Past Projects‎ > ‎

2014-15

Brennan Clark and 
Prashant Godishala - Cellular and Molecular Biology

This study shows that lengthening the culture period for pre-transplantation adult porcine islets from 7 days to 30 days significantly lessens the chance of immunorejection after xenotransplantation for type 1 diabetes. Immunofluorescence staining was chosen to analyze antigen expression, islet viability, and insulin production after culture periods of 7, 15, and 30 days. Because an inexpensive humidity chamber to provided 95% humidity was not available, an inexpensive humidity chamber was engineered from materials purchased at Target and found in the laboratory. Additionally, silane gold-coated slides were optimized for immunofluorescence staining. Immunocytochemistry staining showed that after 30 days, there was a significant reduction in Gal+ cells and in CD45+, Class I, and Class II antigens (p < 0.05), all of which are associated with immunorejection of porcine islets. Additionally, there was a significant increase in insulin production over a 30-day culture period (p < 0.05), as well as a significant reduction in mononuclear cells that are responsible for immunorejection. Furthermore, viability staining showed that a culture period of 30 days provides a rehabilitative environment for islets that experience stress after extraction (p < 0.05). Finally, RT-PCR was done to validate results. RT-PCR showed no significant change in gene expression of insulin over a 30-day culture period (p > 0.05). As a result of our work, our research site is now running quality control trials to determine efficacy of incubating all pre-transplantation adult porcine islets for 30 days.

Jack Dickinson and Andrew Sit - 

In this project, a state-of-the art spaceflight fabrication tracking system was engineering that provides real-time data to efficiently track and manage inventory components that are being used to manufacture the James Webb Space Telescope and the Geostationary Operational Environmental Satellite Series-R. Radio frequency identification (RFID) hardware was chosen over universal product code (UPC) barcoding and global positioning systems (GPS) based on a paper by Ong & Forgione (2013) that indicated RFID has great potential to “improve accountability and maintenance of…aviation parts that cost $5,000 or more,” which was our goal. Because commercial middleware required to run RFID hardware is expensive, an inexpensive workaround was devised by programming two layers of middleware that were integrated into a back-end database, designed using Microsoft Access. Blender, which is open-source three-dimensional graphics software, was used to integrate three-dimensional modeling into the system. Furthermore, for ease of use by Millennials, the Splashtop remote desktop application from Apple was integrated into the system to enable access to the database from mobile devices. A cost-benefit analysis of the system showed a profit margin of 180% over ten years. The system is engineered so that it can be replicated to support large-scale and advanced aerospace programs with the goal of expanding the use of RFID technology in the aerospace industry.

Siddarth Eswarachari and Moira Southern - Bioinformatics

Carcinogenic aromatic hydrocarbons are being released into water supplies in increasing concentrations each year. One technique to bioremediate these carcinogens is to bioengineer bacteria to catabolize (degrade) the aromatic hydrocarbons. The bacteria Pseudomonas putida F1 shows promise in bioremediation of aromatic hydrocarbons; however, before P. putida F1 can be bioengineered to catabolize aromatic hydrocarbons, ways in which P. putida F1 catabolize “more appetizing” aromatic nutrients, such as homogentisate, must be found. We focused on the homogentisate catabolic pathway because this pathway is the central route into which the aromatic amino acids L-phenylalanine and L-tyrosine funnel in the bacteria P. putida U and P. putida KT2440, which are closely related to P. putida F1. Using the online databases FASTA in GenBank, BLAST, T-COFFEE, and WebLogos, we compared protein sequences for P. putida F1 to other closely related bacterial strains that included P. putida KT2440, P. aeruginosa PAO1, P. fluorescens Pf0-1, and Comamonas testosterone CNB-1 to identify substrates and enzymes in the pathway by which homogentisate is catabolized by P. putida F1. We then used TMHMM and PSORTb to determine the transporter and regulatory proteins for the homogentisate pathway in P. putida F1. E-values were used to validate best-fit protein sequences, showing 100% accuracy for choices of enzymes identified for the homogentisate pathway in P. putida F1. Consequently, our results were published on GENI-ACT, which is a database that contains genomic information on 2775 bacteria.

Darartu Gamada and Zhuang (Maxwell) Miao - Computational Biology and Bioinformatics

We investigated mechanisms by which organic triblock copolymers can serve as molecular band-aids in sealing cardiac muscle-cell membranes that have been damaged by Duchenne muscular dystrophy. Molecular dynamics simulations were carried out on copolymers and their hydrophilic and hydrophobic subunits at varying physical and chemical compositions to examine effects of molecular structure and temperature on the mechanism by which the copolymers insert into healthy, partially damaged, and fully damaged muscle-cell membranes. The collapse or expansion of the copolymers was monitored by examining the radius of gyration and solvent accessible surface area of copolymers during the insertion processes, a technique that is novel to the field.

Results showed that copolymers significantly increase in size after insertion into damaged membranes (p < 0.05), and elevated temperatures significantly enhance insertion (p < 0.05). Additionally, short-chained triblock copolymers show the most potential in repairing partially damaged membranes while larger triblock hydrophobic copolymers are most effective in repairing fully damaged membranes. This suggests that short-chain triblock copolymers are most effective in early stages of muscular dystrophy while longer-chain triblock copolymers are most effective in the later more severe stages of muscular dystrophy. Results also suggest that hydrophobic subunits of triblock copolymers are the driving force behind insertion while the hydrophilic subunits cause the expansion of the copolymers within damaged membranes. Furthermore, none of the copolymers studied inserted into undamaged membranes at any temperatures simulated, suggesting that the copolymers may safely serve as molecular bandages without affecting undamaged tissue.

Sofie Kim and Jake Levy - Chemistry

Because of its ability to serve as a linkage within larger molecules, 1,2,3-triazole derivatives are widely used in pharmaceuticals that require linkages to facilitate stability, utility, and permeability. This study reports the first documented synthesis and optimization of the 4,5- (bis)trimethylsilyl-5-ethynyl-1,2,3-triazole; the 5-ethynyl-1,2,3-triazole; and the 4-trimethylsilyl- 5-ethynyl-1,2,3-triazole, using environmentally friendly and cost-effective synthesis protocols.

Synthesis of the 4,5-(bis)trimethylsilyl-5-ethynyl-1,2,3-triazole involved a 1,3-dipolar cycloaddition reaction between benzyl azide and 1,4(bistrimethylsilyl)-1,3-butadiyne (BTMSBD). Optimization of this reaction, which involved testing varying quantities of BTMSBD, which gave a maximized yield of 82%. This synthesis was followed by a silver tetrafluoraborate-, copper(II) sulfate-, and sodium L-ascorbate(+)-catalyzed desilylation of the 4,5-(bis)trimethylsilyl-5-ethynyl-1,2,3-triaozle to synthesize the 5-ethynyl-1,2,3-triazole. Optimization of the 5-ethynyl-1,2,3-triazole involved varying time, temperature, and reagent quantities, and gave a maximized yield of 73%. During optimization of the 5-ethynyl-1,2,3-triazole, a less expensive silver ion catalyst, silver nitrate, was tested, which resulted in the unexpected synthesis of 4-trimethylsilyl-5-ethynyl-1,2,3-triazole. The 4-trimethylsilyl-5-ethynyl-1,2,3-triazole was optimized in terms of reagent quantities, time, and temperature to give a maximized yield of 92%. These results are important in the context of chemoselectivity, illuminating that small differences in time, temperature, and amounts and choice of reagents can lead to significant distinctions in the final product. The optimized protocols developed for the three 5-substituted-1,2,3-triazoles reported in this paper will be published and then added to a chemical library to enable syntheses of additional 1,2,3-triazole derivatives for use in pharmaceutical drug discovery.

Grace Kirkpatrick and Annie McFarland - Biology

    This study reports significant damage to dendrites (neural information receivers in synapses) in the CA1 stratum radiatum region of the hippocampal due to hyperglycemia. Hyperglycemia is a disorder caused by abnormally high blood glucose levels (> 125 mg/dL) that puts 80% of extremely preterm babies born each year at risk for decreased brain mass, intraventricular hemorrhage, blindness, and possible damage to the hippocampus, which is the area of the brain responsible for memory and spatial reasoning (1).

    Fluorescent immunohistochemistry was selected to stain for greater sensitivity of dendrite visualization in tissues of the CA1 stratum radiatum region of the hippocampus from a hyperglycemic rat model. When some tissues stained poorly with the primary antibody anti-beta tubulin, chosen for its structural component of microtubules and critical role in axon growth and maintenance, a small-scale test was developed using the primary antibody microtubule associated protein-2 (MAP2) to isolate the staining problem. Results suggested that MAP2 was more effective in staining. Then, Adobe Photoshop CS6 image analysis was pioneered in the lab to quantify fluorescence from stained dendrites by fluorescence of tagged dendrites in pixesl, rather than numbers of dendrites expressed relative to number of neurons (i.e. dendrites/neuron). To eliminate bias, Photoshop CS6 analyses were blinded. Adobe Photoshop CS6 worked well, effectively eliminating the need to manually count dendrites, thus decreasing error and time needed for analysis. Results showed significant damage to dendrites in tissues of the CA1 stratum radiatum region of the hippocampus due to hyperglycemia (p = 0.024).

Rustam Kosherbay - Computer Science

The purpose of this study was to program a flight simulator with a virtual drone that generates energy-efficient flight paths over farm fields to monitor agricultural crops. 3D models of the virtual drone and a virtual agricultural field were developed in Blender using low-polygon 3D modeling style to minimize random-access memory (RAM) use and were then imported into Unity. A navigational algorithm for the virtual drone was developed employing Monte Carlo methods, specifically simulated annealing. Mathematical functions were developed to achieve maximal field coverage with minimal energy consumption. Results demonstrated that the distance-traveled and area-of-vision functions operate simultaneously to identify flight paths with minimal energy costs and maximum coverage over agricultural fields. The navigational algorithm works to minimize energy spent by the drone by modifying the flight path such that the ideal score of the flight path approaches zero. By randomizing the initial flight paths using Monte-Carlo methods, the algorithm improves progressively as it operates.

Tests of the system showed that the sinusoidal patterns farmers typically use to plant and harvest crops are significantly less effective than logarithmic spiral patterns or computer-randomized patterns (p < 0.05). However, there was no significant difference among the flight paths that began with logarithmic spirals or computer randomized paths (p > 0.05). The most efficient flight path overall was achieved with a flight pattern that was initially computer randomized with a-value of 0.7, which gives 70% priority to distance-traveled and 30% priority to area-of-vision.  

Easton McChesney and Wolfgang Ofstedal - Engineering: Materials and Bioengineering

Studies by Lee et al. (2008) and Park et al. (2007) formed the basis of our study. Lee et al. reported that crystalline organic semiconducting materials have higher electron mobility than amorphous organic semiconductors that are currently used in organic electronics. Based on the study by Park et al. (2007) that showed organic semiconducting anthracene has only one accessible crystal structure at room temperature and pressure, we choose to experiment with anthracene since we did not have the capability to differentiate between polymorphs using a polarized microscope.

For our methods, we chose to use melt-process crystallization to engineer anthracene crystals based on surface crystallization techniques used by Wu & Yu (2006) to crystallize the organic semiconductor indomethacin. We varied cooling rates of the molten anthracene based on work by Manoury et al. (2007) that studied cooling rates to crystallize 5,5’-bis(4-hexylphenyl)-2,2’-bithiophene, which is not a semiconductor. Finally, based on a study by Kéna-Cohen, Davanço, & Forrest (2008) that developed anthracene crystals using melt processing with a gold-plated capillary-force apparatus, we decided to employ a simpler, less expensive apparatus to incorporate capillary force into the melt-crystallization process.

Dr. R. Holmes served as our mentor. N. Morgan and T. Fielitz provided training on the ultraviolet-visible spectrometry and X-ray diffraction processes we used to analyze crystals, the sonication process that we used to clean substrates, and the polarized optical microscope that we used to analyze crystals. In addition, Ms. L. Fruen and the Breck Science Research team gave suggestions on our paper and guidance throughout the extent of our study. The University of Minnesota Department of Chemical Engineering and Materials Science provided funding, materials, and equipment for our work.

Evelyn McChesney and Maddy McCue - Biomedical Engineering

Three avirulent strains of bacteria were engineered to produce antimicrobial peptides (AMPs) that are capable of replacing antibiotics in treating bacterial infections. Two major problems associated with using AMPs were addressed. First, because AMPs are broken down by enzymes in the stomach, enzyme-resistant probiotic bacteria were engineered as delivery systems. Second, the two main categories of AMPs—gram-positive bacteriocins and gram-negative microcins—require different delivery systems. The gram-positive bacteria Lactococcus lactis IL1403 was chosen as a delivery system for bacteriocin Abp118 because it is an AMP that has activity against gram-positive pathogens. The gram-negative bacteria Escherichia coli MC4100 and E. coli BL21 were chosen as delivery systems for microcin V and microcin J25 they are AMPs that have activity against gram-negative pathogens.

Bacteriocin Abp118 from the engineered L. lactis IL1403 showed antimicrobial activity against the gram-positive pathogens Enterococcus faecium E8-9, Ent. faecium 7a, and Ent. faecalis OGR1F. Microcin V from E. coli MC4100 showed long-term (38 hours) antimicrobial activity against the pathogen Listeria monocytogenes. Microcin J25 from the engineered E. coli BL21 showed short-term (19 hours and 20 minutes) antimicrobial activity against the gram-negative pathogen Salmonella enterica serotype Tennessee, and long-term (38 hours) antimicrobial activity against the pathogen Listeria monocytogenes.

AMP-producing bacteria grew in chemically simulated conditions that mimicked the gastrointestinal tract. These results suggest that engineered bacteria show promise as alternatives to conventional antibiotics in targeting pathogens.

Luke Onopa  - Water Pollution and Water Quality

Triclosan, an antibiotic found in hand disinfectants, and perfluorooctane sulfonate, an organofluorine compound found in food wrappers and textiles, are entering wastewater in increasingly disturbing concentrations. This study showed that a combination of these pollutants significantly disrupts methanogens (Methanopyrus kandleri), which are anaerobic digesters that play a critical role in anaerobic wastewater treatment. Methods used include populating methanogens in the presence of triclosan with and without perfluorooctane sulfonates. A simple water-displacement method was developed to measure total methane (biogas) production by methanogens over the course of four weeks. Results showed that low concentrations of the antibiotic triclosan alone do not disrupt function of methanogens (p > 0.05), while high concentrations of triclosan do significantly disrupt methanogens (p < 0.05). Additionally, when methanogens were in an environment with perfluorooctane sulfonate alone, the methanogen function was not significantly disrupted (p < 0.05). However, methanogen function was significantly disrupted in an environment of perfluorooctane sulfonate mixed with a high concentration of triclosan (p < 0.05), as well when in an environment of perfluorooctane sulfonate mixed with a low concentration of triclosan (p < 0.05), suggesting that perfluorooctane sulfonate has a significant biomagnification effect on triclosan. These results suggest that triclosan and perfluorooctane sulfonate that are entering wastewater in increasingly disturbing numbers may significantly disrupt anaerobic digesters that play an indispensable role in anaerobic wastewater treatment. This has critical implications on the future ecology of lakes and rivers as well as on the safety of drinking supplies.

Karesten Salveson and Eve Zelickson - 

The purpose of our study was to investigate the affinity of zebra mussels (Dreissena polymorpha) towards fresh water macrophytes and determine locations on the macrophytes where the mussels prefer to settle. Samples of three macrophytes—coontail (Ceratophyllum demersum), Eurasian watermilfoil (Myriophyllum spicatum), and clasping leaf pondweed (Potamogeton richardsonii)—were sampled via scuba diving from three different bays (Gray’s Bay, Cook’s Bay, and North Arm) in Lake Minnetonka, MN, over the course of 12 weeks during the summer of 2014. The mussel density (mussels/g dry mass) was determined for each species of macrophytes as well as on each section (top, bottom, and middle) of the macrophytes. Zebra mussels showed significant preference for Eurasian watermilfoil compared to coontail and clasping leaf pondweed (p < 0.05). Preference for the top section of the macrophytes was significantly less than for the bottom and middle sections (p < 0.05). Our study is novel in the field of D. polymorpha biology.

Grant Two Bulls - 

This study examined impacts of an early 19th century Mdewakanton Dakota settlement on the ecology of Lake Calhoun in Minneapolis, MN. Lacustrine coring was used to take two core samples from Lake Calhoun at 44.94362 latitude and -93.31303 longitude and at 44.94192 latitude and -93.31292 longitude, each representing over 400 years of sediment accumulation. Because there were no funds available for Pb-210 dating of the cores, a technique called loss-on-ignition dating was run using a prototcol outlined by Engstrom, Balogh, & Swain (2007). By taking core samples at the same longitude and latitude in Lake Calhoun as Engstrom did, it was possible to compare loss-on-ignition results to loss-on-ignition/lead-210 data reported by Engstrom to date the cores.

Pollen present in the cores was counted based on work by McLauchlan (2003) that showed pollen counts can be employed to determine quantitative measures of the prevalence of different types of plants in a core sample. Pollen counts showed that the shoreline around Lake Calhoun was predominately oak at the time of the settlement (AD 1830-40), with no significant change in oak pollen in lake sediments over that period of time (p = 0.096 ). However, results did show a significant increase in ragweed (p = 0.039) and grass pollen, which includes corn, (p = 0.040) at the time of the settlement compared to dates before the settlement.

 This study succeeded in using two highly reproducible methods for examining historical lake ecology over time that may prove useful in future studies. Pollen types were identified in sediment from Lake Calhoun, and the loss-on-ignition data matched well to data reported in the study by Engstrom, Balogh, & Swain (1).


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