in spintronics, quantum computation, and other applications one can only conjecture at present. This project will train graduate and undergraduate students in scientific and engineering research, with undergraduate students preparing samples and investigating transistors properties for spin lattices. Graduate students will broadly disseminate this knowledge by creating an interactive exhibit in the new "Utah Museum of Science and Technology".

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DESCRIPTION (provided by applicant): Project Summary This application for a Mentored Research Scientist Development Award (K01) describes a training program and research project that provides the candidate with the necessary skills and laboratory-based techniques to conduct clinical research studies and to make the transition to an independent researcher. Candidate: The candidate's long term goal is to develop his own research program focusing on the structural and functional alterations in human skeletal muscle that result from aging, disuse and disease. The candidate is a Research Associate at the University of Vermont with a background in engineering. He has, for the first time, applied the novel technique of sinusoidal analysis to single human skeletal muscle fibers;thereby allowing the first examinations of the molecular determinants of contractile function in humans. Environment: The University of Vermont is ideally suited to the candidate's proposed research and training. It includes a cohesive group of clinicians and basic science researchers engaged in clinical research and muscle physiology that examines function at the whole body, whole muscle, single fiber and single molecule levels. Research: The objective of the proposed research study is to characterize the molecular mechanisms underlying age-related changes in single human skeletal muscle fiber function. We hypothesize that aging impairs single fiber function by: 1) altering myosin kinetics (increasing myosin attachment time and decreasing myosin rate of force production) and 2) decreasing myosin heavy chain content. To test these hypotheses, contractile performance and myofibrillar protein expression from single skeletal muscle fibers will be obtained from young (21-35 yrs old) and elderly (65-75 yrs old) volunteers. The proposed studies will represent the first comprehensive examination of the mechanisms underlying human skeletal muscle contractile dysfunction with aging at the molecular level. Relevance: Understanding age-related skeletal muscle contractile dysfunction at the level of the myosin- actin cross-bridge is a necessary step towards developing more effective pharmacological and lifestyle countermeasures to correct sarcopenia that are directed specifically at molecular defects.

PROJECT SUMMARY In general, men and women experience differing degrees of age-related decreases in physical function, with women having a greater prevalence of functional limitations and disability. A key predictor of this decrease in functional capacity is the reduction in leg muscle maximal power (product of force and velocity), which can be improved with exercise training. However, the development of exercise interventions to optimally improve skeletal muscle function in older adults has been difficult, in part because we now know that men and women respond differently to the same exercise training stimulus. In fact, the fundamental mechanisms by which habitual exercise improves physical function in older adults are still not well understood. The proposed studies, which build upon our recent work, are designed to address these knowledge gaps by examining the molecular and cellular mechanisms underlying the response to two distinct exercise training paradigms, and determining how these responses differ between older men and women. We hypothesize that molecular, cellular and whole muscle contractile performance will be most improved in men by traditional low-velocity, high-load resistance training, and in women by high-velocity, low-load power training. Moreover, sex-specific structural responses in myofilament remodeling, protein expression and post- translational modifications will explain these sex-specific performance adaptations to each modality. To test our hypotheses, data will be gathered from 50 healthy, sedentary older men and women (65-75 years) prior to and following a 16-week unilateral exercise training program in which one leg undergoes resistance training and the other power training. The Specific Aims of this project are to identify the sex-specific effects of low-velocity resistance training versus high-velocity power training on: Aim 1) skeletal muscle function at the molecular, cellular and whole muscle levels, and Aim 2) protein expression and modification as well as size at the molecular and cellular levels. Our within subject, unilateral intervention design provides a powerful model to minimize the effects of between-subject variability, and our translational approach will take advantage of our unique expertise with state-of-the-art measures from the molecular to whole body levels. Our results will challenge conventional wisdom by determining the sex-specific responses in intrinsic skeletal muscle adaptations to different exercise training programs. We will advance scientific knowledge by providing critically- needed information regarding the specific molecular and cellular determinants that support exercise-induced improvements in muscle performance. This knowledge will have a significant positive impact on the clinical care of older adults by providing novel insight about optimal exercise interventions to improve skeletal muscle function in each sex, and by identifying potential new therapeutic targets for pharmaceutical interventions. Thus, this project is highly relevant to the mission and vision of NIA to support biological research to mitigate conditions associated with aging that may limit health and independence in older adults.