Bioinformatic analysis for exploring relationships between genes and gene products

To carry out their specific roles in the cell, genes and gene products often work together in groups, forming many relationships among themselves and with other molecules. Such relationships include physical protein-protein interaction relationships, regulatory relationships, metabolic relationships, genetic relationships, and much more. With advances in science and technology, some high throughput technologies have been developed to simultaneously detect tens of thousands of pairwise protein-protein interactions and protein-DNA interactions. However, the data generated by high throughput methods are prone to noise. Furthermore, the technology itself has its limitations, and cannot detect all kinds of relationships between genes and their products. Thus there is a pressing need to investigate all kinds of relationships and their roles in a living system using bioinformatic approaches, and is a central challenge in Computational Biology and Systems Biology. This dissertation focuses on exploring relationships between genes and gene products using bioinformatic approaches. Specifically, we consider problems related to regulatory relationships, protein-protein interactions, and semantic relationships between genes. A regulatory element is an important pattern or "signal", often located in the promoter of a gene, which is used in the process of turning a gene "on" or "off". Predicting regulatory elements is a key step in exploring the regulatory relationships between genes and gene products. In this dissertation, we consider the problem of improving the prediction of regulatory elements by using comparative genomics data. With regard to protein-protein interactions, we have developed bioinformatics techniques to estimate support for the data on these interactions. While protein-protein interactions and regulatory relationships can be detected by high throughput biological techniques, there is another type of relationship called semantic relationship that cannot be detected by a single technique, but can be inferred using multiple sources of biological data. The contributions of this thesis involved the development and application of a set of bioinformatic approaches that address the challenges mentioned above. These included (i) an EM-based algorithm that improves the prediction of regulatory elements using comparative genomics data, (ii) an approach for estimating the support of protein-protein interaction data, with application to functional annotation of genes, (iii) a novel method for inferring functional network of genes, and (iv) techniques for clustering genes using multi-source data.

Elemental analysis of glass and ink by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and laser induced breakdown spectroscopy (LIBS)

The necessity of elemental analysis techniques to solve forensic problems continues to expand as the samples collected from crime scenes grow in complexity. Laser ablation ICP-MS (LA-ICP-MS) has been shown to provide a high degree of discrimination between samples that originate from different sources. In the first part of this research, two laser ablation ICP-MS systems were compared, one using a nanosecond laser and another a femtosecond laser source for the forensic analysis of glass. The results showed that femtosecond LA-ICP-MS did not provide significant improvements in terms of accuracy, precision and discrimination, however femtosecond LA-ICP-MS did provide lower detection limits. In addition, it was determined that even for femtosecond LA-ICP-MS an internal standard should be utilized to obtain accurate analytical results for glass analyses. In the second part, a method using laser induced breakdown spectroscopy (LIBS) for the forensic analysis of glass was shown to provide excellent discrimination for a glass set consisting of 41 automotive fragments. The discrimination power was compared to two of the leading elemental analysis techniques, μXRF and LA-ICP-MS, and the results were similar; all methods generated >99% discrimination and the pairs found indistinguishable were similar. An extensive data analysis approach for LIBS glass analyses was developed to minimize Type I and II errors en route to a recommendation of 10 ratios to be used for glass comparisons. Finally, a LA-ICP-MS method for the qualitative analysis and discrimination of gel ink sources was developed and tested for a set of ink samples. In the first discrimination study, qualitative analysis was used to obtain 95.6% discrimination for a blind study consisting of 45 black gel ink samples provided by the United States Secret Service. A 0.4% false exclusion (Type I) error rate and a 3.9% false inclusion (Type II) error rate was obtained for this discrimination study. In the second discrimination study, 99% discrimination power was achieved for a black gel ink pen set consisting of 24 self collected samples. The two pairs found to be indistinguishable came from the same source of origin (the same manufacturer and type of pen purchased in different locations). It was also found that gel ink from the same pen, regardless of the age, was indistinguishable as were gel ink pens (four pens) originating from the same pack.

The development of optimized high performance liquid chromatography systems for the analysis of improvised explosives

Existing instrumental techniques must be adaptable to the analysis of novel explosives if science is to keep up with the practices of terrorists and criminals. The focus of this work has been the development of analytical techniques for the analysis of two types of novel explosives: ascorbic acid-based propellants, and improvised mixtures of concentrated hydrogen peroxide/fuel. In recent years, the use of these explosives in improvised explosive devices (IEDs) has increased. It is therefore important to develop methods which permit the identification of the nature of the original explosive from post-blast residues. Ascorbic acid-based propellants are low explosives which employ an ascorbic acid fuel source with a nitrate/perchlorate oxidizer. A method which utilized ion chromatography with indirect photometric detection was optimized for the analysis of intact propellants. Post-burn and post-blast residues if these propellants were analyzed. It was determined that the ascorbic acid fuel and nitrate oxidizer could be detected in intact propellants, as well as in the post-burn and post-blast residues. Degradation products of the nitrate and perchlorate oxidizers were also detected. With a quadrupole time-of-flight mass spectrometer (QToFMS), exact mass measurements are possible. When an HPLC instrument is coupled to a QToFMS, the combination of retention time with accurate mass measurements, mass spectral fragmentation information, and isotopic abundance patterns allows for the unequivocal identification of a target analyte. An optimized HPLC-ESI-QToFMS method was applied to the analysis of ascorbic acid-based propellants. Exact mass measurements were collected for the fuel and oxidizer anions, and their degradation products. Ascorbic acid was detected in the intact samples and half of the propellants subjected to open burning; the intact fuel molecule was not detected in any of the post-blast residue. Two methods were optimized for the analysis of trace levels of hydrogen peroxide: HPLC with fluorescence detection (HPLC-FD), and HPLC with electrochemical detection (HPLC-ED). Both techniques were extremely selective for hydrogen peroxide. Both methods were applied to the analysis of post-blast debris from improvised mixtures of concentrated hydrogen peroxide/fuel; hydrogen peroxide was detected on variety of substrates. Hydrogen peroxide was detected in the post-blast residues of the improvised explosives TATP and HMTD.

Fiscal decentralization and development: An analysis of city governments in Argentina and Mexico, 1980--2010

This dissertation examines local governments' efforts to promote economic development in Latin America. The research uses a mixed method to explore how cities make decisions to innovate, develop, and finance economic development programs. First, this study provides a comparative analysis of decentralization policies in Argentina and Mexico as a means to gain a better understanding of the degree of autonomy exercised by local governments. Then, it analyzes three local governments each within the province of Santa Fe, Argentina and the State of Guanajuato, Mexico. The principal hypothesis of this dissertation is that if local governments collect more own-source tax revenue, they are more likely to promote economic development and thus, in turn, promote growth for their region. ^ By examining six cities, three of which are in Santa Fe—Rosario, Santa Fe (capital) and Rafaela—and three in Guanajuato—Leon, Guanajuato (capital) and San Miguel de Allende, this dissertation provides a better understanding of public finances and tax collection efforts of local governments in Latin America. Specific attention is paid to each city's budget authority to raise new revenue and efforts to promote economic development. The research also includes a large statistical dataset of Mexico's 2,454 municipalities and a regression analysis that evaluates local tax efforts on economic growth, controlling for population, territorial size, and the professional development. In order to generalize these results, the research tests these discoveries by using statistical data gathered from a survey administered to Latin American municipal officials. ^ The dissertation demonstrates that cities, which experience greater fiscal autonomy measured by the collection of more own-source revenue, are better able to stimulate effective economic development programs, and ultimately, create jobs within their communities. The results are bolstered by a large number of interviews, which were conducted with over 100 finance specialists, municipal presidents, and local authorities. The dissertation also includes an in-depth literature review on fiscal federalism, decentralization, debt financing and local development. It concludes with a discussion of the findings of the study and applications for the practice of public administration.^

Dynamic modeling and analysis of single-stage boost inverters under normal and abnormal conditions

Inverters play key roles in connecting sustainable energy (SE) sources to the local loads and the ac grid. Although there has been a rapid expansion in the use of renewable sources in recent years, fundamental research, on the design of inverters that are specialized for use in these systems, is still needed. Recent advances in power electronics have led to proposing new topologies and switching patterns for single-stage power conversion, which are appropriate for SE sources and energy storage devices. The current source inverter (CSI) topology, along with a newly proposed switching pattern, is capable of converting the low dc voltage to the line ac in only one stage. Simple implementation and high reliability, together with the potential advantages of higher efficiency and lower cost, turns the so-called, single-stage boost inverter (SSBI), into a viable competitor to the existing SE-based power conversion technologies.^ The dynamic model is one of the most essential requirements for performance analysis and control design of any engineering system. Thus, in order to have satisfactory operation, it is necessary to derive a dynamic model for the SSBI system. However, because of the switching behavior and nonlinear elements involved, analysis of the SSBI is a complicated task.^ This research applies the state-space averaging technique to the SSBI to develop the state-space-averaged model of the SSBI under stand-alone and grid-connected modes of operation. Then, a small-signal model is derived by means of the perturbation and linearization method. An experimental hardware set-up, including a laboratory-scaled prototype SSBI, is built and the validity of the obtained models is verified through simulation and experiments. Finally, an eigenvalue sensitivity analysis is performed to investigate the stability and dynamic behavior of the SSBI system over a typical range of operation. ^

Sediment and soil organic matter source assessment as revealed by the molecular distribution and carbon isotopic composition of n-alkanes

The assessment of organic matter (OM) sources in sediments and soils is a key to better understand the biogeochemical cycling of carbon in aquatic environments. While traditional molecular marker-based methods have provided such information for typical two end member (allochthonous/terrestrial vs. autochthonous/microbial)-dominated systems, more detailed, biomass-specific assessments are needed for ecosystems with complex OM inputs such as tropical and sub-tropical wetlands and estuaries where aquatic macrophytes and macroalgae may play an important role as OM sources. The aim of this study was to assess the utility of a combined approach using compound specific stable carbon isotope analysis and an n-alkane based proxy (Paq) to differentiate submerged and emergent/terrestrial vegetation OM inputs to soils/sediments from a sub-tropical wetland and estuarine system, the Florida Coastal Everglades. Results show that Paq values (0.13–0.51) for the emergent/terrestrial plants were generally lower than those for freshwater/marine submerged vegetation (0.45–1.00) and that compound specific δ13C values for the n-alkanes (C23 to C31) were distinctively different for terrestrial/emergent and freshwater/marine submerged plants. While crossplots of the Paq and n-alkane stable isotope values for the C23n-alkane suggest that OM inputs are controlled by vegetation changes along the freshwater to marine transect, further resolution regarding OM input changes along this landscape was obtained through principal component analysis (PCA), successfully grouping the study sites according to the OM source strengths. The data show the potential for this n-alkane based multi-proxy approach as a means of assessing OM inputs to complex ecosystems.

Switching patterns and steady-state analysis of grid-connected and stand-alone single-stage boost-inverters for PV applications

Renewable or sustainable energy (SE) sources have attracted the attention of many countries because the power generated is environmentally friendly, and the sources are not subject to the instability of price and availability. This dissertation presents new trends in the DC-AC converters (inverters) used in renewable energy sources, particularly for photovoltaic (PV) energy systems. A review of the existing technologies is performed for both single-phase and three-phase systems, and the pros and cons of the best candidates are investigated. In many modern energy conversion systems, a DC voltage, which is provided from a SE source or energy storage device, must be boosted and converted to an AC voltage with a fixed amplitude and frequency. A novel switching pattern based on the concept of the conventional space-vector pulse-width-modulated (SVPWM) technique is developed for single-stage, boost-inverters using the topology of current source inverters (CSI). The six main switching states, and two zeros, with three switches conducting at any given instant in conventional SVPWM techniques are modified herein into three charging states and six discharging states with only two switches conducting at any given instant. The charging states are necessary in order to boost the DC input voltage. It is demonstrated that the CSI topology in conjunction with the developed switching pattern is capable of providing the required residential AC voltage from a low DC voltage of one PV panel at its rated power for both linear and nonlinear loads. In a micro-grid, the active and reactive power control and consequently voltage regulation is one of the main requirements. Therefore, the capability of the single-stage boost-inverter in controlling the active power and providing the reactive power is investigated. It is demonstrated that the injected active and reactive power can be independently controlled through two modulation indices introduced in the proposed switching algorithm. The system is capable of injecting a desirable level of reactive power, while the maximum power point tracking (MPPT) dictates the desirable active power. The developed switching pattern is experimentally verified through a laboratory scaled three-phase 200W boost-inverter for both grid-connected and stand-alone cases and the results are presented.

Geographic provenancing of unprocessed cotton using elemental analysis and stable isotope ratios

Cotton is the most abundant natural fiber in the world. Many countries are involved in the growing, importation, exportation and production of this commodity. Paper documentation claiming geographic origin is the current method employed at U.S. ports for identifying cotton sources and enforcing tariffs. Because customs documentation can be easily falsified, it is necessary to develop a robust method for authenticating or refuting the source of the cotton commodities. This work presents, for the first time, a comprehensive approach to the chemical characterization of unprocessed cotton in order to provide an independent tool to establish geographic origin. Elemental and stable isotope ratio analysis of unprocessed cotton provides a means to increase the ability to distinguish cotton in addition to any physical and morphological examinations that could be, and are currently performed. Elemental analysis has been conducted using LA-ICP-MS, LA-ICP-OES and LIBS in order to offer a direct comparison of the analytical performance of each technique and determine the utility of each technique for this purpose. Multivariate predictive modeling approaches are used to determine the potential of elemental and stable isotopic information to aide in the geographic provenancing of unprocessed cotton of both domestic and foreign origin. These approaches assess the stability of the profiles to temporal and spatial variation to determine the feasibility of this application. This dissertation also evaluates plasma conditions and ablation processes so as to improve the quality of analytical measurements made using atomic emission spectroscopy techniques. These interactions, in LIBS particularly, are assessed to determine any potential simplification of the instrumental design and method development phases. This is accomplished through the analysis of several matrices representing different physical substrates to determine the potential of adopting universal LIBS parameters for 532 nm and 1064 nm LIBS for some important operating parameters. A novel approach to evaluate both ablation processes and plasma conditions using a single measurement was developed and utilized to determine the "useful ablation efficiency" for different materials. The work presented here demonstrates the potential for an a priori prediction of some probable laser parameters important in analytical LIBS measurement.

A preliminary analysis of the correlation of food-web characteristics with hydrology and nutrient gradients in the southern Everglades

We estimated trophic position and carbon source for three consumers (Florida gar, Lepisosteus platyrhincus; eastern mosquitofish, Gambusia holbrooki; and riverine grass shrimp, Palaemonetes paludosus) from 20 sites representing gradients of productivity and hydrological disturbance in the southern Florida Everglades, U.S.A. We characterized gross primary productivity at each site using light/dark bottle incubation and stem density of emergent vascular plants. We also documented nutrient availability as total phosphorus (TP) in floc and periphyton, and the density of small fishes. Hydrological disturbance was characterized as the time since a site was last dried and the average number of days per year the sites were inundated for the previous 10 years. Food-web attributes were estimated in both the wet and dry seasons by analysis of δ15N (trophic position) and δ13C (food-web carbon source) from 702 samples of aquatic consumers. An index of carbon source was derived from a two-member mixing model with Seminole ramshorn snails (Planorbella duryi) as a basal grazing consumer and scuds (amphipods Hyallela azteca) as a basal detritivore. Snails yielded carbon isotopic values similar to green algae and diatoms, while carbon values of scuds were similar to bulk periphyton and floc; carbon isotopic values of cyanobacteria were enriched in C13compared to all consumers examined. A carbon source similar to scuds dominated at all but one study site, and though the relative contribution of scud-like and snail-like carbon sources was variable, there was no evidence that these contributions were a function of abiotic factors or season. Gar consistently displayed the highest estimated trophic position of the consumers studied, with mosquitofish feeding at a slightly lower level, and grass shrimp feeding at the lowest level. Trophic position was not correlated with any nutrient or productivity parameter, but did increase for grass shrimp and mosquitofish as the time following droughts increased. Trophic position of Florida gar was positively correlated with emergent plant stem density.

``This is your brain on drugs'': The public policy of anti -drug programming. An exploratory analysis of media and other sources of drug information for adolescents

In the 1980s, government agencies sought to utilize research on drug use prevention to design media campaigns. Enlisting the assistance of the national media, several campaigns were designed and initiated to bring anti-drug use messages to adolescents in the form of public service advertising. This research explores the sources of information selected by adolescents in grades 7 through 12 and how the selection of media and other sources of information relate to drug use behavior and attitudes and perceptions related to risk/harm and disapproval of friends' drug-using activities.^ Data collected from 1989 to 1992 in the Miami Coalition School Survey provided a random selection of secondary school studies. The responses of these students were analyzed using multivariate statistical techniques.^ Although many of the students selected media as the source for most of their information on the effects of drugs on the people who use them, the selection of media was found to be positively related to alcohol use and negatively related to marijuana use. The selection of friends, brothers, or sisters was a statistically significant source for adolescents who smoke cigarettes, use alcohol or marijuana.^ The results indicate that the anti-drug use messages received by students may be canceled out by media messages perceived to advocate substance use and that a more persuasive source of information for adolescents may be friends and siblings. As federal reports suggest that the economic costs of drug abuse will reach an estimated $150 billion by 1997 if current trends continue, prevention policy that addresses the glamorization of substance use remains a national priority. Additionally, programs that advocate prevention within the peer cluster must be supported, as peers are an influential source for both inspiring and possibly preventing drug use behavior. ^

A meta -analysis of empirical research studies on resilience among students at -risk for school failure

Being at-risk is a growing problem in the U.S. because of disturbing societal trends such as unemployment, divorce, substance abuse, child abuse and neglect, and the new threat of terrorist violence. Resilience characterizes individuals who rebound from or adapt to adversities such as these, and academic resilience distinguishes at-risk students who succeed in school despite hardships. ^ The purpose of this research was to perform a meta-analysis to examine the power of resilience and to suggest ways educators might improve academic resilience, which was operationalized by satisfactory test scores and grades. In order to find all studies that were relevant to academic resilience in at-risk kindergarten through 12th-grade students, extensive electronic and hardcopy searches were conducted, and these resulted in a database of 421 articles. Two hundred eighty seven of these were rejected quickly, because they were not empirical research. Upon further examination, another 106 were rejected for not meeting study protocol criteria. Ultimately, 28 studies were coded for study level descriptors and effect size variables. ^ Protective factors for resilience were found to originate in physical, psychological, and behavioral domains on proximal/intraindividual, transitional/intrafamilial, or distal/extrafamilial levels. Effect sizes (ESs) for these were weighted and the means for each level or category were interpreted by commonly accepted benchmarks. Mean effect sizes for proximal (M = .27) and for transitional (M = .15) were small but significant. The mean effect size for the distal level was insignificant. This supported the hypotheses that the proximal level was the source of most protective factors for academic resilience in at-risk students followed by the transitional level. The distal effect size warranted further research particularly in light of the small number of studies (n = 11) contributing effect sizes to that category. A homogeneity test indicated a search for moderators, i.e., study variables affecting outcomes, was justified. “Category” was the largest moderator. Graphs of weighted mean effect sizes in the physical, psychological, and behavioral domains were plotted for each level to better illustrate the findings of the meta-analysis. Suggestions were made for combining resilience development with aspects of positive psychology to promote resilience in the schools. ^

Method development for the analysis of smokeless powders and organic gunshot residue by ultra performance liquid chromatography with tandem mass spectrometry

The goal of this project was to develop a rapid separation and detection method for analyzing organic compounds in smokeless powders and then test its applicability on gunshot residue (GSR) samples. In this project, a total of 20 common smokeless powder additives and their decomposition products were separated by ultra performance liquid chromatography (UPLC) and confirmed by tandem mass spectrometry (MS/MS) using multiple reaction monitoring mode (MRM). Some of the targeted compounds included diphenylamines, centralites, nitrotoluenes, nitroglycerin, and various phthalates. The compounds were ionized in the MS source using simultaneous positive and negative electrospray ionization (ESI) with negative atmospheric pressure chemical ionization (APCI) in order to detect all compounds in a single analysis. The developed UPLC/MS/MS method was applied to commercially available smokeless powders and gunshot residue samples recovered from the hands of shooters, spent cartridges, and smokeless powder retrieved from unfired cartridges. Distinct compositions were identified for smokeless powders from different manufacturers and from separate manufacturing lots. The procedure also produced specific chemical profiles when tested on gunshot residues from different manufacturers. Overall, this thesis represents the development of a rapid and reproducible procedure capable of simultaneously detecting the widest possible range of components present in organic gunshot residue.^

Advanced Placement and American education: A Foucauldian analysis of the Advanced Placement Program of the college board

Advanced Placement is a series of courses and tests designed to determine mastery over introductory college material. It has become part of the American educational system. The changing conception of AP was examined using critical theory to determine what led to a view of continual success. The study utilized David Armstrong's variation of Michel Foucault's critical theory to construct an analytical framework. Black and Ubbes' data gathering techniques and Braun and Clark's data analysis were utilized as the analytical framework. Data included 1135 documents: 641 journal articles, 421 newspaper articles and 82 government documents. ^ The study revealed three historical ruptures correlated to three themes containing subthemes. The first rupture was the Sputnik launch in 1958. Its correlated theme was AP leading to school reform with subthemes of AP as reform for able students and AP's gaining of acceptance from secondary schools and higher education. The second rupture was the Nation at Risk report published in 1983. Its correlated theme was AP's shift in emphasis from the exam to the course with the subthemes of AP as a course, a shift in AP's target population, using AP courses to promote equity, and AP courses modifying curricula. The passage of the No Child Left Behind Act of 2001 was the third rupture. Its correlated theme was AP as a means to narrow the achievement gap with the subthemes of AP as a college preparatory program and the shifting of AP to an open access program. ^ The themes revealed a perception that progressively integrated the program into American education. The AP program changed emphasis from tests to curriculum, and is seen as the nation's premier academic program to promote reform and prepare students for college. It has become a major source of income for the College Board. In effect, AP has become an agent of privatization, spurring other private entities into competition for government funding. The change and growth of the program over the past 57 years resulted in a deep integration into American education. As such the program remains an intrinsic part of the system and continues to evolve within American education. ^

Social Network Structure as a Critical Success Condition for Open Source Software Project Communities

In recent years, a surprising new phenomenon has emerged in which globally-distributed online communities collaborate to create useful and sophisticated computer software. These open source software groups are comprised of generally unaffiliated individuals and organizations who work in a seemingly chaotic fashion and who participate on a voluntary basis without direct financial incentive. The purpose of this research is to investigate the relationship between the social network structure of these intriguing groups and their level of output and activity, where social network structure is defined as 1) closure or connectedness within the group, 2) bridging ties which extend outside of the group, and 3) leader centrality within the group. Based on well-tested theories of social capital and centrality in teams, propositions were formulated which suggest that social network structures associated with successful open source software project communities will exhibit high levels of bridging and moderate levels of closure and leader centrality. The research setting was the SourceForge hosting organization and a study population of 143 project communities was identified. Independent variables included measures of closure and leader centrality defined over conversational ties, along with measures of bridging defined over membership ties. Dependent variables included source code commits and software releases for community output, and software downloads and project site page views for community activity. A cross-sectional study design was used and archival data were extracted and aggregated for the two-year period following the first release of project software. The resulting compiled variables were analyzed using multiple linear and quadratic regressions, controlling for group size and conversational volume. Contrary to theory-based expectations, the surprising results showed that successful project groups exhibited low levels of closure and that the levels of bridging and leader centrality were not important factors of success. These findings suggest that the creation and use of open source software may represent a fundamentally new socio-technical development process which disrupts the team paradigm and which triggers the need for building new theories of collaborative development. These new theories could point towards the broader application of open source methods for the creation of knowledge-based products other than software.

A content analysis of food and nutrition television advertisements

Television (TV) reaches more people than any other medium which makes it an important source of health information. Since TV ads often offer information obliquely, this study investigated implied health messages found in food and nutrition TV ads. The goals were to determine the proportion of food and nutrition ads among all TV advertising and to use content analysis to identify their implied messages and health claims. A randomly selected sample of TV ads were collected over a 28-day period beginning May 8, 1987. The sample contained 3547 ads; 725 (20%) were food-related. All were analyzed. About 10% of food-related TV ads contained a health claim. Twenty-five representative ads of the 725 food ads were also reviewed by 10 dietitians to test the reliability of the instrument. Although the dietitians agreed upon whether a health claim existed in a televised food ad, their agreement was poor when evaluating the accuracy of the claim. The number of food-related ads dropped significantly on Saturday, but the number of alcohol ads rose sharply on Saturday and Sunday. Snack ads were shown more often on Thursday, but snack commercials were also numerous on Saturday morning and afternoon, as were cereal ads. Ads for snack foods accounted for the greatest proportion of ads (20%) while fast food accounted for only 7%. Alcohol constituted about 9% of all food and nutrition ads.