Browsing by Subject "Biotechnology"
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Item Advanced Problem Solving in the Biotherapeutics Industry: Parameters influencing the delivery of a novel cell therapy product and exploration of a new method for determining activity of Clostridium histolyticum collagenase, a wound debridement enzyme(2015-05-01) Harris, Melanie A.; Jerry W. Simecka; Patricia A. GwirtzBiotechnology is a multi-faceted industry with many unique challenges that require knowledge in a broad range of topics. When working in the wound care field it is necessary to not only create a product in the laboratory, but also effectively bring it to the patient. This task requires many skilled people who can test it for efficacy, design and conduct clinical trials, confirm quality and consistency, design packaging, consider transportation issues and so on. The following investigation focuses on the testing of a cellular product and its accompanying device under various conditions as well as the exploration of a new assay capable of the activity of a wound debridement enzyme. The results of the product/device testing have generally confirmed the comparability of the cellular product devices as well as their resistance to various temperatures encountered in the clinical environment. A new modified assay for the testing of collagenase has been established as precise and comparable to current methods, though it requires more testing to confirm robustness.Item ELUCIDATING THE DIVERSITY OF MICROBIAL RETTING COMMUNITIES ON HIBISCUS CANNABINUS USING NEXT-GENERATION SEQUENCING(2014-03) Visi, David K.; Allen, Michael S.Purpose (a): Global environmental concerns have led to a growing interest in renewable resources such as plant-based fibers. Beyond textiles and cordage, plant fibers have the potential for incorporation into renewable, bio-based composite materials for the building and manufacturing sectors. Successful commercialization of fiber production requires optimization of fiber extraction. Retting is the traditional method of fiber extraction, whereby endogenous microorganisms break down heteropolysaccharides to release fiber bundles. Previous studies have analyzed the retting solution for its bacterial constituents, but none have followed changes in the microbial community through the retting process. This research aims to track the bacterial components of the retting community through time, and determine the effects of bacterial augmentation with isolated pectinolytic bacteria using next generation sequencing of 16S rRNA gene amplicons. Methods (b): Batch C1 included plant material and an inoculum of pond water represented a “traditional” retting environment, while C2 contained autoclaved pond water to represent the endogenous microorganisms associated with the plant material. Experimental batch E1 included the addition of three pectinolytic bacterial isolates: Bacillus DP1, Paenibacillus DP2, and Bacillus K1. Total DNA was extracted from the surface-adhering biofilm bacteria and used for PCR with full-length 16S primers 27F and 1492R. Amplification products were used as templates for a nested PCR with primers 786F and 939R targeting variable region 5 of the 16S molecule. The nested 16S rRNA amplicons were then sequenced on the next-generation Ion Torrent PGM platform. Results (c): The E1 environment showed a marked increase in phylum Firmicutes (55 to 94%), while phylum Proteobacteria showed a progressive decrease from Day 1 to 4 (36% to 5%). This was correlated with easy separation of fibers by mechanical movement. At a finer taxonomic level, E1 showed a rapid loss of inoculated Bacillus species DP1 and K1, and a more gradual loss of the family Paenibacillaceae 1 (P. DP2) as the time course progressed. In contrast, C1 was co-dominated by phyla Firmicutes and Proteobacteria, while C2 was composed in large part by the phylum Bacteroidetes. Additionally, comparison of the microbial communities under the different conditions revealed differences in diversity and composition at day 4 time points between the three conditions. Conclusions (d): Introduction of pectinolytic bacteria into the batch reactions increased production rate and increased fiber quality. Introduction of Paenibacillus DP2 is likely the driving force behind the community shifts detected in E1, which warrants further study to determine the mechanism of action. The findings confirmed previous studies that suggest a gradual replacement of aerobic organisms to an environment that is dominated by strict anaerobes. Moreover, the efforts shed light on conditions and mechanisms for the manipulation of microbiomes. These approaches may have relevance to the treatment of dysbiosis in the gut flora in humans and the treatment of related diseases.Item Optimization of Spermatozoa Capture During the Differential Extraction Process for STR Typing with the Potential for Automation(2002-05-01) Marshall, Pamela L.; Eisenberg, Arthur; Martin, Michael W.; Wordinger, Robert J.Marshall, Pamela. Optimization of Spermatozoa Capture During the Differential Extraction Process for STR Typing With the Potential for Automation. Master of Science (Forensic Genetics). May, 2002. In 1998, within the United States, it is estimated that a rape occurred every 2.3 minutes. In 1995, according to the Bureau of Justice Statistics, an estimated 350,000 rapes or sexual assaults (R/SA0 were experienced by persons age 12 or older. Of the estimated 100,000 R/SA reported, there were only approximately, 25,000 cases analyzed by crime labs nationwide. The majority of crime laboratories throughout the U.S., especially those in major metropolitan cities, have a significant backlog of unresolved R/SA cases. With the implementation of the Convicted Offender Database (CODIS), it is essential that all R/SA cases by analyzed, especially those lacking a known suspect. The comparison of the short tandem repeat (STR) profiles derived from sperm DNA recovered from evidentiary material with CODIS samples would provide the police with critical investigative leads resulting in the identification of the assailant. The goal of this research is to develop a cellular sorting method for the isolation of sperm cells from sexual assault samples which will: 1) take advantage of differentiating features (extracellular antigenic sites) for complete separation of cell types, 2) provide a more efficient means of sperm recovery, increasing DNA yield from the male fraction, and 3) ensure the DNA isolation process is compatible with the amplification of the CODIS core STR loci. Overall, the proposed technique will increase the probability of success in the analysis of sexual assault case samples. (NIJGrant #: 2000-IJ-CX-K009).