Selecting individual virus particles in micrographs, or boxing, is done frequently by hand, but the automation of this process would allow for greater efficiency during the process of 3-D reconstruction. Two methods of semi-automated boxing, autoboxing and autoboxing from references, were tested, both of which are two automatic boxing settings found as a part of the program Electron Micrograph Analysis (EMAN), version 1.9. Autoboxing from references appears to pick up slightly less undesirable particles, each of which were reviewed manually after automatic selection. After boxing, selected virus particles can then be used to form a 3-D reconstruction of the virus, whose accuracy is dependent on the quality of particles selected, and help with both virus analysis and virus-like particle (VLP) creation. Particles selected from autoboxing from references, however, appear to form less accurate reconstructions of viruses after 3-D reconstruction and 10 cycles of refinements.

The design of a conventional syringe includes a dead space between the needle and barrel of the syringe called the hub. This space collects liquid that once drawn up, cannot be plunged out. As a result, the substance remaining in the hub is inevitably wasted and difficult to account for. Little information is known about the true amount of hub loss that is expected from a given vial of controlled substances. We calculated and measured the hub volume using a variety of different syringes and vial volumes to determine the anticipated substance loss. Additionally, we tested the effects of interpersonal variability, number of syringes used per vial, volume drawn up in each syringe, and other real world conditions. Prior to finding the hub loss per vial, we calculated the hub volume of three different syringe and needle types. The two conventional 1ml syringe types used had means of 0.068ml and 0.066ml of hub loss. The hubless tuberculin syringe had a mean of 0ml of hub loss. After calculating the hub volume of each syringe, we tested expected hub loss from 10ml and 20ml vials. The trend of these tests showed an increase in hub loss as the number of syringes used increased. When 50 syringes were used to draw out half of a 10ml vial, there was a mean of 3.3ml of hub loss. When 10 syringes were used to draw out half of a 10ml vial, there was a mean of 0.5ml of hub loss. Of the recorded hub loss, the range of all datasets ranged from 0.1 to 2.9. These wide ranges display inconsistent amounts of hub loss per vial. The inconsistency may have been due to real world variables such as imprecise starting vial volumes, interpersonal variability, air bubbles, and unstable pressure within vials. The aim of this study was to establish a reasonable range of hub loss to monitor the usage of controlled substances. Based on our data, a reasonable range of hub loss depends on the number of syringes used to draw from a single vial, with the volume per draw not significantly affecting hub loss. As an example, a reasonable amount of hub loss from a vial when 10 syringes are used is 0.1ml-1.07ml. When 50 syringes are used, 1.99ml-4.53ml. The study may also be used to improve laboratory protocols so hub loss can be minimized.

While a great deal is known about the effects of plant circadian clocks on general biological functions, the molecular mechanisms behind this regulation are somewhat uncertain. Sunflowers with known point mutations were obtained, primers were designed, and restriction enzymes selected for each mutant. The primers were tested on wild-type DNA to determine whether they effectively amplified DNA fragments around the desired point mutations. Since the mutations are in genes associated with circadian clocks, this work will help facilitate experiments in this field in the future.

In order to defend itself from various pathogens, plants use various methods including ROS secretion by RbohD in order to eliminate pathogens. Various signaling cascades and protein interactions can lead to ROS secretion, yet only few have been studied. Some of the unstudied protein interactions are the ones between RLCKs and RbohD, yet here we provide this information through a method known as yeast two hybrid. These findings will highlight the direct roles that RLCKs play in ROS secretion.

The worm C. elegans is a transparent, free-living nematode that has shown to be an efficient and effective model organism. Since 65% of human disease genes have homologues on the C. elegans genome, it has great potential to be an invaluable research organism in the health and biomedical sciences. Researchers studying the C. elegans worm, however, have no cost-effective method of distinguishing homologous chromosomes. This becomes a problem when investigating its reproductive processes such as chromosomal loss in aneuploid individuals. In our project, we propose a cost-effective method of distinguishing homologous chromosomes through size differences on specific regions on the genome which are the result of indel polymorphisms. These size differences can be amplified through PCR and distinguished through gel electrophoresis. We analyzed a dataset of all indel polymorphisms referenced to the N2 strain on significant number of C. elegans strains. We were able to find six strain pairs which had size polymorphisms on all six chromosomes. Laboratory work confirmed our method as a viable, cost-effective solution to distinguishing homologous chromosomes. Our research aids in better understanding the reproductive processes of C. elegans worms which may increase our capacity to utilize it as an effective research organism.

As the most harvested crop in the United States, maize (Zea mays) serves as a source of food, livestock feed, and biofuel. Pests, pathogens, and abiotic stresses contribute to significant crop losses, and the stresses, particularly drought, have only been exacerbated by climate change and rising global temperatures. All higher plants including maize produce an array of specialized metabolites that coordinate the plant’s interaction with the environment. Among these metabolites, diterpenes constitute the largest and most diverse group of compounds and have critical functions in the defense of maize against pest and pathogens. Recently, two diterpene synthase (diTPS) enzymes were identified in maize and shown to play a role in these stress responses: kaurene synthase 2 (KS2) and kaurene synthase 4 (KS4). While both proteins utilize the same substrate, ent-copalyl diphosphate (ent-CPP), which is produced by the class II diTPS Anther Ear2 (An2), the two enzymes produce different products. Investigation of the structural-functional differences of these two enzymes through expression in Escherichia coli (E. coli) and activity analysis with gas chromatography/mass spectrometry (GC/MS) enabled the identification of several amino acid residues that are important for the distinct enzyme functions. These findings contribute to our understanding of the chemical diversity of plant diterpene metabolism, and may be an opportunity for improving stress resistance in maize.

Tyrosine sulfation is a permanent posttranslational modification to a protein that regulates protein-protein interactions. This project focuses on predicting potential tyrosine sulfation sites in type II cadherins, transmembrane proteins dependent on calcium-ion binding. Evidence from statistical data, 3-D protein modeling, and conservation data supports that tyrosine sulfation could occur in type II cadherins. Two of the predicted potential tyrosine sulfation sites showed to contain known calcium-binding sites. However, a study has shown tyrosine sulfation to decrease calcium-ion binding in a particular peptide hormone. If this study is true for all proteins, the location of tyrosine sulfation sites near calcium-binding sites would be impractical. Further research would be needed to clarify if tyrosine sulfation actually occurs in cadherins and the effect it might have on cadherin function.