David Moffett - US grants

This research is directed at understanding the nature of three transport processes of the intestine of lepidopteran insects for potassium, chloride, and bicarbonate, and the role of these processes in the ionic and acid-base balance of the whole animal. An important goal is to identify the processes by which the animal resists the potassium load presented by the plant diet and the mechanisms of alkalinization of the contents in the anterior parts and recovery of base in the posterior parts of the gut. The complexity of the tissue necessitates separation of the basal and apical membranes and study of their transport mechanisms in isolation. The immediate goals of this project are, first, to isolate the basal membrane components responsible for potassium entry by one of the following methods: incorporation into lipid bilayers, study in membrane vesicles, or patch-clamping of dissociated cells in primary culture. Second, using similar techniques, possible chloride/bicarbonate exchange will be investigated in apical membranes of both columnar cells and goblet cells. Third, the relationships between electrochemical driving forces and transport rates for both chloride and hydrogen ions in isolated gut will be measured using ion-specific intracellular electrodes. The effect of dietary potassium on acid-base, potassium, and chloride balances will also be measured in the intact animal. The results of these studies will provide information on the basic mechanisms by which insects regulate the ionic composition and acid-base balance of their body fluids. It will help to explain how butterfly and moth larvae are able to resist the load of potassium provided by their diet of plant leaves. New knowledge of the physiology of these transport systems may, in the future, suggest new approaches for the control of these insects.

In contrast to the acid stomach of vertebrates, the anterior stomach of mosquito larvae is very alkaline (pH10). More acidic conditions are restored in the posterior stomach. These observations suggest that, while the animal is feeding, the stomach epithelium continuously recycles alkali between the gut of the animal and its physiological interior. Disturbance that causes the animal to stop feeding and initiate escape behavior interrupts gut alkalinization, suggesting a tight neural and/or endocrine control of the process. It is already clear that some transport proteins characteristic of other well-studied acid or alkali secreting cells are present in the cells of the mosquito larval stomach. Although this group of researchers has identified a vacuolar-type H+ ATPase and at least one anion exchanger, other transporters involved in this system are not known. The gut is innervated by axons that express immunoreactivity to the neurotransmitter serotonin and to nitric oxide synthase, the enzyme responsible for synthesis of the transmitter nitric oxide. The gut epithelium contains endocrine cells that show immunoreactivity to peptide hormones belonging to the FMRFamide family. The effects of serotonin on the anterior stomach have been partly described, and it appears that it, plus an additional messenger or messengers, constitute a sufficient stimulatory signal for gut alkalinization. It is probable that there are also inhibitory signals that come from the CNS and/or the gut itself. The major questions of the project are aimed at the cellular mechanisms that drive alkalinization of the anterior gut, reacidification in the posterior stomach, and the identity of the neural and endocrine control pathways. Development of isolated, perfused preparations of the stomach by this team of investigators was a critical basis for the projected studies, because they allow experiments that would be impossible in the whole animal: electrophysiological characterization of transport mechanisms by measurement of the transepithelial voltage of the tissue, the intracellular voltages and ionic concentrations using ion-specific intracellular microelectrodes, and the transepithelial ionic fluxes using isotopes. The perfused preparations also will be used to assay the activities of candidate control neuropeptides. In a parallel experimental approach, the presence and cellular locations of known transport proteins will be determined by fluorescence immunohistochemistry, a technique in which antibodies generated against defined molecular targets are localized by fluorescently tagged secondary antibodies. Mosquitoes are by far the world's most medically significant insects. They are potential vectors for approximately 100 arboviruses that cause human disease, including yellow fever, dengue and a number of forms of encephalitis. They also transmit nematodes that cause elephantiasis, and plasmodia that cause malaria. The larval mosquito is an aquatic form that feeds on detritus. Although the larval form does not transmit disease, it may be more vulnerable to 3rd generation control measures than adults, because larvae are generally concentrated in aquatic breeding sites whereas the winged adults disperse widely. Gut alkalinization is believed to protect the animal from infection by killing ingested microbes and viruses. Weakening of this mechanism by a specific attack on the cellular processes, or their control signals, could make the larvae more susceptible to endemic or applied microbial pathogens. This project could provide the knowledge base for such an approach.

AST-0520928--Dennison/UNC Asheville

This project will utilize the two 26 m radio telescopes at the Pisgah Astronomical Research Institute to revitalize the field of long-term monitoring of flux variability in cm-wavelength radio sources. The results will determine or constrain intrinsic characteristics of potential variable sources such as active galactic nuclei, and can help characterize the ionized interstellar medium in our own galaxy which imparts scintillation and, on rare occasions, extreme lensing effects that impact the flux of background radio sources. The Pisgah facility has an established record of involving undergraduates in its research programs, including those from historically non-research-oriented institutions in North Carolina and South Carolina.

[unreadable] DESCRIPTION (provided by applicant): The most prominent organ of larval mosquitoes is the stomach, which makes up the major part of the gastrointestinal tract. This organ is of prominent importance for ionic, volume and acid/base homeostasis. The anterior stomach actively secretes alkali equivalents, generating a compartment of extremely high pH which is of importance for digestion, but also for the susceptibility to biological control agents such as Bacillus thuringienis endotoxin. In the posterior stomach the pH returns to almost neutral values by recovery of alkali equivalents. Most likely, this cycle involves a high turnover of acid/base equivalents as well as of sodium and chloride ions. The processes of ion transport, their regulation and their coordination with peristalsis in the stomach of larval mosquitoes are poorly understood. The project is directed at understanding these processes in two mosquito species: the yellow fever mosquito (Aedes aegypti) and the malaria mosquito of subsaharan Africa Anopheles gambiae. Specifically we will determine: 1. the ion transport mechanisms involved in alkalization of the anterior stomach. 2. the ion transport mechanisms involved in reacidification of the posterior stomach. 3. the mechanisms of regulation of ion transport in the anterior and posterior stomach. 4. the mechanisms of coordination of ion transport with peristalsis in the anterior and posterior stomach. The studies will be carried out, using mainly five different methodological approaches: 1.) Electrophysiological characterization of ion transport across isolated stomach segments and its regulation, using conventional transepithelial techniques and microelectrodes 2.) Analyses of luminal pH changes with isolated stomach segments, using pH indicators and/or pH sensitive microelectrodes in the perfusate 3.) Histochemical and immunhistochemical localization of involved transporters and regulative units/systems 4.) Biochemical determination of transporter activities and their regulation by hormonal/non-hormonal factors 5.) Analyses of peristalsis and its regulation with electrophysiological techniques and image analyses. The results of this project will provide information about specific targets for the development of mosquito control agents. [unreadable] [unreadable] [unreadable]

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

Dr. Brian Dennison (University of North Carolina at Asheville) and his team will use the Dedicated Interferometer for Rapid Variability (DIRV) at the Pisgah Astronomical Research Institute (PARI) to observe intraday variability (IDV) of compact extra-galactic radio sources, and simultaneously search for rare extreme scattering events (ESEs). These events are caused by intervening structures in the interstellar medium with significant density enhancements on quite small (approximately astronomical unit) scales. Interleaved with observations of extragalactic sources, a single 26-meter antenna of the interferometer will be used to monitor the intensities and rotational dynamics of pulsars. Both 26-meter antennas will host an unpointed, simultaneous search for dispersed millisecond bursts (DMBs). The research program involves commissioning DIRV, developing and installing hardware for the simultaneous "piggyback" search for DMBs, and carrying out the overall observational program. This research addresses several outstanding problems in astrophysics. The physical nature and origin of the interstellar structures responsible for ESEs are unknown. Only by discovering additional events, can the team then pursue specific concurrent observations designed to elucidate the nature and likely origin of these structures. Another major astrophysical problem arises from the single DMB discovered and credibly documented. Although theories are now being put forward to explain this event, there is a lack of observations of additional DMBs necessary to confirm the phenomenon, and elucidate its properties. Because the sole documented burst was rapid, strong, and evidently extragalactic in origin, it suggests an extremely energetic, compact source. Both ESEs and DMBs are unexplained astrophysical phenomena and this research program aims to gain insight into both. In either case, the potential exists for a transformative insight expanding our understanding of the universe.

Time-intensive radio observations are not feasible within the national observatory system, in which time on highly versatile telescopes is apportioned among a large international user community. The observations to be carried out under this program add a unique and necessary component to the overall national effort in astronomy. This work also complements the setting provided by the national observatories by providing undergraduate students with an intensive hands-on research experience in which instrumentation problems are solved in the context of overarching scientific goals. Undergraduate students from three universities (University of North Carolina-Asheville, Furman University, and University of North Carolina-Pembroke) will participate in all phases of this research through an established intern program. They will work in a multi-disciplinary, collaborative environment in which instrumentation issues are central to the scientific goals.