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The sustainability of the beef industry has become a point of national interest, particularly the investment of land and water resources. Our objective was to estimate how much land and irrigation water are required to maintain a simulated Angus cow-calf operation in the North Central Great Plains (NCGP) for an average year. A stochastic model was used, which enabled consideration of biological variation. The model computed 100 iterations of a 24-year timeframe (1995–2018). The simulated herd had 100 breeding females with replacement heifers being retained annually. The nutrients required to maintain a body condition score 5 for each individual animal, adjusting for temperature and physiological state, were calculated. A stocking rate of 3.3 hectares (ha) per cow-calf pair and mature cow weight of 600 kg was set, which is representative of the NCGP. Replacement heifers were assumed to be 65% of mature cow weight and allotted 1.22 ha. Bred heifers were assumed to be 85% of mature cow weight and allotted 1.81 ha. The herd was assumed to be grazing from May 1 to October 31. A supplemented ration of 60% alfalfa and 40% corn was provided if an individual’s nutritional needs were not met. Animals were assumed to be delivered a base ration from November 1 to April 30, which consisted of 73% alfalfa, 19% wheat straw, and 8% corn. The amount of irrigation necessary to grow feed was determined by estimating evapotranspiration of each crop then subtracting the amount of precipitation during the growing season. Average crop yield was determined using county level data from the UDSA NASS to estimate how much land would be needed for feed production. Sustaining a 100 head cow-calf herd in the NCGP for an average year requires 103.5 million liters for irrigation, 1288.5 ha for crop production, 357 ha grazing land.

Leishmaniasis is caused by the flagellated protozoan Leishmania, and is a neglected tropical disease (NTD), as defined by the World Health Organisation (WHO). Bisulphite conversion technology converts all genomic material to a simplified form during the lysis step of the nucleic acid extraction process, and increases the efficiency of multiplex quantitative polymerase chain reaction (qPCR) reactions. Through utilization of qPCR real-time probes, in conjunction with bisulphite conversion, a new duplex assay targeting the 18S rDNA gene region was designed to detect all Leishmania species. The assay was validated against previously extracted DNA, from seven quantitated DNA and cell standards for pan-Leishmania analytical sensitivity data, and 67 cutaneous clinical samples for cutaneous clinical sensitivity data. Specificity was evaluated by testing 76 negative clinical samples and 43 bacterial, viral, protozoan and fungal species. The assay was also trialed in a side-by-side experiment against a conventional PCR (cPCR), based on the Internal transcribed spacer region 1 (ITS1 region). Ninety-seven percent of specimens from patients that previously tested positive for Leishmania were positive for Leishmania spp. with the bisulphite conversion assay, and a limit of detection (LOD) of 10 copies per PCR was achieved, while the LOD of the ITS1 methodology was 10 cells/1000 genomic copies per PCR. This method of rapid, accurate and simple detection of Leishmania can lead to improved diagnosis, treatment and public health outcomes.

The aim of this thesis is the development and implementation of a controlled testing platform for the Robust Intelligent Sensing and Controls (RISC) Lab at Utah State University (USU). This will be an open source adaptable expandable robotics platform usable for both education and research. This differs from the many other platforms developed in that the entire platform software will be made open source. This open source software will encourage collaboration among other universities and enable researchers to essentially pick up where others have left off without the necessity of replicating months or even years of work. The expected results of this research will create a foundation for diverse robotics investigation at USU as well as enable attempts at novel methods of control, estimation and optimization. This will also contribute a complete software testbed setup to the already vibrant robotics open source research community. This thesis first outlines the platform setup and novel developments therein. The second stage provides an example of how this has been used in education, providing an example curriculum implementing modern control techniques. The third section provides some exploratory research in trajectory control and state estimation of the tip of an inverted pendulum atop a small unmanned aerial vehicle as well as bearing-only cooperative localization experimentation. Finally, a conclusion and future work is discussed.

Canonical models of rational choice fail to account for many forms of motivated adaptive behaviors, specifically in domains such as food selections. To describe behavior in such emotion- and reward-laden scenarios, researchers have proposed dual-process models that posit competition between a slower, analytic faculty and a fast, impulsive, emotional faculty. In this paper, we examine the assumptions and limitations of these approaches to modeling motivated choice. We argue that models of this form, though intuitively attractive, are biologically implausible. We describe an approach to motivated choice based on sequential sampling process models that can form a solid theoretical bridge between what is known about brain function and environmental influences upon choice. We further suggest that the complex and dynamic relationships between biology, behavior, and environment affecting choice at the individual level must inform aggregate models of consumer choice. Models using agent-based complex systems may further provide a principled way to relate individual and aggregate consumer choices to the aggregate choices made by businesses and social institutions. We coin the term "brain-to-society systems" choice model for this broad integrative approach.

Background It is well established that human papillomavirus (HPV) infection is highly related to the development of precursor lesions of cervical cancer and uterine cancers. However, for a pre-cancerous lesion to develop, a persistent infection with a high-risk type HPV is necessary. The Digene Hybrid Capture II (hcII) assay is the only FDA approved method used in conjunction with cytology for HPV screening of women older than 30. The hcII has moderate sensitivity (64.7%) and is dependent on the cellular content of samples, rendering occasionally false positive and false negative results. Objective This study aims to evaluate the performance of a new HPV diagnostic kit (High-Risk HPV detection kit, manufactured by Human Genetic Signatures (HGS), Sydney, Australia). Methods The method under evaluation was assessed by comparing the results obtained from testing 834 cervical specimens with the HGS method and the Digene hcII method, using genotyping as the reference standard. Results Results of the study showed that the specificity and positive predictive value of the HGS High-Risk HPV detection test are significantly greater than those of the Digene hcII test. Overall the HGS HPV assay provides a more accurate system for the detection of high-risk HPV strains, with simpler technical use compared with PCR-sequencing methods.

The growing importance of nanotechnology in industry and society has not been accompanied by a widespread understanding of the subject among the general public. Simple questions to initially probe the smallest thing that people can see and can think of reveals a divide in the understanding of the general public. A survey of 1500 individuals ranging in age from 6 to 74 has revealed a lack of knowledge of nanotechnology and especially a lack of understanding of the context of nanotechnology in the world that is too small to see. Survey findings are corroborated by in-depth interviews with 400 adults in studies of nanoscience literacy commisioned by University of California, Berkeley and Cornell in 2002 and 2004, respectively. In general, with the exception of 14–28 year olds, over 60% of respondents say they have never heard of nano or nanotechnology. The results suggest that the general public, especially middle-school children, has no firm foundation to understand nanotechnology and likely will continue to be equally impressed by credible scientific information as well as pure fictional accounts of nanotechnology.

Inhibin is composed of an alpha- and a beta-subunit. Transgenic studies assigned a tumor-suppressive role to the inhibin alpha-subunit, and in human prostate cancer inhibin alpha-subunit gene expression was down-regulated. This study examined the inhibin alpha-subunit gene promoter and gene locus to determine whether promoter hypermethylation or LOH occurred in DNA from prostate cancer. The 5'-untranslated region of the human inhibin alpha-subunit gene was sequenced and shown to be highly homologous to the bovine, rat, and mouse inhibin alpha-subunit promoter sequences. A 135-bp region of the human promoter sequence that continued a cluster of CpG sites was analyzed for hypermethylation. Significant (P0.001) hypermethylation of the inhibin alpha-subunit gene promoter occurred in DNA from Gleason pattern 3, 4, and 5 carcinomas compared with nonmalignant tissue samples. A subset of the carcinomas with a cribriform pattern were unmethylated. LOH at 2q32-36, the chromosomal region harboring the inhibin alpha-subunit gene, was observed in 42% of prostate carcinomas. These data provide the first demonstration that promoter hypermethylation and LOH are associated with the inhibin alpha-subunit gene and gene locus in prostate cancer.

The molecular basis for downregulation of the KAI1 metastasis suppressor gene in invasive and metastatic human cancers is unknown. We have used bisulphite methylation analysis of DNA from paraffin-embedded invasive bladder tumour samples and from bladder cancer cell lines to determine if hypermethylation of a CpG island within the KAI1 promoter is responsible for this effect. Representative invasive tumour cell lines were also exposed to 5-aza-2-deoxycytidine. We found no evidence for hypermethylation of the CpG island and suggest that mechanisms other than promoter hypermethylation are responsible for reduced KAI1 expression in invasive bladder tumours and tumour cell lines.

Nucleic acid sequence capture techniques are used to improve both the sensitivity and specificity of PCR for the diagnosis of plant, animal and human diseases. Hybridization capture-PCR (HC-PCR) was first reported as a method for the detection of Mycobacterium avium subsp. paratuberculosis in 1995 and was successfully trialed on a small number of faecal samples from cattle with Johne's disease. A locally optimized HC-PCR method was evaluated on faeces from infected and non-infected animals. However, sample to sample cross contamination during the DNA purification step highlighted that the original format of the test was unsuitable for routine diagnostic use. Here, we report modifications and optimization of HC-PCR, particularly with respect to DNA purification from faeces, hybridization and capture steps. We also identified procedurally sensitive critical points in the test during capture and washing of magnetic beads. Southern blotting was omitted from the protocol to preserve specificity but this resulted in analytical sensitivity of 5000 organisms per 200 mg faecal sample. Nevertheless, HC-PCR detected M.paratuberculosis in pellets from infected sheep diluted at rates of up to 1 in 100 in normal faeces, suggesting that the technique should be evaluated further for low-cost diagnosis in flocks/herds using pooled samples.

Glutathione-S-Transferases (GSTs) comprise a family of isoenzymes that provide protection to mammalian cells against electrophilic metabolites of carcinogens and reactive oxygen species. Previous studies have shown that the CpG-rich promoter region of the pi-class gene GSTP1 is methylated at single restriction sites in the majority of prostate cancers. In order to understand the nature of abnormal methylation of the GSTP1 gene in prostate cancer we undertook a detailed analysis of methylation at 131 CpG sites spanning the promoter and body of the gene. Our results show that DNA methylation is not confined to specific CpG sites in the promoter region of the GSTP1 gene but is extensive throughout the CpG island in prostate cancer cells. Furthermore we found that both alleles are abnormally methylated in this region. In normal prostate tissue, the entire CpG island was unmethylated, but extensive methylation was found outside the island in the body of the gene. Loss of GSTP1 expression correlated with DNA methylation of the CpG island in both prostate cancer cell lines and cancer tissues whereas methylation outside the CpG island in normal prostate tissue appeared to have no effect on gene expression.

Methylation analysis of individual cytosines in genomic DNA can be determined quantitatively by bisulphite treatment and PCR amplification of the target DNA sequence, followed by restriction enzyme digestion or sequencing. Methylated and unmethylated molecules, however, have different sequences after bisulphite conversion. For some sequences this can result in bias during the PCR amplification leading to an inaccurate estimate of methylation. PCR bias is sequence dependent and often strand-specific. This study presents a simple method for detection and measurement of PCR bias for any set of primers, and investigates parameters for overcoming PCR bias.

We aimed to streamline the diagnosis of gastrointestinal disease by producing multiplexed real time polymerase chain reaction (PCR) panels employing universal sample processing for DNA and RNA containing pathogens. A total of 487 stored, previously characterised stool samples comprising bacterial, viral, protozoan and Clostridium difficile positive samples were tested using four multiplexed real time PCR panels. A further 81 pre-selected clinical samples from a teaching hospital were included to provide an independent validation of assay performance. Improved sensitivity was achieved using the protozoan panels and 16 more mixed infections were observed compared to tests with conventional methods. Using the C. difficile panels, 100% sensitivity was achieved when compared to the gold standard of toxigenic culture. In addition, hypervirulent strains including ribotype 027 could be identified directly from primary sample without the need for ribotyping methods. Bacterial and viral panels detecting Salmonella, Shigella, Campylobacter, Yersinia enterocolitica, Listeria monocytogenes, norovirus groups I and II, rotavirus A, astrovirus, sapovirus, rotavirus B, adenovirus and adenovirus 40/41 performed as well as conventional methods, whilst allowing detection in 3 hours from processing to result. Multiplex real time PCR panels with universal sample preparation allow streamlined, rapid diagnosis of gastrointestinal pathogens whilst extending the characterisation of pathogens present in stool samples from affected patients.

Polymerase chain reaction (PCR) has been widely applied to the detection of microorganisms. Overall sensitivity of PCR tests may be substantially reduced due to a large excess of nontarget DNA and inhibitory substances in the sample. We used a 5'-biotinylated 513-bp probe from the 3' region of the IS 900 element specific for Mycobacterium paratuberculosis (Mptb) to capture target Mptb DNA from crude sample DNA extracts. Captured target DNA was separated using streptavidin-coated magnetic particles (Dynal). Since the IS 900 element shares homology over this region with IS 902 in Mycobacterium avium subsp. silvaticum (Mavs), target DNA from this other pathogen was also retained. Highly specific PCR for the detection of either organism directed to the 5' regions of IS 900 or IS 902 was then performed directly on the solid phase. Hybridization capture of target DNA using sequence adjacent to the desired specific PCR site applied to Mptb increased overall sensitivity of detection in tissue and fecal extracts 10- to 100-fold. False positives due to contamination artifact were substantially excluded since the capture probe did not retain amplicons from the detection PCR. Development of the method to involve covalent 5' immobilization of capture probes on heat-resistant polymers should, in the future, provide a simple system with broad potential applications.

The method of moments in conjunction with the maximum entropy method of reconstructing density distributions is applied to the energy dependent neutron diffusion equation to solve for neutron flux within a critical assembly. The energy dependent neutron diffusion equation (EDNDE) is converted into a moment equation which is solved analytically for a bare spherical critical assembly of pure 235U in the radial direction. The normalized energy dependent neutron diffusion moments (NEDNDM) generated analytically is verified to NEDNDM, as calculated by Monte Carlo N Particle 5 version 1.40 (MCNP5) and Attila-7.1.0-beta version (Attila). The normalized NEDNDM are validated with the bare spherical critical assembly experiment, named GODIVA. The NEDNDM are then put into the maximum entropy method to solve for neutron flux within the two critical assemblies (100% 235U and GODIVA) and the neutron flux is verified with MCNP5 and Attila and validated with GODIVA. The analytic NEDNDM values fall between the NEDNDM from MCNP5 (lower bound) and Attila (upper bound). The error is taken to be relative to the Monte Carlo simulation. The error range is from 0% to 14%. The error range of the NEDNDM compared to NEDNDM from GODIVA is 0% to 24%. The verification and validation error of the maximum entropy method is 12% to 25% where MCNP5 is taken to be the comparison standard. The error range of the reconstructed flux validated with GODIVA is 0% to 10%. The error range of the neutron flux spectrum from MCNP5 compared to GODIVA is 0%-20% and the Attila error range compared to the GODIVA is 0%-35%. The method of moments coupled with the maximum entropy method for reconstructing flux is shown to be a fast reliable method, compared to either Monte Carlo methods (MCNP5) or 30 multienergy group methods (Attila) and to GODIVA the bare sphere critical assembly experiment.

The modulation of DNA-protein interactions by methylation of protein-binding sites in DNA and the occurrence in genomic imprinting, X chromosome inactivation, and fragile X syndrome of different methylation patterns in DNA of different chromosomal origin have underlined the need to establish methylation patterns in individual strands of particular genomic sequences. We report a genomic sequencing method that provides positive identification of 5-methylcytosine residues and yields strand-specific sequences of individual molecules in genomic DNA. The method utilizes bisulfite-induced modification of genomic DNA, under conditions whereby cytosine is converted to uracil, but 5-methylcytosine remains nonreactive. The sequence under investigation is then amplified by PCR with two sets of strand-specific primers to yield a pair of fragments, one from each strand, in which all uracil and thymine residues have been amplified as thymine and only 5-methylcytosine residues have been amplified as cytosine. The PCR products can be sequenced directly to provide a strand-specific average sequence for the population of molecules or can be cloned and sequenced to provide methylation maps of single DNA molecules. We tested the method by defining the methylation status within single DNA strands of two closely spaced CpG dinucleotides in the promoter of the human kininogen gene. During the analysis, we encountered in sperm DNA an unusual methylation pattern, which suggests that the high methylation level of single-copy sequences in sperm may be locally modulated by binding of protein factors in germ-line cells.

Culture of mesenchymal stem cells (MSCs) under conditions promoting proliferation and differentiation, while supporting genomic and epigenetic stability, is essential for therapeutic use. We report here the extent of genome-wide DNA gains and losses and of DNA methylation instability on 170 cancer-related promoters in bone marrow (BM) MSCs during culture to late passage in medium containing fetal bovine serum (FBS) or autologous serum (AS). Comparative genomic hybridization indicates that expansion of BMMSCs elicits primarily telomeric deletions in a subpopulation of cells, the extent of which varies between donors. However, late passage cultures in AS consistently display normal DNA copy numbers. Combined bisulfite restriction analysis and bisulfite sequencing show that although DNA methylation states are overall stable in culture, AS exhibits stronger propensity than FBS to maintain unmethylated states. Comparison of DNA methylation in BMMSCs with freshly isolated and cultured adipose stem cells (ASCs) also reveals that most genes unmethylated in both BMMSCs and ASCs in early passage are also unmethylated in uncultured ASCs. We conclude that (i) BMMSCs expanded in AS or FBS may display localized genetic alterations, (ii) AS tends to generate more consistent genomic backgrounds and DNA methylation patterns, and (iii) the unmethylated state of uncultured MSCs is more likely to be maintained in culture than the methylated state.

This research was undertaken to investigate the effects of solid modeling and visualization on technical problem solving. The participants were 47 students enrolled in solid modeling classes at Southeast Missouri State University. The control and experimental groups consisted of 23 and 24 randomly assigned students respectively. This study was a posttest only design that used logistic regression to analyze the results. Both groups were required to take the Purdue Spatial-Visualization Test/Visualization of Rotations (PSVT/TR). Participants in the control group used only sketching to design their solutions while participants in the experimental group used parametric solid modeling software to design their solutions. All participants then constructed prototypes of their designs. The prototype was evaluated to determine if it successfully met the design specifications. The findings revealed that visualization was a significant predictor of technical problem solving as defined by successful prototype construction (p=.021). There was no significant difference between the sketching and solid modeling design methods used for technical problem solving (p=.752). The interaction between the method of design, solid modeling or sketching, was analyzed to determine if using solid modeling would offset low visualization scores It was found that the interaction was not significant (p=.393). Ph. D.

It is frequently useful to determine the methylation state of samples containing limited amounts of DNA such as from embryos, or from fixed tissue samples in which DNA is degraded or difficult to isolate. By modification of the standard protocols for DNA preparation and bisulfite treatment, it is possible to obtain DNA methylation sequence data for such samples. We present methods for bisulfite treatment of embryos, fixed sections, and samples obtained by laser capture microdissection, and discuss the additional experimental considerations required when working with small numbers of cells or degraded DNA samples.

What defines the boundaries between methylated and unmethylated domains in the genome is unclear. In this study we used bisulfite genomic sequencing to map the boundaries of methylation that flank the 5′- and 3′-ends of the CpG island spanning the promoter region of the glutathione S-transferase (GSTP1) gene. We show that GSTP1 is expressed in a wide range of tissues including brain, lung, skeletal muscle, spleen, pancreas, bone marrow, prostate, heart, and blood and that this expression is associated with the CpG island being unmethylated. In these normal tissues a marked boundary was found to separate the methylated and unmethylated regions of the gene at the 5′-flank of the CpG island, and this boundary correlated with an (ATAAA)19–24 repeated sequence. In contrast, the 3′-end of the CpG island was not marked by a sharp transition in methylation but by a gradual change in methylation density over about 500 base pairs. In normal tissue the sequences on either side of the 5′-boundary appear to lie in separate domains in which CpG methylation is independently controlled. These separate methylation domains are lost in all prostate cancer whereGSTP1 expression is silenced and methylation extends throughout the island and spans across both the 5′- and 3′-boundary regions.

Summary: The technique of representation difference analysis PCR has been applied to find genes specific to Mycobacterium avium subsp. paratuberculosis. This generated a 671 bp fragment which was used to isolate a larger genetic element found in the enteric pathogens M. avium subsp. paratuberculosis and M. avium subsp. silvaticum but which was absent from the very closely related and relatively benign M. avium subsp. avium. This element, designated GS, is greater than 6·5 kbp in length and has a G+C content 9 mol% lower than other genes from this species. There is a previously uncharacterized insertion sequence associated with one end. The GS element encodes five ORFs in M. avium subsp. paratuberculosis and M. avium subsp. silvaticum, all of which have counterparts encoded in Mycobacterium tuberculosis. Database searches revealed homologues for these ORFs in a number of bacterial species, predominantly Gram-negative organisms, including a number of enteric pathogens. These homologous genes encode functions related to LPS or extracellular polysaccharide biosynthesis. This element has a number of features in common with pathogenicity islands such as its low G+C content, an association with a putative insertion sequence and a grouping of genes of related function with a possible link to virulence. No direct link to pathogenicity has been shown but GS may belong to a group of related ‘genetic islands’ and represents the first such element to be identified in mycobacteria.

The Mycobacterium avium subsp. paratuberculosis (formerly Mycobacterium paratuberculosis) atypical insertion sequence, IS900, encodes a novel gene on the complementary strand to the putative transposase, p43. This gene requires a promoter, ribosome binding site (RBS) and termination codon to be acquired upon insertion into the M. avium subsp. paratuberculosis genome and hence is designated the hed (host expression-dependent) gene of IS900. Analysis of IS900 insertion sites suggests that this element targets translation initiation signals in M. avium subsp. paratuberculosis, specifically inserting between the RBS and start codon of a putative gene sequence. This aligns the hed initiation codon adjacent to a functional RBS and possibly downstream of an active promoter, driving expression of Hed protein. We have confirmed this unique targeting process by detecting expression of hed in M. avium subsp. paratuberculosis at the level of transcription by reverse transcription-PCR. Further, two Hed-specific antibodies detected Hed translation products in Western blots of protein extracts from M. avium subsp. paratuberculosis. A recombinant form of Hed expressed and purified from Escherichia coli will facilitate studies of IS900 transposition and will also be assessed as a diagnostic antigen for M. avium subsp. paratuberculosis disease. Implications of IS900 insertion in M. avium subsp. paratuberculosis pathogenicity are discussed.