Master of Science Department

thesisThe mammalian Endosomal Sorting Complex Required for Transport (ESCRT) pathway mediates the final membrane fission step of cytokinesis, known as abscission. Prior to abscission, the ESCRT machinery is recruited to the intercellular bridge that connects nascent daughter cells. Once there, subunits of the ESCRT-III complex bind the plasma membrane and oligomerize into membrane-bound filaments. These filaments constrict the membrane, resulting in membrane fission and release of two independent daughter cells. Here, I describe the creation of siRNA/shRNA depletion and rescue assays for testing abscission functions of the ESCRT-III proteins IST1 and CHMP2A. siRNA or shRNA treatments were used to deplete endogenous IST1 or CHMP2A protein levels, and exogenous wild-type or mutant constructs were tested for their ability to "rescue" the resulting abscission defects, as assayed by flow cytometry or morphological assays. These assays confirmed that both IST1 and CHMP2A are required for abscission, and that exogenous wild-type IST1 and CHMP2A can rescue the defects induced by depletion of the cognate proteins. The IST1 and CHMP2A depletion/rescue assays provided a means for testing whether different IST1 and CHMP2A mutants can function in abscission. As an initial proof of principle, I tested whether mutations in conserved basic patches in the N-terminal alpha helix inhibit the ability of IST1 and CHMP2A to function in abscission. Tests were performed because previous studies had implicated these basic patches in ESCRT-III membrane binding. Consistent with this hypothesis, mutations in the basic residues of IST1 abolished abscission functions; however, the CHMP2A data were ambiguous. Thus, my work has confirmed that the ESCRT-III proteins IST1 and CHMP2A are both required for the abscission step of cytokinesis, and have provided an assay system that can be used to test the functionality of mutant proteins

Multi-attribute criteria applied to electric generation energy system analysis LDRD.

This report began with a Laboratory-Directed Research and Development (LDRD) project to improve Sandia National Laboratories multidisciplinary capabilities in energy systems analysis. The aim is to understand how various electricity generating options can best serve needs in the United States. The initial product is documented in a series of white papers that span a broad range of topics, including the successes and failures of past modeling studies, sustainability, oil dependence, energy security, and nuclear power. Summaries of these projects are included here. These projects have provided a background and discussion framework for the Energy Systems Analysis LDRD team to carry out an inter-comparison of many of the commonly available electric power sources in present use, comparisons of those options, and efforts needed to realize progress towards those options. A computer aid has been developed to compare various options based on cost and other attributes such as technological, social, and policy constraints. The Energy Systems Analysis team has developed a multi-criteria framework that will allow comparison of energy options with a set of metrics that can be used across all technologies. This report discusses several evaluation techniques and introduces the set of criteria developed for this LDRD

Lipidomics Reveals Multiple Pathway Effects of a Multi-Components Preparation on Lipid Biochemistry in ApoE*3Leiden.CETP Mice

Background: Causes and consequences of the complex changes in lipids occurring in the metabolic syndrome are only partly understood. Several interconnected processes are deteriorating, which implies that multi-target approaches might be more successful than strategies based on a limited number of surrogate markers. Preparations from Chinese Medicine (CM) systems have been handed down with documented clinical features similar as metabolic syndrome, which might help developing new intervention for metabolic syndrome. The progress in systems biology and specific animal models created possibilities to assess the effects of such preparations. Here we report the plasma and liver lipidomics results of the intervention effects of a preparation SUB885C in apolipoprotein E3 Leiden cholesteryl ester transfer protein (ApoE*3Leiden.CETP) mice. SUB885C was developed according to the principles of CM for treatment of metabolic syndrome. The cannabinoid receptor type 1 blocker rimonabant was included as a general control for the evaluation of weight and metabolic responses. Methodology/Principal Findings: ApoE*3Leiden.CETP mice with mild hypercholesterolemia were divided into SUB885C-, rimonabant- and non-treated control groups. SUB885C caused no weight loss, but significantly reduced plasma cholesterol (-49%, p <0.001), CETP levels (-31%,

Context Matters: The Illusive Simplicity of Macaque V1 Receptive Fields

Even in V1, where neurons have well characterized classical receptive fields (CRFs), it has been difficult to deduce which features of natural scenes stimuli they actually respond to. Forward models based upon CRF stimuli have had limited success in predicting the response of V1 neurons to natural scenes. As natural scenes exhibit complex spatial and temporal correlations, this could be due to surround effects that modulate the sensitivity of the CRF. Here, instead of attempting a forward model, we quantify the importance of the natural scenes surround for awake macaque monkeys by modeling it non-parametrically. We also quantify the influence of two forms of trial to trial variability. The first is related to the neuron’s own spike history. The second is related to ongoing mean field population activity reflected by the local field potential (LFP). We find that the surround produces strong temporal modulations in the firing rate that can be both suppressive and facilitative. Further, the LFP is found to induce a precise timing in spikes, which tend to be temporally localized on sharp LFP transients in the gamma frequency range. Using the pseudo R[superscript 2] as a measure of model fit, we find that during natural scene viewing the CRF dominates, accounting for 60% of the fit, but that taken collectively the surround, spike history and LFP are almost as important, accounting for 40%. However, overall only a small proportion of V1 spiking statistics could be explained (R[superscript 2]~5%), even when the full stimulus, spike history and LFP were taken into account. This suggests that under natural scene conditions, the dominant influence on V1 neurons is not the stimulus, nor the mean field dynamics of the LFP, but the complex, incoherent dynamics of the network in which neurons are embedded.National Institutes of Health (U.S.) (K25 NS052422-02)National Institutes of Health (U.S.) (DP1 ODOO3646

Shifts of Gamma Phase across Primary Visual Cortical Sites Reflect Dynamic Stimulus-Modulated Information Transfer

Distributed neural processing likely entails the capability of networks to reconfigure dynamically the directionality and strength of their functional connections. Yet, the neural mechanisms that may allow such dynamic routing of the information flow are not yet fully understood. We investigated the role of gamma band (50–80 Hz) oscillations in transient modulations of communication among neural populations by using measures of direction-specific causal information transfer. We found that the local phase of gamma-band rhythmic activity exerted a stimulus-modulated and spatially-asymmetric directed effect on the firing rate of spatially separated populations within the primary visual cortex. The relationships between gamma phases at different sites (phase shifts) could be described as a stimulus-modulated gamma-band wave propagating along the spatial directions with the largest information transfer. We observed transient stimulus-related changes in the spatial configuration of phases (compatible with changes in direction of gamma wave propagation) accompanied by a relative increase of the amount of information flowing along the instantaneous direction of the gamma wave. These effects were specific to the gamma-band and suggest that the time-varying relationships between gamma phases at different locations mark, and possibly causally mediate, the dynamic reconfiguration of functional connections

Bulletin No. 295 - Comparison of Two Methods of Determining Wool Density

Density of the fleece is one of the primary factors determining the wool-producing capacity of the sheep. Sheepmen have attempted to evaluate density by handling the fleece. This method is not sufficiently refined for use in experimental work, and may be subject to errors and misinterpretations. For example, if two fleeces are alike in density and diameter of fibers, yet differ in length, the longer fleece will feel less dense. If, on this basis, the sheep with the fleece that appeared most dense were selected for breeding, selection would be away from the longer fleece. Any method that can be used to measure accurately the density of the fleece will be very useful in inheritance and wool production studies