Charles Wilson - US grants

Wilson
MCB 9876350

1. Technical: This study explores the folded structures of RNAs and their
interactions with other molecules. Aptamers isolated from random sequence
pools, including RNAs that specifically bind the enzymatic cofactors
cyanocobalamin and biotin and the chromophore malachite green, will serve
as model systems. Focus is placed on biochemical characterization of these
molecules and on the preparation of crystals and solution samples that will
enable their analysis by x-ray diffraction and NMR spectroscopy. The
structures of cyanocobalamin and biotin aptamer crystals will be determined
by X-ray crystallography and the isotopically-labeled malachite green
aptamer RNA by solution NMR spectroscopy. This is to understand the
specific atomic interactions that stabilize RNA folding, the variety of
structural folds available to RNA, the involvement of metals and waters in
RNA function, and the nature of conformational changes in RNA that result
from its interaction with other molecules. The educational component of
this project aims to use structural biology for introducing students to the
physical sciences through a combination of both classroom and laboratory
instruction. New and modified curricula in eukaryotic molecular biology
and structural biology, supplemented by the development of computer-based
educational materials, will be developed to establish the relationship
between macromolecular structure and function. Selected number of
students will be encouraged to engage in the research.

2. Non-technical: The capacity for ribonucleic acids (RNAs) to fold into
specific conformations and thereby obtain functional properties (e.g. the
ability to bind molecules or to catalyze reactions) has been increasingly
appreciated, in part through experiments in which RNAs have been evolved de
novo from pools of random sequence molecules. This recognition has fueled
speculation about the involvement of RNAs in many biological processes
(e.g. ribosome-directed protein synthesis) and supported theories favoring
an RNA-based biology as a precursor to modern life. Our understanding of
the structural basis for RNA function, critical for addressing these
issues, remains poor in part because few RNA structures have been
determined at atomic resolution. In vitro-evolved ligand-binding RNAs will
serve as model systems for understanding RNA structure and its relationship
to RNA function. X-ray crystallography and NMR spectroscopy will be used
in this study to determine high resolution models for three different
RNA-ligand complexes. Analysis of these structures will reveal the
mechanisms by which specific RNA sequences adopt unique structures and how
these structures enable specific interactions with other molecules.
Integrating these studies with teaching, this study provides undergraduate
students with basic skills in structural biology. Enhancements to existing
eukaryotic molecular biology curricula will emphasize the concept that
biological processes are driven by molecular interactions specified by the
folded conformations of macromolecules. A new course in structural biology
will expand upon this theme with the additional goal of providing
interdisciplinary training in applied physical and computational sciences.

neurophysiology; neuroanatomy; nervous system disorder; biomedical resource; clinical research;

The local infrasound environment in Alaska is rich in both natural and human-generated sources. Identifying the source and localizing it is a challenge with many different applications. The Infrasound group at UAF has operated arrays in Alaska and Antarctica for several years. The primary arrays are comprised of eight microphones arranged to obtain wide-band operation with no spatial aliasing over the frequency band of interest. These arrays are operated and maintained by the UAF group as part of the International Monitoring System (IMS) of the United Nations Comprehensive Nuclear Test-Ban Treaty Office (CTBTO). A second array of four microphones is operated by the UAF group as a research array. The group has used this array to test methods of wind-noise reduction, microphone performance and array performance under varying array geometries. As part of the process of identifying infrasound signal sources a prototype locator algorithm was developed that allows estimation of the range to the signal source for locations of the signals source of up to 10 to 15 times the array diameter. The locator algorithm has been used extensively in cases where signal-to-noise levels are high and it appears to be robust. Investigators will continue to study the method and investigate its behavior under a range of signal-to-noise conditions, as well as develop an estimator of the variance in the estimates of range and azimuth.

The description and classification of signal sources will improve the ability to discriminate against known natural and man-made sources in signal detection operations. The development of an algorithm to estimate source range to accompany the traditional azimuth estimates will also benefit the general area of source localization using acoustic methods. This algorithm should be transferable to any detection program using arrays of sensors.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. "Systems Pathophyisology: A Clinical Neurophyiology Program for the Partial Epilepsies" Anatomical correlates of high frequency oscillations recorded in the epileptic mesial temporal lobe Fast Ripple (FR) oscillations in the EEG frequency range of 200 to 500 Hz have been identified in epileptogenic hippocampus, entorhinal cortex and subiculum of mesial temporal lobe epilepsy patients (Staba et al., J, Neurophysiol, 2002, 88: 1743-1752). We wish to determine if mesial temporal lobe (MTL) atrophy (as indicated by volumetric measurement) might form an anatomical substrate for this abnormal activity, and to learn if anatomical changes outside of the MTL may also be present in patients with temporal lobe epilepsy and limbic FR oscillations. We currently have data from 27 patients (15 males and 10 females with a median age of 35 years ranging from 17 to 51 years) with medically intractable seizures and monitored with depth electrodes for diagnostic determination of the seizure onset location. Spontaneous interictal wide band activity recorded from microelectrodes in hippocampus, entorhinal cortex and subiculum allowed us to determine distribution and rate of FR. Volumetric analysis of the hippocampus, amygdala and entorhinal cortex has been carried out using standard criteria based on guidelines developed at Montreal Neurological Institute (Cendes et al., Neurology, 1993, 43: 719-725;Bernasconi et al., Neurology, 1999, 52: 1870-1876). Mean volumes of the MTL structures were correlated with the recorded FR rates detected in 57 microelectrodes in the epileptogenic (side of seizure onset) and contralateral MTL. There was an inverse correlation between FR rates and hippocampal volumes that was significant (p<.0009) showing a strong relationship between higher rates of FR in atrophic hippocampus. Given the significantly higher FR rate in the epileptogenic MTL containing atrophic hippocampi and amygdala, we now wish to determine 1) whether the presence of this electrophysiological marker may correlate with anatomical changes outside of the mesial temporal lobe, and 2) what differences exist between the brains of this population of epileptic patients and brains from an age- and gender-matched normal population. To answer these questions, we wish to employ MRIcro and STM software along with MatLab to establish norms with a 5:1 ratio using normal T1 weighted MRI[unreadable]s from ICBM, and then compare the normal brain template with our patient population. By evaluating which regions from epileptic brains show significant variance from normal brains we hope to further identify anatomical changes associated with temporal lobe epilepsy. I have attached an Excel file containing 50 female and 75 male T1 weighted MRIs from the ICBM data base that we would like to obtain for this study. The presence of FR in our recordings from adjacent entorhinal cortex and subiculum as well as hippocampus suggests that a network supporting synchronization of high frequency oscillations exists. The anatomical evidence from this study may lead to a better understanding of how those networks engage the rest of the brain to support propagation of seizures outside of the limbic system and to motor areas that create the severe behavioral consequences associated with secondarily generalized epileptic seizures.