Milk oligosaccharides play a prebiotic role in the growth and establishment of a balanced gut flora in infants, selectively enriching the beneficial and protective bacteria that promote healthy immune and cognitive development. By acting as anti-pathogenic agents, milk oligosaccharides hold great promise as high quality supplements that can help support the health of any population with comprised immune systems. In order to provide sufficient quantities of oligosaccharides for clinical and functional testing, this work optimized several steps of activated carbon oligosaccharide filtration from human milk permeates. For measurement of process efficiency, a carbohydrate assay was first developed by treating milk products treated with Carrez clarification. Carbohydrate adsorption tests on activated carbon followed by total carbohydrate and lactose quantification showed that ratios of activated carbon to carbohydrate from 5-10 had better carbohydrate retention. In addition, permeates filtered with finer mesh charcoal (20-500) at a charcoal/carbohydrate ratio of 10 had the majority of carbohydrates adsorbed. Optimized conditions found in this study can be applied to purification from bovine milk whey permeate, a dairy industry waste stream. With further refinements to other steps of the purification technique, activated carbon filtration can be adapted as an effective method for mass extraction of health promoting oligosaccharides.
Uridine 5’diphopho-α-D-galactopyranoside (UDP-Gal) is a sugar nucleotide commonly used by animals, plants, and bacteria as the activated donor of galactose (Gal). The salvage pathway is the simplest route to synthesize UDP-Gal and involves three different enzymes: Streptococcus pneumoniae TIGR4 galactokinase (SpGalK), Bifidobacterium longum UDP-sugar pyrophosphorylase (BLUSP), and Pasteurella multocida inorganic pyrophosphatase (PmPpA)1. The optimum conditions for the entire reaction pathway in a one-pot multienzyme system were determined. Then, using these conditions, synthesis of UDP-Gal was scaled-up to the gram-scale. Production of UDP-Gal was successfully attempted using 1 g of Gal and a reaction yield of 70% was obtained. No isolation yield was determined because of incomplete purification.
The posttranslational modification of tyrosine sulfation is critical for the binding affinity and peptide specificity of many G-protein coupled receptors (GPCRs). Using a log-odds position specific scoring matrix (PSSM), 64 tyrosine sulfation sites in 31 different peptide binding GPCRs were scored. Higher scores indicated a higher degree of similarity between confirmed sulfation sites and that tyrosine. While our PSSM did not account for characteristics such as clustering and conservation, the sites nonetheless exhibited these characteristics. Most predicted sites were located in the binding pockets of GPCRs, which is also consistent with confirmed tyrosine sulfation sites. Binding affinity or peptide specificity decreased when our tyrosines were mutated into alanine or phenylalanine, indicating the important role of the tyrosine in ligand binding.
Gene Expression Differences Between Fayoumi and Leghorn Chicken and their Impact on NDV Infection Resistance.
NDV, Newcastle Disease Virus, is a negative sense single RNA strand virus that infects many wild Avian species. While NDV poses no major threat to humans directly, it causes major losses in the poultry industry and disrupts the poultry food supply. It has been shown that differences in immune response has associated genetic factors that can affect susceptibility or resistance. Determining what those genetic factors are in chicken response to NDV can greatly increase our knowledge of host-pathogen interaction, but also improve genetic selection for resistant birds. And so, the goal of this project was to determine difference in gene expression between two distinct chicken breeds, Fayoumi and Leghorn. Past research has demonstrated that Fayoumi are more resistant to viral infections while Leghorn are more susceptible (Wang, 2014). Chickens were hatched and subjected to heat treatment and viral infection. Samples were collected at 3 different time points: 2 dpi, 6 dpi, and 10 dpi and mRNA was extracted. cDNA was generated and analyzed using qPCR. Analysis of the data demonstrates that Fayoumi gene expression for TLR3, TLR7, IRF7, CD8A in spleen and TLR3, PRKCD, and CD8A in lung is greater when compared to Leghorn. The difference in expression of these genes between these two lines are possibly associated with why Fayoumi has a greater resistance to NDV infection, however further studies must be conducted to show a direct connection between this difference in gene expression and the genetic mechanism used to cause the difference in resistance between Fayoumi and Leghorn.
Shade avoidance is a series of plant responses elicited when a plant experiences a reduced ratio of red to far-red (R:FR) light. In this study we investigate the genetic basis of shade avoidance by examining hypocotyl and root growth of Arabidopsis thaliana in four natural variants and three mutant lines. The four natural variants were Columbia (Col), Kondara (KON), KNO-18, and RRS-10. The three mutant lines, numbered 19, 38, and 52, were TDNA lines with insertions in the gene of interest. The average hypocotyl length was found to be longer in the shade conditions for every genotype. From the difference between the average hypocotyl length in sun and shade conditions, it was found that 19 may be a high responder, 38 responds similarly to wild-type Col, and 52 may be a low responder. After 3 days of shade treatment, there was little difference between the average root length between genotypes or between plants grown in sun or shade conditions. However, primary root length measured after 10 days of light treatment revealed that the average root length was longer in the shade conditions for every genotype. This suggests that roots show increased growth in response to shade. These results are relevant because knowledge gained from this experiment can be utilized in the agricultural industry when deciding crop spacing and land conservation.
Examination of posttranscriptional regulation and de novo transcription during early embryonic development using RNA-Seq
Background: Embryonic genome activation (EGA) is a critical time during mammalian development during which an embryo initiates transcription of many important genes after a long period of transcriptional silence and dependence on stored transcripts in the oocyte. In bovine, EGA typically occurs at the 8-16 cell stage. Before EGA, the oocyte and early embryo regulate protein production using post-transcriptional mechanisms like cytoplasmic poly-adenylation. The global targets of this regulation are not very well understood. Despite the fact that the oocyte is mostly transcriptionally silent, there is some evidence of de novo transcription between fertilization and EGA. In fact, it has been suggested that there is a minor EGA earlier in development at the 4-cell stage. This study focuses on that transcription and on the post-transcriptional regulation of stored maternal mRNAs. Results: Our results support and characterize the notion of EGA. They yielded inconclusive results with respect to cytoplasmic poly-adenylation and post-transcriptional modification. They also suggest that androgen receptors that play a role in oocyte maturation may also play a role in signaling between embryos and the mother during early embryonic development.
Movement of dissolved reactive phosphorous from soils to bodies of water in relation to eutrophication
Dissolved reactive phosphorous (DRP), or soluble reactive phosphorous (SRP), is a nutrient that can flow from agricultural land to bodies of water, causing eutrophication, which poses a threat to the health and efficiency of waterways. In order to prevent eutrophication from harming bodies of water, it is crucial to understand the movement of these nutrients, which was the main objective of this research.
Levels of DRP/SRP were analyzed by studying the sorption and desorption capacities of soils from different areas using the Bridgham et al (2001) method. This study found that sorption and desorption capacities varied between soil samples taken at different locations, indicating the level of effectiveness of different areas at retaining high amounts of phosphorous while releasing very low levels.
The information from this study can be used to evaluate potential areas for restoration based on the soil’s sorption and desorption abilities. In restoring wetland buffers, it is possible to benefit the environment by protecting water quality, using agricultural land more efficiently, managing waterways, and reducing or preventing eutrophication.
Protein synthesis is an important facet of any biological process. In the cardiac muscle, it is especially important in determining the state and condition of the muscle. One cardiovascular condition that is closely related to protein synthesis is cardiac hypertrohy. Cardiac hypertrophy is a thickening of the heart muscle, which results in a decrease in size of the chamber of the heart, including the left and right ventricles. This hypertrophy is a common known response to the increased workload of the heart that is associated with hypertension. The larger heart and cardiac cell mass means that more protein is necessary to make up the additional mass of the heart. This project investigated which proteins are specifically synthesized for the sake of contributing to cardiac hypertrophy and exactly how the rate of protein synthesis increases when cells are induced with hypertrophy by the cancer drug Doxorubicin. Using Click-iT AHA Protein Labeling Kits, and SDS-PAGE Gels, we were able to determine that the rate of protein synthesis increases by an average of 30.26%. In addition, we determined that Meclofenamate sodium, a Non-Steroidal Anti-Inflamatory Drug (NSAID), decreased the rate of protein synthesis in cardiac cells while simultaneously decreasing protein degradation, an important step in determining how exactly NSAIDs affect cardiotoxicity and induce cardiovascular disease.
Identification and Quantification of Fusarium oxysporum f. sp. vasinfectum Infestation in Soil with a Real Time Polymerase Chain Reaction Assay
A real-time PCR protocol was developed to detect specific concentrations of Fusarium oxysporum f. sp. vasinfectum (FOV) race 4 in soil samples, using publicly available primers specific to FOV race 4. Environmental samples of equivalent spore concentrations from a range of 10^3-10^6 spores/ml were detected with this protocol. Soispore concentrations in this range have been shown to cause Fusarium wilt; this protocol detected both artificially spiked samples as well as environmental samples within this critical range. Pending further validation, this protocol will serve as an appropriate means of testing soil and determining soil spore load.
Restoration of native grasslands in California is expensive, mostly due to the high cost of seeds. Although restoration can increase biodiversity and ecosystem services, ecosystem services do not generate revenue. In order to reconcile economics and the need for biodiversity in California annual grasslands, this study focuses on discovering how a grass species’ status as an annual or perennial plant affects biodiversity and relates to cost. We studied several restoration methods based on strip seeding, where strips of various widths were alternatively seeded or left unseeded. We hypothesized that over time seeds from the seeded strips would establish in the unseeded strips, a cost-efficient means of restoration. Requiring $825 and $1250 respectively, the 33% seeded and 50% seeded (wide) treatment demonstrated potential as cost efficient means of habitat restoration. The 33% treatment resulted in approximately 12.5 species at the plot level for species richness, while the 50% wide treatment yielded 12.4 species. Botanical composition of seeded and unseeded strips indicated that strip seeding is a successful low-cost method for Elymus glaucus, but other native perennial grasses failed to establish in unseeded strips.
Understanding the Structural and Molecular Basis of Postharvest Chilling Injury in Tomato Fruit (Solanum lycopersicum L.)
Postharvest Chilling Injury (PCI) is a devastating disorder that leads to loss of produce. In this study two approaches were taken to better understand the development of PCI in tomato (Solanum lycopersicum L.) a species susceptible to PCI. First, the spatio-temporal development of PCI symptoms in developing fruit was assessed using Magnetic Resonance Imaging (MRI). Second, the CBF1 gene from Arabidopsis thaliana was cloned so that the gene may be engineered into tomato fruit to improve the tomato fruit response to chilling. MRI data indicates that chilling injury affects each tomato tissue type (the pericarp, the locular tissue and the columella) differently with greater effects seen in green fruit due to their continuation of ripening and various metabolic processes. A 780 bp fragment corresponding to the coding sequence (CDS) of CBF1 was amplified from Arabidopsis genomic DNA and attempts to sub-clone into pGEM-T Easy Vector® are underway.
In N.tabacum, chloroplastic protein NRIP1 mediates the innate immune receptor recognition of p50, the viral effector protein of the Tobacco Mosaic Virus. The presence of stromules was observed extending from the stroma of the chloroplasts towards the nucleus. Researchers discovered that ferritin, another chloroplastic protein, does not complete a similar process to NRIP1, instead, it is always located in small quantities in the nucleus that do not appear to change during N-mediated defense.
Recommended Methods Such As Folding, Recutting, and Bagging Techniques on Leaves to Measure Stem Water Potential in Cherries, Walnuts, Pistachios, and Grapes
According to the soil-plant-atmosphere-continuum (SPAC) model, water from soil is under tension within a plant, which allows for water to be transported from the root to the leaf of a plant. Water potential is a way of quantifying the tension in the plant, which directly correlates to its water stress. The Scholander pressure chamber was used to test different methods of finding stem water potential in plants and its impact on the measurement taken. This research was done on adjacent leaves, testing for the difference in stem water potential in leaves that were folded and unfolded, and between those cut directly from a branch by its petiole and those with petioles recut after being pulled off a branch. Additionally, research was done on pistachio trees by comparing branches versus individual leaflets, as there is a problem with latex being mistaken for water. Adjacent leaves were placed into light-proof bags and placed into a pressure chamber, where pressure was slowly added until water visibly left the xylem of the leaf. It was found that folding and recutting generally did not have an impact on the measured stem water potential, which will allow farmers to adapt these methods to their convenience instead of worrying about whether or not they need to fold their leaves or cut a leaf only once to fit into the pressure chambers. With pistachio trees, bagging an entire branch produces very close results to bagging just a terminal leaflet, which will allow agriculturalists to pick which method to use that would produce the clearest measure of water potential. In the future, this research can possibly be used to determine a universal and most accurate method of finding stem water potential.
NSAIDs are the most commonly used drugs around the world. They are linked to many side effects such as heart attack and stroke. More specifically, NSAIDs affect the proteasome, a multimeric enzyme that degrades damaged and old protein. Without proteasomes, the body would not be able to function. Indomethacin is a common NSAID used to treat pain, inflammation, arthritis, and patent ductus arteriosis. It has been shown to cause gastrointestinal problems, however, the effects of the drug on proteasomes has not been studied. Due to the high abundance of proteasome activity in the heart, eight mice were injected with indomethacin to investigate the effects of the drug on proteasome activity in the heart. Using western blots, biological assays, and data analysis on homogenized heart samples, it was determined that the control mice and indomethacin injected mice had similar proteasome activity levels. However, trends suggest that proteasomes were being slightly effected by the drug.
Lucy Cui is working with Professor Eduardo Blumwald in the Plants Science Department to genetically engineer crops to be more drought and salt tolerant. A model plant, Brachypodium, is used in place of wheat, rice, corn, barley, oats, and other monocots, to determine which genes to turn on in order to prolong crop life and produce greater yields with less water. Another model plant,Arabidopsis, is used to understand the effect of genes that control pH in dicots, or flowering plants, on plant development. With the growing human population and loss of quality agricultural land, it is important to create these crops in order to continually feed the demanding population despite the harsh, arid conditions and to efficiently use the limited amount of agricultural land.