Phyllis Robinson - US grants

The molecular details of phototransduction have been intensively studied over the last ten years and many features of the processes underlying activation have been well characterized. In contrast, the details of the mechanism underlying inactivation and light-activation are not as well understood . The broad aim of the research outlined in this proposal is to elucidate the molecular mechanism of photopigment deactivation by determining the precise role of rhodopsin phosphorylation in this process and identifying the binding sites on the rhodopsin molecule for both rhodopsin kinase and arrestin. The experimental approach will be to use site-directed mutagenesis of rhodopsin in conjunction with an in vitro reconstitution assay system. There are three specific aims of this proposal. First, to identify the amino acid residues whose phosphorylations are essential for deactivation. Second, to determine the amino acid residues that affect the binding of arrestin and rhodopsin kinase to rhodopsin and to determine if these residues also alter the binding of transducin. Third, to determine if constitutively active rhodopsin mutants [opsin molecules that in vitro activate transducin in the absence of chromophore and the absence of light] are inactivated by the same mechanisms as wild type rhodopsin. The experiments described in this grant are significant for they are aimed at understanding the molecular details that underlie photopigment deactivation. In addition, it appears that some forms of retinal degeneration maybe caused by persistent activation of the visual transduction cascade. Therefore, insights into the molecular details of rhodopsin deactivation will certainly lead to an understanding of pathologies that result in constitutive activity and retinal degeneration.

The scientific exploration of vision and particularly color vision has its historical roots as far back as the eighteenth century, when scientists such as Dalton and Young attempted to explain color vision and color blindness. Color vision allows an animal to make distinctions based on chromatic information rather than luminosity. Color vision helps an animal identify important objects in their environment The study color vision in aquatic mammals provides an unique opportunity to examine the adaptation of the mammalian visual system to the unique properties of an aquatic visual envirom-nent. We have recently demonstrated that the bottlenose dolphin (Tursiops truncatus) lacks the common cone-based dichromatic form of color vision typical of most terrestrial mammals. This is an exciting finding, and it is the beginning of a comprehensive molecular study of the visual pigments of aquatic mammals for two different mammalian orders and one suborder (whales, seals and manatees) have independently adapted to life in an aquatic environment. This research project will determine if all aquatic mammals: Cetacea, Pinnipedia and Sirenia have a visual system similar to that of the dolphin and express only two spectrally distinct classes of visual pigments: one rhodopsin and one cone pigment and hence lack typical mammalian dichromatic color vision. This comparative study of color vision in aquatic mammals will examine the adaptive processes that occurred as these distinct orders and suborder of mammals with different terrestrial ancestors moved from a land to an aquatic environment.
This research project will allow my laboratory to continue to successfully train both women and minority science students. Of the ten minority and women undergraduates who have worked in my laboratory, two are enrolled in MD/Ph.D. programs, five are currently in graduate school and two are in medical school.

The goal of this project is to contribute to the development of a national science and engineering academic workforce that includes the full participation of women at all levels of faculty and academic leadership, particularly at the senior academic ranks, through the transformation of institutional practices, policies, climate, and culture.

The University of Maryland, Baltimore County (UMBC) proposes to implement a five-year program to remove barriers, transform the culture of the university, and expand opportunities for women. This transformation will increase the representation and advancement of women, and in particular underrepresented women, in academic STEM careers, thereby contributing to the development of a more diverse science and engineering workforce. Through changes in policies and practices that affect the recruitment, selection, promotion, and transition of STEM women faculty to leadership positions, UMBC seeks to ensure women will be represented in numbers equal to or higher than parity with the pools from which they are selected. Within five years, all policies, procedures, partnerships, and processes will be in place and in motion to ensure this goal is achieved within ten years.
Objective 1: With a focus on all STEM departments, UMBC will work collaboratively to develop and institutionalize new policies, practices, and resources that will effectively encourage the recruitment, selection, and hiring of women, and particularly minority women, to the faculty at all ranks.
UMBC will identify and develop networking opportunities from which to recruit women graduate and post-doctoral students to the campus, create a new program called Faculty Horizons, modeled on UMBC's successful Graduate Horizons to recruit female faculty to the campus, and define challenging yet attainable goals for each department to recruit, select, and hire a diverse group of women faculty.
Objective 2: Establish a system of targeted programs to create a clear and understandable pathway in support of women's efforts in science, technology, engineering, and mathematics to achieve tenure and promotion, and transition to leadership positions at the university.
Planned programs include a formalized mentoring program, structured leadership experiences, a new faculty track for lecturers who have demonstrated excellence in applied education projects, and time for faculty to balance family and medical needs with preparation for promotion, tenure, and advancement without penalty to their careers.
Objective 3: Develop programs for constituency building, such that all levels of administration and each department support all aspects of the project and create new partnerships to connect UMBC to the broader community of research universities leading in the development and advancement of STEM women faculty.
Over five years, UMBC will create and institutionalize the complex system of support needed for sustainability, from the grass roots through the top administration. UMBC will identify or hire an individual who will report directly to the president, work collaboratively with STEM faculty, and provide the leadership needed to set the course and maintain it. She/he will work to develop and implement an effective model for gender awareness training that addresses the unique culture of STEM in the academy and develop partnerships with other research institutions across the country to both gather and disseminate effective and sustainable practices.
An extensive evaluation process has been formulated to guide the transformation process through outcome assessment, feedback, and continual program improvement.
UMBC will chart new territory in supporting women's career options and advancement and believes that these new opportunities will benefit both men and women in STEM, across the campus, and across the country. The ADVANCE team is supported by all levels of faculty, staff, and administration under the leadership of the PI, the president of the university. The full commitment of the university assures that adequate resources are available to achieve the goal and objectives.
UMBC is committed to creating a diverse environment for all members of its community. At a minimum, women faculty will be represented in proportion to the available pool of candidates from which it draws. By increasing STEM faculty women's numbers and involvement, the university will better support its female students who in turn will play an important role in addressing the nation's critical workforce shortages.

Many aspects of mammalian physiology and behavior exhibit a daily 24 hour rhythm. These daily oscillations are circadian rhythms and are controlled by a brain structure known as the suprachiasmatic nucleus (SCN). The mammalian circadian clock is constantly being reset by the onset of environmental light. Light entrainment of the clock requires input from the retina, which communicates with the SCN via the axonal projections of a small subset of retinal ganglion cells (RGCs). Surprisingly, rod and cone photoreceptors are not required; instead, RGCs that project to the SCN appear to function as autonomous circadian photoreceptors as they exhibit light responses independent of rod- and cone-driven synaptic input. Interestingly, the action spectrum of the light-evoked depolarization of these photosensitive retinal ganglion cells is described by a photopigment with a wavelength of maximum absorbance of 484 nm.

Melanopsin, a novel opsin-like protein expressed in some of the retinal ganglion cells that project to the SCN, appears to be the elusive circadian photopigment based on several lines of evidence. Melanopsin is expressed in the light-sensitive RGCs and disruption of the melanopsin gene in mice abolishes the intrinsic light response of the SCN-projecting RGCs and impairs circadian entrainment. However, these elegant experiments do not address the question of whether melanopsin is a photopigment directly responsible for generating the light response, or simply an isomerase required for chromophore regeneration on a separate photopigment. Data from the Robinson laboratory was the first to demonstrate that melanopsin does indeed form a functional photopigment. This led to the hypothesis that melanopsin has a unique role in mammalian RGCs involved in circadian photoentrainment and the light-activated melanopsin triggers a G-protein cascade that underlies the photic response generated by these melanopsin-containing RGCs. Based on its homology with invertebrate opsins, the prediction is that melanopsin activates a Gq-based signaling pathway. This grant proposes to use molecular and biochemical approaches to further characterize melanopsin.

The proposed research is significant because melanopsin is a novel and newly discovered mammalian retinal visual pigment involved in circadian photoentrainment. It appears to have many properties similar to visual pigments that have been characterized in rhabdomeric invertebrate eyes. This makes this pigment unique among mammalian visual pigments and fascinating to study. The approaches proposed in this grant are novel. The Robinson lab is the only laboratory that has taken a decidedly molecular and biochemical approach to study this visual pigment, and is poised to make significant progress on understanding this visual pigment within the next two years.

Broader Impacts:
The P.I of this grant is acutely aware of NSF's commitment to education and the development of underrepresented groups in the United States' Scientific workforce in the 21 st century. The P.I is currently a co-P.I. on a NSF institutional ADVANCE award to UMBC which as an institution is committed to the inclusion of both women and minorities in science. The P.I. has established an inclusive laboratory environment where underrepresented students, both graduate and undergraduate, have a positive research experience. Part of research described in this grant will be conducted by an African-American male. When possible, these undergraduates will include talented minority and women undergraduates identified form the nationally recognized Meyerhoff Scholars Program. The P.I. will also continue the practice of disseminating research results to the public through public lectures and visits to local high schools.

This project is supporting a new cohort of Meyerhoff scholars who have demonstrated financial need and are majoring in programs offered by the College of Natural and Mathematical Sciences (CNMS) - comprised of the departments of Biology, Biochemistry and Molecular Biology, Chemistry, Mathematics/Statistics, and Physics. The project is seeking to retain 90% of these students in CNMS majors into the junior year. Furthermore, the project expects each scholar to complete at least one summer of significant research activity by the beginning of the junior year, with at least 70% of these students entering graduate or professional school at the time of their graduation. The merit of the project rests on its grounding in the almost two decades old Meyerhoff Program at the University of Maryland Baltimore County (UMBC). Outcomes and process evaluation and research are being conducted by the original evaluation team for the Meyerhoff Program. The project's broader impacts are being felt through the significant increase in the number of underrepresented minority science and math Ph.D. candidates that is expected. The strong track record of the Meyerhoff Program supports this ambitious goal.

Evolution of sensory systems, and specifically the evolution of visual systems, is a research area of fundamental significance to ecologists, physiologists, neurobiologists, and evolutionary biologists. In particular, the evolution of visual pigments (the molecules that absorb light and in many ways determine primary features of visual sensitivity and color vision) has long served as a model system for understanding how genes and proteins evolve, how structural features of proteins affect function, and how sensory systems evolutionarily adjust to meet the functional requirements of different species. Research on stomatopod crustaceans, or mantis shrimps, has been central to this work. These marine animals have evolved a uniquely complex visual system, one that is unparalleled in the animal kingdom. They possess the most complicated systems of color vision of any animals, based on 16 or more different visual pigments (greater than the number known for any other animal group by a factor of more than two). These pigments are the light-capturing molecules in photoreceptor classes specialized to sense wavelengths of light from the deep ultraviolet to the far red. Stomatopods, therefore, have been favored subjects for studies of color vision, visual adaptation to habitat, color and polarizational signaling, and evolution of visual systems. Their visual pigments are often viewed as models of molecular machines for understanding protein design and function in all organisms.

This project brings together a team of vision scientists, molecular biologists, and evolutionary biologists to investigate how mantis shrimp vision has evolved at the molecular level, to learn how their numerous classes of photoreceptors have become specialized over evolutionary time, and to discover how the evolutionary advances made by stomatopods actually operate to improve visual function. Because stomatopods have so many different types of visual pigments, all based on a single class of proteins called opsins, they provide a natural laboratory within which to study diversification of function and to learn how changes in these opsin proteins down to the level of specific amino acid substitutions foster the evolution of new visual pigments, produce new spectral classes, and tune the visual functions of different species to particular habitats. Using the opsins in retinas of selected species of mantis shrimps as a model system, the research of this project will characterize the genetic diversity within and among species and between habitats, identify the molecular mechanisms used to tune their visual pigments, and establish how the modern diversity evolved. The work will involve cutting-edge approaches, including genetic sequencing, cellular identification of gene expression, computational assessment of phylogenetic diversification, and molecular modeling. The results will serve as new and significant underpinnings for advances in understanding visual function and for inspiring advances in medical and technological aspects of vision science. The work will involve the training of post-doctoral fellows, graduate students and undergraduates as part of the project, and the PIs will give public lectures at schools and non-scientific professional meetings and plan to contribute to textbook development and museum exhibitions.

DESCRIPTION (provided by applicant): Many aspects of mammalian physiology and behavior exhibit a daily 24 hour rhythm. These daily oscillations are circadian rhythms and are controlled by a brain structure known as the suprachiasmatic nucleus (SCN). The mammalian circadian clock is constantly being reset by the onset of environmental light. Light entrainment of the clock requires input from the retina, which communicates with the SCN via the axonal projections of a small subset of retinal ganglion cells (RGCs). Surprisingly, rod and cone photoreceptors are not required; instead, RGCs that project to the SCN appear to function as autonomous circadian photoreceptors as they exhibit light responses independent of rod- and cone-driven synaptic input. These SCN-projecting RGCs also express melanopsin, a novel vertebrate opsin, which is necessary for initiating the light response in these cells. Among all known vertebrate opsins, melanopsin is unique; it shows greater sequence similarity to invertebrate rhabdomeric photoreceptor opsins than to other vertebrate opsins. The current understanding of melanopsin's biochemical properties is rudimentary and its in vivo second messenger system has yet to be conclusively established and extensively characterized. The overall goal of the proposed research is to characterize the spectral and biochemical properties of melanopsin and to determine the biochemical pathway that mediates the intrinsic light responses of SCN-projecting RGCs. We hypothesize that melanopsin forms a photopigment that has biochemical characteristics similar to visual pigments associated with rhabdomeric photoreceptors and activates a Gq-based signaling pathway. Furthermore, we hypothesize that melanopsin undergoes light-dependent modifications that contribute to light adaptation of the melanopsin-dependent signaling cascade. To test these hypotheses, we propose an interdisciplinary approach combining biochemistry, electrophysiology, molecular genetics and behavioral studies. This approach is designed to determine melanopsin's photochemistry, to elucidate the nature of the second messenger pathway activated by melanopsin, and to determine if there are light-dependent post-translational modifications of melanopsin. PUBLIC HEALTH RELEVANCE: Many aspects of mammalian physiology and behavior exhibit a daily 24 hour rhythm. These daily oscillations are circadian rhythms and are controlled by a brain structure known as the suprachiasmatic nucleus. The mammalian circadian clock is constantly being reset by the onset of environmental light. Light entrainment of the clock requires input from the retina, which communicates with the suprachiasmatic nucleus via the axonal projections of a small subset of retinal ganglion cells. Surprisingly, classical photoreceptors, rods and cones are not necessary; instead, suprachiasmatic nucleus - projecting retinal ganglion cells function as autonomous photoreceptors. These ganglion cells also express a novel vertebrate visual pigment, known as melanopsin, which is necessary for initiating the light response in these cells. The proposed research project aims to characterize melanopsin. Understanding the signaling cascade activated by melanopsin is of great interest and significance for the field of sensory biology and circadian rhythms. In addition, disorders of the circadian system often accompany neurodegenerative diseases, sleep disorders, and blindness, and are more commonly experienced as a result of transmeridian travel and shift work. In the future, elucidation of the melanopsin-based signaling cascade should allow us to develop successful pharmacological treatments for these disorders.

? DESCRIPTION (provided by applicant): The purpose of this application is to obtain continued funding for five years to support research training of 40 trainees per year in the MARC U*STAR Program at UMBC. The aim is to maximize the access to research careers for students from groups underrepresented in biomedical research. The MARC U*STAR Program consists of a carefully designed combination of activities to assure: (i) excellence in academic science, (ii) understanding and adapting to the intensity and rigor of contemporary scientific research, (iii) student interest in pursuing a PhD degree and a career as a researcher in the biomedical, behavioral, and mathematical sciences, and (iv) ongoing counseling of students in strategies of learning and time management, as well as career choices, selection of graduate school, and developing successful admission applications. The program will also provide comprehensive financial support to ensure that trainees can focus on preparing for a smooth and successful transition from UMBC to competitive graduate programs at highly ranked institutions. Students with relevant majors will be recruited from UMBC and community colleges during their second sophomore semester, and successful applicants will be appointed to the program at the beginning of the following summer. The Program begins with a summer research internship and continues in the junior and senior academic years with academically challenging courses plus 8-10 hours per week in research with a faculty mentor at UMBC or a nearby institution. Trainees must also enroll in required development courses (i.e., scientific writing, and research ethics and policy), designed specifically for MARC trainees. In the summer between the junior and senior year the students will do a second summer research internship. At least one of the summer research internships must be at an institution other than UMBC. Trainees will also meet with successful faculty from competitive institutions with top-level graduate programs. Admission eligibility will require a 3.2 cumulative GPA, a minimum of 60 college-credits, and commitment to a research career in biomedical, behavioral, or mathematics research. Trainees must comply with program requirements to remain in the program. Program design and participant selection will be guided by the MARC Steering Committee and administered by the MARC Program Director (who has trained more than 100 undergraduate students in his lab) with the assistance of the full-time MARC Assistant Director. The efficacy of the program will be monitored regularly by analysis of performance and student and alumni surveys.

ABSTRACT This is an application for an Administrative Supplement to the University of Maryland Baltimore County ?s (UMBC) Undergraduate Research Training Initiative for Student Enhancement (U-RISE) grant to purchase for UMBC U-RISE scholars a software site license for LabArchives? electronic laboratory notebooks (ELNs) and the computer tablets required to run the software. This initiative will enhance our U- RISE undergraduates? biomedical research training by adding instruction in the use of ELNs, the laboratory notebook of the 21st century. UMBC is a young (founded in 1966), mid-size public research university with an enrollment of 13,767 students. UMBC has developed from a commuter campus into a strong public research university whose vision ?redefines excellence in higher education through an inclusive culture that connects innovative teaching and learning, research across disciplines, and civic engagement? (UMBC Mission Statement). It has built a reputation as a national ?powerhouse in fostering diversity in the sciences? (Keeley, J. 2014). A key component contributing to this strength has been UMBC?s partnership with NIH/NIGMS to provide academic research training programs for underrepresented groups (URGs) via the MARC U*STAR Program (Maximizing Access to Research Careers Undergraduate Student Training in Academic Research), from 1997 ? 2020, and the U-RISE Program (funded 4/2020). In its 23 years of operation, the MARC U*STAR Program trained over 450 undergraduates with 66% enrolling in Ph.D. or M.D./Ph.D. programs. Almost 150 of the program alumni have earned a Ph.D. or M.D./Ph.D. to date. The new U-RISE Program incorporates the successful elements of UMBC?s MARC U*STAR Program, as well as, new innovative additions such as a unit on rigor and reproducibility and a mentor training workshop that reflect the evolving nature of biomedical sciences and the imperative to meet the needs of URG students. This supplement aims to add another innovative element to the research-training environment for UMBC?s U-RISE Scholars, augmenting their technical, operational and professional development.

Abstract: Electron paramagnetic resonance (EPR) spectroscopy can provide information about the structure and dynamics of biomacromolecules in physiologically relevant conditions. Its inherently high sensitivity is still inadequate for some mass-limited samples -- for example, membrane proteins -- which are notoriously difficult to express, purify, and crystallize. This project aims to decrease the limit of detection for inductive-detection EPR spectroscopy at room temperature, so that low-yield biomacromolecular samples can be studied under physiologically relevant conditions. To achieve this objective, we will use a novel resonator design that bridges the gap between planar microresonators and conventional cavity resonators. Our novel planar inverse anapole microresonator design provides nanoliter active volumes combined with high quality factors, providing a projected improvement of two orders of magnitude in sensitivity at room temperature. Our first aim is to design and fabricate microresonators for operation at 9 GHz and 34 GHz. First, we will carry out finite element simulations of the field distributions and reflection coefficients for resonators coupled to waveguides. Based on these results, we will optimize the device geometry and dimensions to obtain nanoliter magnetic-field hotspots and high quality-factors. We will fabricate these optimized resonators at the NIST Nanofabrication facility. Next, we will characterize the microresonators and integrate them into a commercial 9 GHz and home-built 34 GHz EPR spectrometer. Our second aim is to demonstrate the viability of these resonators for structural biology EPR spectroscopy experiments. To do this, we will first design and fabricate microfluidic devices capable of localizing nanoliter sample volumes in the magnetic hotspot volume of the microresonator. To validate the device performance, we will use a concentration series of spin-labeled peptides. Finally, to demonstrate applicability to a biomacromolecular sample, we will study the Gprotein coupled receptor melanopsin. If successfully implemented, this resonator design will achieve an unprecedented sensitivity for EPR spectroscopy, broadening its applicability to low-yield biomacromolecular samples whose structures are currently poorly understood.

ABSTRACT The purpose of this application is to obtain funding for five years to support research training of 35 trainees per year in the Undergraduate Research Initiative for Student Enhancement (U-RISE) program at UMBC. The aim of U-RISE is to develop a diverse pool of undergraduates who complete their baccalaureate degree, and transition into and complete biomedical, research-focused higher degree programs (e.g., Ph.D. or M.D./Ph.D.) and pursue biomedical research careers. The U-RISE trainee pool will include students underrepresented in science, technology, engineering and mathematics (STEM); specifically, racial and ethnic minorities, students of low socioeconomic status, and students with disabilities. The program will provide a combination of activities to assure: (i) excellence in academic science, (ii) understanding and adapting to the intensity and rigor of contemporary scientific research, (iii) student interest in pursuing a Ph.D. degree and a career as a researcher in the biomedical sciences, as well as (iv) selection of graduate school, and developing successful admission applications, and (v) an emphasis on student mental health and wellness. U-RISE at UMBC will provide comprehensive financial support to ensure that trainees can focus on preparing for a smooth and successful transition from UMBC to competitive graduate programs at highly ranked institutions. Students with relevant majors will be recruited from UMBC and community colleges during the second semester of their sophomore year, and successful applicants will be appointed to U-RISE at the beginning of the following summer. Admission eligibility will require a 3.2 cumulative GPA, a minimum of 60 college-credits, and commitment to a research career in biomedical research. Trainee participation will begin with a summer research internship and continue in the junior and senior academic years with academically challenging courses, plus 8-10 hours per week in research with a faculty mentor at UMBC or a nearby institution. Trainees must also enroll in required development courses (i.e., scientific writing, and research ethics and policy), designed specifically for U-RISE participants. Trainees must comply with program requirements to remain in good standing. Program design and participant selection will be guided by the U-RISE Advisory Committee and administered by the U-RISE Program Director (who has trained more than 50 undergraduate students in her lab) with the assistance of the full-time U-RISE Associate Director. The efficacy of the program will be monitored regularly by analysis of performance and student and alumni surveys.