NMR, Metabonomics and Molecular Profiles: Applications to the Quality Assessment of Foodstuffs

Nuclear Magnetic Resonance (NMR) is a branch of spectroscopy that is based on the fact that many atomic nuclei may be oriented by a strong magnetic field and will absorb radiofrequency radiation at characteristic frequencies. The parameters that can be measured on the resulting spectral lines (line positions, intensities, line widths, multiplicities and transients in time-dependent experi-ments) can be interpreted in terms of molecular structure, conformation, molecular motion and other rate processes. In this way, high resolution (HR) NMR allows performing qualitative and quantitative analysis of samples in solution, in order to determine the structure of molecules in solution and not only. In the past, high-field NMR spectroscopy has mainly concerned with the elucidation of chemical structure in solution, but today is emerging as a powerful exploratory tool for probing biochemical and physical processes. It represents a versatile tool for the analysis of foods. In literature many NMR studies have been reported on different type of food such as wine, olive oil, coffee, fruit juices, milk, meat, egg, starch granules, flour, etc using different NMR techniques. Traditionally, univariate analytical methods have been used to ex-plore spectroscopic data. This method is useful to measure or to se-lect a single descriptive variable from the whole spectrum and , at the end, only this variable is analyzed. This univariate methods ap-proach, applied to HR-NMR data, lead to different problems due especially to the complexity of an NMR spectrum. In fact, the lat-ter is composed of different signals belonging to different mole-cules, but it is also true that the same molecules can be represented by different signals, generally strongly correlated. The univariate methods, in this case, takes in account only one or a few variables, causing a loss of information. Thus, when dealing with complex samples like foodstuff, univariate analysis of spectra data results not enough powerful. Spectra need to be considered in their wholeness and, for analysing them, it must be taken in consideration the whole data matrix: chemometric methods are designed to treat such multivariate data. Multivariate data analysis is used for a number of distinct, differ-ent purposes and the aims can be divided into three main groups: • data description (explorative data structure modelling of any ge-neric n-dimensional data matrix, PCA for example); • regression and prediction (PLS); • classification and prediction of class belongings for new samples (LDA and PLS-DA and ECVA). The aim of this PhD thesis was to verify the possibility of identify-ing and classifying plants or foodstuffs, in different classes, based on the concerted variation in metabolite levels, detected by NMR spectra and using the multivariate data analysis as a tool to inter-pret NMR information. It is important to underline that the results obtained are useful to point out the metabolic consequences of a specific modification on foodstuffs, avoiding the use of a targeted analysis for the different metabolites. The data analysis is performed by applying chemomet-ric multivariate techniques to the NMR dataset of spectra acquired. The research work presented in this thesis is the result of a three years PhD study. This thesis reports the main results obtained from these two main activities: A1) Evaluation of a data pre-processing system in order to mini-mize unwanted sources of variations, due to different instrumental set up, manual spectra processing and to sample preparations arte-facts; A2) Application of multivariate chemiometric models in data analy-sis.

Morphological and physiological responses of apple trees under photoselective colored nets

In recent years and thanks to innovative technological advances in supplemental lighting sources and photo-selective filters, light quality manipulation (i.e. spectral composition of sunlight) have demonstrated positive effects on plant performance in ornamentals and vegetable crops. However, this aspect has been much less studied in fruit trees due to the difficulty of conditioning the light environment of orchards. The aim of the present PhD research was to study the use of different colored nets with selective light transmission in the blue (400 – 500 nm), red (600 – 700 nm) and near infrared (700 – 1100 nm) wavelengths as a tool to the light quality management and its morphological and physiological effects in field-grown apple trees. Chapter I provides a review the current status on physiological and technological advances on light quality management in fruit trees. Chapter II shows the main effect of colored nets on morpho-anatomical (stomata density, mesophyll structure and leaf mass area index) characteristics in apple leaves. Chapter III provides an analysis about the effect of micro-environmental conditions under colored nets on leaf stomatal conductance and leaf photosynthetic capacity. Chapter IV describes a study approach to evaluate the impact of colored nets on fruit growth potential in apples. Summing up results obtained in the present PhD dissertation clearly demonstrate that light quality management through photo-selective colored nets presents an interesting potential for the manipulation of plant morphological and physiological traits in apple trees. Cover orchards with colored nets might be and alternative technology to address many of the most important challenges of modern fruit growing, such as: the need for the efficient use of natural resources (water, soil and nutrients) the reduction of environmental impacts and the mitigation of possible negative effects of global climate change.

The prevalence of food insecurity and associated factors among households with children in Head Start programs in Houston, Texas and Birmingham, Alabama

The purposes of this study were to determine the prevalence of food insecurity and factors associated with food insecurity among households with children enrolled in Head Start programs in Houston, Texas, and Birmingham, Alabama. This cross-sectional study utilized data gathered from 688 households recruited by convenience sample from two Head Start districts in each city. Interviewers collected data from primary caregivers on demographic characteristics, dietary intake, and the six-item USDA food security module. Chi-square and logistic regression analysis were used to determine the association of food security and demographic characteristics. Comparison of means was used to analyze the association between the child's fruit and vegetable intake and the household's food security status. The prevalence of food insecurity among the sample was 34.9% (95% CI: 31.3%, 38.5%). Characteristics associated with food insecurity were the caregiver's national origin (Foreign-born (ref.) v. U.S.-born, adjusted OR = 0.36, 95% CI: 0.14, 0.94), gender of the child (male (ref.) v. female, adjusted OR = 1.44, 95% CI: 1.03, 2.01), and city of residence (Birmingham (ref.) v. Houston, adjusted OR = 0.20, 95% CI: 0.10, 0.39). Children in food insecure households consumed more daily servings of fruits and vegetables on average (mean = 2.44) than children in food secure households (mean = 2.16, p = 0.04). ^

Determining the feasibility and acceptability of a garden-based nutrition curriculum for preschoolers

Obesity rates around the nation have risen to epidemic proportions. Rates of childhood obesity are at very high levels with 24.4% of preschool-aged children in the U.S. currently considered as overweight or obese. The percentage of childhood obesity is much higher in the southern part of the United States as compared to the rest of the nation. Minority populations, especially African American and Hispanic, are affected more than other ethnic groups. Obesity prevention programs are needed targeting young children <6 years of age from minority populations. Currently, there are few obesity prevention programs that have been implemented and evaluated in children <6 years of age. Gardening programs have been successful in improving the health status of elementary school children by increasing fruit and vegetable intake and increasing preferences for healthier food choices. However, there is no evidence of the feasibility and acceptability of a garden-based obesity prevention program among preschoolers. This pretest study, a classroom-based gardening curriculum program with 16 lesson plans and coordinating activities for preschool age children (3-5 years old) enrolled in Head Start, provides the opportunity to address this need. The study included 103 preschoolers from two centers and 9 teachers or teachers' aides. Qualitative data on feasibility and acceptability was collected from process evaluation forms of individual lesson plans and focus groups with teachers. Teacher questionnaires assessed individual teacher characteristics and provided feedback regarding the curriculum. Quantitative measures of teachers' self-efficacy, attitudes, and knowledge pertaining to nutrition were analyzed from pre and post-test surveys. Results revealed this preschool garden-based nutrition curriculum was both feasible and acceptable. The program improved teacher's self-efficacy, knowledge, and attitudes about nutrition, with teacher's confidence in ability to teach a gardening curriculum increasing from a mean score of 2.14 to 3.00 from pre to post test (P value = 0.0046). These results indicate implementing garden-based nutrition lessons within preschools is achievable. Employing garden-based nutrition lessons in the classroom is the first step in teaching children about nutrition and gardening concepts. Constructing gardening beds for more hands-on learning is the next proposed step in the larger parent study of this program.^

Effects of a health advocacy program to promote physical activity and nutrition behaviors among adolescents: An evaluation of the Young Leaders for Healthy Change program

Background. This study was designed to evaluate the effects of the Young Leaders for Healthy Change program, an internet-delivered program in the school setting that emphasized health advocacy skills-development, on nutrition and physical activity behaviors among older adolescents (13–18 years). The program consisted of online curricular modules, training modules, social media, peer and parental support, and a community service project. Module content was developed based on Social Cognitive Theory and known determinants of behavior for older adolescents. ^ Methods. Of the 283 students who participated in the fall 2011 YL program, 38 students participated in at least ten of the 12 weeks and were eligible for this study. This study used a single group-only pretest/posttest evaluation design. Participants were 68% female, 58% white/Caucasian, 74% 10th or 11th graders, and 89% mostly A and/or B students. The primary behavioral outcomes for this analysis were participation in 60-minutes of physical activity per day, 20-minutes of vigorous- or moderate- intensity physical activity (MVPA) participation per day, television and computer time, fruit and vegetable (FV) intake, sugar-sweetened beverage intake, and consumption of breakfast, home-cooked meals, and fast food. Other outcomes included knowledge, beliefs, and attitudes related to healthy eating, physical activity, and advocacy skills. ^ Findings. Among the 38 participants, no significant changes in any variables were observed. However, among those who did not previously meet behavioral goals there was an 89% increase in students who participated in more than 20 minutes of MVPA per day and a 58% increase in students who ate home-cooked meals 5–7 days per week. The majority of participants met program goals related to knowledge, beliefs, and attitudes prior to the start of the program. Participants reported either maintaining or improving to the goal at posttest for all items except FV intake knowledge, taste and affordability of healthy foods, interest in teaching others about being healthy, and ease of finding ways to advocate in the community. ^ Conclusions. The results of this evaluation indicated that promoting healthy behaviors requires different strategies than maintaining healthy behaviors among high school students. In the school setting, programs need to target the promotion and maintenance of health behaviors to engage all students who participate in the program as part of a class or club activity. Tailoring the program using screening and modifying strategies to meet the needs of all students may increase the potential reach of the program. The Transtheoretical Model may provide information on how to develop a tailored program. Additional research on how to utilize the constructs of TTM effectively among high school students needs to be conducted. Further evaluation studies should employ a more expansive evaluation to assess the long-term effectiveness of health advocacy programming.^

Structural conservations and diversities of dsRNA viruses: Structural studies of the cytoplasmic polyhedrosis virus by electron cryomicroscopy and 3D reconstruction

The Reoviridae virus family is a group of economically and pathologically important viruses that have either single-, double-, or triple-shelled protein layers enclosing a segmented double stranded RNA genome. Each virus particle in this family has its own viral RNA dependent RNA polymerase and the enzymatic activities necessary for the mature RNA synthesis. Based on the structure of the inner most cores of the viruses, the Reoviridae viruses can be divided into two major groups. One group of viruses has a smooth surfaced inner core, surrounded by complete outer shells of one or two protein layers. The other group has an inner core decorated with turrets on the five-fold vertices, and could either completely lack or have incomplete outer protein layers. The structural difference is one of the determinant factors for their biological differences during the infection. ^ Cytoplasmic polyhedrosis virus (CPV) is a single-shelled, turreted virus and the structurally simplest member in Reoviridae. It causes specific chronic infections in the insect gut epithelial cells. Due to its wide range of insect hosts, CPV has been engineered as a potential insecticide for use in fruit and vegetable farming. Its unique structural simplicity, unparalleled capsid stability and ease of purification make CPV an ideal model system for studying the structural basis of dsRNA virus assembly at the highest possible resolution by electron cryomicroscopy (cryoEM) and three-dimensional (3D) reconstruction. ^ In this thesis work, I determined the first 3D structure of CPV capsids using 100 kV cryoEM. At an effective resolution of 17 Å, the full capsid reveals a 600-Å diameter, T = 1 icosahedral shell decorated with A and B spikes at the 5-fold vertices. The internal space of the empty CPV is unoccupied except for 12 mushroom-shaped densities that are attributed to the transcriptional enzyme complexes. The inside of the full capsid is packed with icosahedrally-ordered viral genomic RNA. The interactions of viral RNA with the transcriptional enzyme complexes and other capsid proteins suggest a mechanism for RNA transcription and subsequent release. ^ Second, the interactions between the turret proteins (TPs) and the major capsid shell protein (CSPs) have been identified through 3D structural comparisons of the intact CPV capsids with the spikeless CPV capsids, which were generated by chemical treatments. The differential effects of these chemical treatment experiments also indicated that CPV has a significantly stronger structural integrity than other dsRNA viruses, such as the orthoreovirus subcores, which are normally enclosed within outer protein shells. ^ Finally, we have reconstructed the intact CPV to an unprecendented 8 Å resolution from several thousand of 400kV cryoEM images. The 8 Å structure reveals interactions among the 120 molecules of each of the capsid shell protein (CSP), the large protrusion protein (LPP), and 60 molecules of the turret protein (TP). A total of 1980 α-helices and 720 β-sheets have been identified in these capsid proteins. The CSP structure is largely conserved, with the majority of the secondary structures homologous to those observed in the x-ray structures of corresponding proteins of other reoviruses, such as orthoreovirus and bluetongue virus. The three domains of TP are well positioned to play multifunctional roles during viral transcription. The completely non-equivalent interactions between LPP and CSP and those between the anchoring domain of TP and CSP account for the unparalleled stability of this structurally simplest member of the Reoviridae. ^

Effects of skin characteristics on impact response and bruising of apples and pears

Skin properties have an important influence on impact parameters and bruising. Skin deformation at puncture (a measure of the turgidity of the fruit skin) is negatively correlated with bruise volume in Golden apples after cold storage.

Bruise susceptibility in pome fruits under different loading and storage conditions

Samples of "Golden" and "Granny Smith" apples and "Conference" and "Doyenne of Cornice" pears have been tested. A great effect of storage conditions has been detected for pear but not for apple varieties. Both apple cultivars show to be equally resistant to quasi-static and to dinamic loading while pear varieties show great differences. All these effects can be quantified in order to describe mathematically species and varieties behavior.

Mechanical segregation of apricot varieties and their ripeness levels at harvest

Some laboratory tests consisting on quasi-static compression and puncture forces carried out on twelve varieties of apricot during 1990 and 1991 were effective in sorting them. These mechanical properties show a high correlation w i th the ethylene production rate per fruit, so allowing to discriminate between ripeness levels at harvest. In this study it is also demonstrated that puncture seems to be the less variable mechanical test. The values (N/mm) obtained with it show a highly significant correlation with compression resistance and with quasi-static compression damage of the fruits.

Effects of precooling and degreening treatments on the susceptibility of peach and citrus to handling damage.

Mechanical damage to fruits results from a combination of fruit properties and of damage inflicting effects by the handling equipment. Treatments were applied to fruits which affect mechanical damage susceptibility: precooling for stone fruits and degreening to citrus fruits. Laboratory tests (compression, impact, tumbling, abrasion) and field tests (damage in the handling lines) were applied to (3) peach, (2) apricot, (2) orange and (1) lemon varieties. Hydroor-air-cooling influence positively peach and apricot firmness and cause a significant reduction in: number of bruised fruits, and size of visible bruise, when combined with a low level of loading during handling. Degreening also affects the resistance of citrus fruits to handling, when compared to green fruits.

Models for the prediction of handling damage in citrus and peaches grading lines, related to their physical properties.

Fruit damage during harvesting and handling is a standing problem, particularly for susceptible fruits like peaches and apricots. The resulting mechanical damage is a combination of fruit properties and damage inflicting effects due to procedures and to the equipment. Nine packing lines in the region of Murcia (SE Spain) have been tested with the aid of two different-size electronic fruits IS-100. Probabilities of impacts above three preset thresholds (50 g's, 100 g's and 150 g's) were calculated for each transfer point. Interaction fruit-packing line tests have been also performed in order to study the real incidence of packing lines on natural produce: apricots (1 variety), peaches (3 v.), lemons (1 v.) and oranges (3 v.). Bruises of handled and not handled samples of fruits were compared.

Vegetable situation.

Mode of access: Internet.

Trade reference roster; containing names of shippers, brokers and jobbers who have signed agreements making their transactions subject to the standard trading rules or who are members of an association that has adopted the said "rules."

Mode of access: Internet.

Information pertaining to farm, home, and community : with special reference to type-of-farming area.

Errata sheets tipped in.

Consumers' taste reactions to three experimental blends of orange and grapefruit juice.

Mode of access: Internet.