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The objective of this study was to encapsulate cell lines that secrete marker proteins that cover a large molecular weight range (M^r from 45,000 to 300,000) and monitor the secretion of the marker proteins from alginate-poly-L-lysine-alginate (APA) microcapsules and hydroxyethyl methacrylatemethyl methacrylate (HEMA-MMA) thermoplastic capsules. Different parameters for the APA microcapsules, such as the duration of poly-L-lysine (PLL) and sodium citrate treatment, the initial cell density for encapsulation was studied, and their effects on secretion rate and cell proliferation were closely examined. Cell lines used for encapsulation secreted human growth hormone (hGH) (M^r 45,000), β-hexosaminidase (β-hexo.) (M^r 120,000) and β-glucuronidase (β-gluc.) (M^r 300,000). Monitoring the secretion rates, as well as the distribution of the marker proteins within the microcapsules following encapsulation enabled the permeability of the membrane to be assessed over one month in culture. Encapsulation of cell lines in both types of capsules was effective in producing viable cells capable of proliferating within a semi-permeable membrane. Encapsulating cells in single-coated APA microcapsules at 4°C, treated with 10 minutes PLL, 20 minutes sodium citrate and at a cell density of 2x10^6 cells/ml alginate was found to provide the most optimal conditions for prolonged viability of and stable secretion by the recombinant cells. Human growth hormone diffused readily across the capsule membrane into the culture media from both APA and HEMA-MMA capsules, at rates similar to the non-encapsulated cells. Human growth hormone did not accumulate in the intracapsular space in significant quantities. β-glucuronidase and β-hexosaminidase could diffuse across APA capsule membrane, but not across HEMA-MMA capsule membrane into surrounding media. β-glucuronidase secretion from APA microcapsules was 8-fold lower than non-encapsulated cells. β-hexosaminidase secretion from APA microcapsules was 4.5-fold lower than non-encapsulated cells. Slight retention of both β-glucuronidase and β-hexosaminidase was observed in the intracapsular space of APA capsules. HEMA-MMA capsules completely blocked the secretion of both β-glucuronidase and β-hexosaminidase out of the capsule. Massive accumulation of both kinds of secretory enzymes was found in the intracapsular space of HEMA-MMA capsules. This indicated APA microcapsules have a molecular weight cut-off of >300,000 whereas HEMA-MMA microcapsules have a molecular weight cut-off of <120,000. / Thesis / Master of Science (MS)

Transposable elements (Transposons; TEs) constitute the majority of DNA in genomes and are a major source of genetic polymorphisms. TEs act as potential regulators of gene expression and lead to phenotypic plasticity in plants and animals. In crops, several TEs were identified to influence alleles associated with important agronomic traits, such as apical dominance in maize and seed number in rice. Crops may harbor more TE-mediated genetic regulations than expected in view of multifunctional TEs in genomes. However, tools that accurately annotate TEs and clarify their associations with agronomic traits are still lacking, which largely limits applications of TEs in crop breeding. Here we 1) evaluate performances of popular tools and strategies to identify TEs in genomes, 2) develop a tool 'DeepTE' to annotate TEs based on deep learning models, and 3) develop a tool 'TE-marker' to identify potential TE-regulated alleles associated with agronomic traits. As a result, we propose a series of recommendations and a guideline to develop a comprehensive library to precisely identify TEs in genomes. Secondly, 'DeepTE' classifies TEs into 15-24 super families according to sequences from plants, metazoans, and fungi. For unknown sequences, this tool can distinguish non-TEs and TEs in plant species. Finally, the 'TE-marker' tool builds a TE-based marker system that is able to cluster rice populations similar to a classical SNP marker approach. This system can also detect association peaks that are equivalent to the ones produced by SNP markers. 'TE-marker' is a novel complementary approach to the classical SNP markers that it assists in revealing population structures and in identifying alleles associated with agronomic traits. / Doctor of Philosophy / Transposable elements (Transposons; TEs) are DNA fragments that can jump and integrate into new positions in the genome. TEs potentially act as regulators of gene expression and alter traits of plants and animals. In crops, several TEs were identified to influence functions of genes that control important agronomic traits, such as branching in maize and seed number in rice. However, tools that identify these associations in the crops are still lacking, which largely limits applications of TEs in crop breeding. Here we evaluated performance of popular tools and strategies that identify TEs, and provide a series of recommendations to efficiently apply these tools to the TE identification. In view of structural and sequence differences, TEs are classified into multiple families. We developed a 'DeepTE' tool to precisely cluster TEs into different families using a deep learning method. Finally, a 'TE-marker' tool was developed to build TE-based genetic markers to identify nearby alleles associated with agronomic traits. Overall, this work could promote the use of TEs as markers in improving quality and yielding crops.

Background: In recent years, research in the field of Augmented Reality (AR) in cultural heritage has been rapidly expanding, due to the advancement of technology and availability of cheaper “off the shelf” hardware. It is, amongst other things, being used as a means to increase availability and regain the public’s interest in cultural heritage.Objectives: This study compares marker-based and marker-less AR in perceived usability and perceived performance through a user study. Methods: With the use of the software Unity3D and Vuforia, two AR applications were implemented. Both applications display a model of an 18th-century Swedish warship, based on a wooden ship model, each using one of the two AR methods. The digital model was remade in Autodesk Maya, to suit the needs of an AR application used on mobile devices. The applications were evaluated in a user study with 14 participants. Each participant was asked to perform a simple task of walking around the displayed ship and then answering a questionnaire on usability. This process was done for both applications, followed by a post-experiment questionnaire on perceived performance where the two methods were compared. Results: The result of the study showed that both applications were perceived as usable and well performing. The result of the usability questionnaire showed that the applications were considered usable, with an average of 90.5 points for marker-based AR and 86.8 points for marker-less AR on a 0-100 point scale. Regarding performance, the marker-based method was perceived as better performing. Conclusions: The participants felt that with just a few instructions, the applications were easy to use, even though 50% of them had no previous experience in using AR, that it could enhance a museum exhibition. Possible further development of the app would be to complete the ship-model by adding more details that are currently missing.

Organophosphate hydrolase (OPH, EC 3.1.8.1) is a bacterial enzyme with a broad spectrum of potential substrates that include organophosphorus pesticides, herbicides, and chemical warfare agents. OPH has been expressed successfully in bacterial, fungal, and insect cell culture systems; however, none of these systems produces amounts of enzyme suitable for applications outside of the research laboratory. Therefore, a transgenic Zea mays L. (maize) system was developed to express OPH as an alternate to the current OPH expression systems. The bacterial gene encoding the OPH protein was optimized for transcriptional and translational expression in maize. The optimized gene was inserted into the maize genome under the control of embryo specific, endosperm specific, and constitutive plant promoters. Select transformants were analyzed for the expression of OPH. Expression was observed in the seeds of plants transformed with each of the three constructs with the highest expression observed with the embryo specific and constitutive promoter constructs. The highest OPH expressing lines of transgenic maize had expression levels higher than those reported for the E. coli expression system. OPH was purified from transgenic maize seed and analyzed for posttranslational modification and kinetic properties. OPH was observed to undergo a glycosylation event when expressed in maize that yielded at least two forms of OPH homogolous dimer. The glycosylated form of OPH bound tightly to the Concanavalin A sepharose and remained active after months of storage at room temperature. OPH activity was checked against a number of organophosphate herbicides. Enzymatic activity was observed against the herbicide Amiprophos-methyl and kinetic properties were measured. Enzymatic activity was also tested against the organophosphate Haloxon. Transgenic maize callus, leaf, and seed tissue could be screened for the presence of the optimized opd gene by enzymatic activity. Comparison of the growth of transgenic and control callus on media containing organophosphates showed that the transgenic callus was resistant to the herbicidal effects of haloxon. Transgenic plants expressing OPH were also resistant to the herbicide bensulide when compared to control plants. This indicates that OPH can be used as a screenable marker in plant systems and may be a potential scorable marker system as well.

Organophosphate hydrolase (OPH, EC 3.1.8.1) is a bacterial enzyme with a broad spectrum of potential substrates that include organophosphorus pesticides, herbicides, and chemical warfare agents. OPH has been expressed successfully in bacterial, fungal, and insect cell culture systems; however, none of these systems produces amounts of enzyme suitable for applications outside of the research laboratory. Therefore, a transgenic Zea mays L. (maize) system was developed to express OPH as an alternate to the current OPH expression systems. The bacterial gene encoding the OPH protein was optimized for transcriptional and translational expression in maize. The optimized gene was inserted into the maize genome under the control of embryo specific, endosperm specific, and constitutive plant promoters. Select transformants were analyzed for the expression of OPH. Expression was observed in the seeds of plants transformed with each of the three constructs with the highest expression observed with the embryo specific and constitutive promoter constructs. The highest OPH expressing lines of transgenic maize had expression levels higher than those reported for the E. coli expression system. OPH was purified from transgenic maize seed and analyzed for posttranslational modification and kinetic properties. OPH was observed to undergo a glycosylation event when expressed in maize that yielded at least two forms of OPH homogolous dimer. The glycosylated form of OPH bound tightly to the Concanavalin A sepharose and remained active after months of storage at room temperature. OPH activity was checked against a number of organophosphate herbicides. Enzymatic activity was observed against the herbicide Amiprophos-methyl and kinetic properties were measured. Enzymatic activity was also tested against the organophosphate Haloxon. Transgenic maize callus, leaf, and seed tissue could be screened for the presence of the optimized opd gene by enzymatic activity. Comparison of the growth of transgenic and control callus on media containing organophosphates showed that the transgenic callus was resistant to the herbicidal effects of haloxon. Transgenic plants expressing OPH were also resistant to the herbicide bensulide when compared to control plants. This indicates that OPH can be used as a screenable marker in plant systems and may be a potential scorable marker system as well.

Retroreflective Raised Pavement Markers (RRPMs) supplement other pavement markings to provide guidance to road users. Previous research concerning durability of the RRPMs suggests that their performance has been degrading over the years. One of the main causes for underperformance of the RRPMs is the lack of appropriate laboratory testing standards that can test the adequacy of the RRPMs to perform in field conditions. There is a need to modify the existing standards or develop new testing procedures that can better simulate field conditions. This requires identifying critical locations and magnitudes of stresses inside the markers during the tire-marker impacts that happen on roads. The goal of this research was to identify critical magnitudes and locations of the stresses in RRPMs during the tire-marker impacts by doing the finite element modeling and simulation of the impacts, and use the information to recommend laboratory testing procedures that could simulate real-world conditions. The researcher modeled and simulated the tire-marker impacts using the finite element tools Hypermesh and LS DYNA. He calibrated the material properties of the marker models to improve the tiremarker model. Based on the tire-marker impact simulations, the researcher concluded that the critical compressive stresses during impacts are located at the edge contacts of retroreflective sides with the top surface. The critical stresses may also occur at lower and upper corners of the marker. The other areas, especially the lower half of the marker, had tensile stresses. Angle of impact was found to be a critical external variable that affected the stresses inside the markers and the marker-pavement interface forces. The researcher then modeled and simulated a few laboratory-testing procedures that could simulate the field performance of the RRPMs. Based on these simulations, the researcher recommended that the ASTM compression test for evaluation of RRPMs be continued or a similar test be developed. He suggested development of one new test (named as offset compression test) that could better replicate the field conditions. He also recommended having a review of the ASTM flexural test.

Borage (Borago officinalis) is an annual herb plant for culinary and medicinal uses. Due to a high level of gamma-linolenic acid (GLA) in its seed oil and the health-related benefits of GLA, borage is commercially cultivated. However, a herbicide-resistant variety has not yet been developed for effective weed management in borage farming. Thus, this thesis aimed to create, identify and characterize ethyl methanesulfonate (EMS) induced borage mutants for herbicide imidazolinone resistance. An EMS-mutagenized borage population was generated by using a series of concentrations of EMS to treat M1 seeds. After screening M2 borage plants with the herbicide, tolerant plants were selected, self-pollinated and grown to their maturity. The offsprings were subjected to herbicide screening again to confirm the phenotype, resulting in identification of two genetically stable imidazolinone-resistant lines. Two acetohydroxyacid synthase (AHAS) genes, AHAS1 and AHAS2, involved in the imidazolinone resistance were isolated and sequenced from both mutant (resistant) and wild type (susceptible) borage plants. Comparison of these AHAS sequences revealed that a single nucleotide substitution occurred in the AHAS1 resulting in an amino acid change from serine (S) in the susceptible plant to asparagine (N) in the first resistant line. The similar substitution was later found in the AHAS2 of the second resistant line. A KASP marker was developed for the AHAS1 mutation to differentiate the homozygous susceptible, homozygous and heterozygous resistant borage plants for the breeding purpose. The in vitro assay showed homozygous resistant borage containing the AHAS1 mutation could retain significantly higher AHAS activity than susceptible borage across different imazamox concentrations. The herbicide dose response test showed that the resistant line with the AHAS1 mutation was tolerant to four times the field applied concentration of the “Solo” herbicide.