Affinity maturation and characterization of novel binders to the HIV-1 TAR element based on the U1A RNA recognition motif

2018 Fall.Includes bibliographical references.The increased understanding of the importance of RNA, both as a carrier of information and as a functional molecule, has led to a greater demand for the ability to target specific RNAs, but the limited chemical diversity of RNA makes this challenging. This thesis documents the use of yeast display to perform affinity maturation for the ability of a protein to bind the TAR element of HIV-1, which is a desirable therapeutic target due to its prominent role in the HIV-1 infection cycle. To accomplish this, we used a "semi-design" strategy—repurposing a natural RNA bind- ing protein to bind a different target—by creating a library based on important binding regions (especially the β2β3 loop) of the U1A RRM. Following selection for TAR binding, a strong consensus sequence in the β2β3 loop emerged. The affinity of certain library members for TAR was measured by ELISA and SPR, and it was determined that the best binder (TBP 6.7) had remarkable affinity (KD = ~500 pM). This TAR binding protein also proved capable of disrupting the Tat–TAR interaction (necessary for HIV-1 replication) both in vitro and in the context of extracellular transcription. Through collaboration, we were able to obtain a co-crystal structure of TBP 6.7 and TAR. This crystal structure showed that the overall structure of TBP 6.7 was largely unchanged from that of U1A, thereby validating our semi-design strategy. We also found that the β2β3 loop played a disproportionately large role in the binding interaction (~2⁄3 of the buried surface area). The importance of this region inspired the creation and characterization of peptide derivatives of the TBP 6.7 β2β3 loop. These β2β3 loop derived peptides maintain affinity for TAR RNA (KD = ~1.8 μM), and can disrupt Tat/TAR-dependent transcription. Ultimately, the project has yielded the most avid known binders of TAR RNA, a potential novel platform of TAR binding peptides, and a crystal structure which will hopefully inform future targeting efforts

Behavior and weight correlates of weight-control efforts in Australian women living in disadvantage : the READI study

BackgroundWith increasing obesity rates worldwide, more and more people are actively attempting to lose weight or avoid weight gain, but relatively little is known about what specific behaviors comprise these efforts and which, if any, are associated with better weight control over time.MethodsThis paper reports relationships between body weight, weight-control efforts and related behaviors over a three-year period in 1,634 Australian women. The women were purposefully recruited from 80 disadvantaged neighborhoods in Victoria, Australia. Weight loss efforts were categorized as trying to lose weight, trying to prevent weight gain and no weight-control efforts. Behavioral correlates examined included different kinds of physical activity and consumption of a number of specific foods types.Results and discussionSelf-reported body weight at baseline was higher in women trying to lose weight. Frequency of consumption of low energy density foods was positively associated with reported weight-control efforts, as was frequency of reported total and leisure-time physical activity. Longitudinal associations between changes in weight-control efforts and changes in behaviors were consistent with the cross-sectional findings. At three-year follow up, however, weight-control efforts were not associated with change in body weight. More detailed analyses of specific food choices suggested that part of the explanation of no effect of reported weight-control efforts and weight over time might be that people are not as well-informed as they should be about the energy density of some common foods. In particular, those reporting engagement in weight-control efforts reported reducing consumption of carbohydrate-containing foods such as bread and potatoes more than is justified by their energy content, while they reported increasing consumption of some high energy density foods (e.g., cheese and nuts).ConclusionIt is tentatively concluded that women living in disadvantaged neighborhoods understand messages about weight-control (more activity and foods with lower fat and lower energy density) but that some foods eaten more by women engaged in weight control may reduce the effectiveness of these efforts.<br /

DETERMINANTS OF FERTILIZER ADOPTION BY AFRICAN FARMERS: POLICY ANALYSIS FRAMEWORK, ILLUSTRATIVE EVIDENCE, AND IMPLICATIONS

Crop Production/Industries,

Electrical Safety for Center Pivot Irrigation Systems (1993)

Many irrigators have received minor tingles while working around electrical irrigation machinery. Under pressure to keep the system running, they tend to ignore warning signs until serious injury occurs. Two electrical inspection surveys (conducted in Nebraska) point out the dangers of such practices

Wolves: Behavior, Ecology, and Conservation: Photo Section I

Wolves in Yellowstone National Park\u27s restored population often must compete with grizzly bears at the carcasses of their kills. Especially after their first feeding at a kill, wolves tend to relinquish their kills to bears. Photograph by Doug Smith. Elk are one of the most common prey of wolves in the northern Rocky Mountains of North America, primarily because in most areas they tend to outnumber other prey. This large bull standing his ground escaped this pack. Photograph by Doug Smith

Is Agricultural Intensification Profitable For Mozambican Smallholders? An Appraisal of the Inputs Subsector and the 1996/97 DNER/Sg2000 Program

Farm Management, Downloads July 2008-June 2009: 5,

What Makes Agricultural Intensification Profitable for Mozambican Smallholders? An Appraisal of the Inputs Subsector and the 1996/97 DNER/SG2000 Program

This report summarizes an appraisal of input utilization and marketing in Mozambique, focusing on the following research questions: (1) What are current smallholder yields for major commodities, and what is the potential for increasing yields through the use of improved technologies? (2) To what extent are improved technologies already being used by smallholders, and is the use of improved technologies profitable? (3) How are improved seeds, fertilizer and pesticides currently produced and distributed? and (4) What are the key constraints and opportunities for increasing the use of improved technologies by smallholders?food security, food policy, improved technologies, Farm Management, Downloads July 2008-July 2009: 9, O31,

What Makes Agricultural Intensification Profitable for Mozambican Smallholders? An Appraisal of the Inputs Subsector and the 1996/97 DNER/SG2000 Program, Volume II: Main Report

Ministry of Agriculture and Rural Development, Directorate of Economics, Republic of Mozambiquefood security, food policy, Mozambique, agriculture, Farm Management, Q18,

Seismic Velocity Database for the New Madrid Seismic Zone and Its Vicinity

Over the last 20 years, researchers at the University of Kentucky have collected seismic-reflection and refraction data to characterize seismic velocity models of the soil-sediment overburden throughout the central United States. The data are in different forms, such as published reports, theses, and journal articles, and in digital form. In order to construct a unified database for easier management, access, and use, Microsoft Access was used to design the data structure and field properties. The database consists of four tables with unified field names, data type, and units. An ArcGIS geodatabase with the same data structure as the Access database was then created for visualization and querying capability. The compiled seismic velocity data used the same data types and units, and were stored in both databases. The databases are flexible, so that new data and data types can be added and joined with existing databases at the Kentucky Geological Survey

Barriers to avoiding fast-food consumption in an environment supportive of unhealthy eating

Objective To investigate factors (ability, motivation and the environment) that act as barriers to limiting fast-food consumption in women who live in an environment that is supportive of poor eating habits.Design Cross-sectional study using self-reports of individual-level data and objectively measured environmental data. Multilevel logistic regression was used to assess factors associated with frequency of fast-food consumption.Setting Socio-economically disadvantaged areas in metropolitan Melbourne, Australia.Subjects Women (n 932) from thirty-two socio-economically disadvantaged neighbourhoods living within 3 km of six or more fast-food restaurants. Women were randomly sampled in 2007&ndash;2008 as part of baseline data collection for the Resilience for Eating and Activity Despite Inequality (READI) study.Results Consuming low amounts of fast food was less likely in women with lower perceived ability to shop for and cook healthy foods, lower frequency of family dining, lower family support for healthy eating, more women acquaintances who eat fast food regularly and who lived further from the nearest supermarket. When modelled with the other significant factors, a lower perceived shopping ability, mid levels of family support and living further from the nearest supermarket remained significant. Among those who did not perceive fruits and vegetables to be of high quality, less frequent fast-food consumption was further reduced for those with the lowest confidence in their shopping ability.Conclusions Interventions designed to improve women\u27s ability and opportunities to shop for healthy foods may be of value in making those who live in high-risk environments better able to eat healthily