Method to Create Arbitrary Sidewall Geometries in 3-Dimensions Using Liga with a Stochastic Optimization Framework

Disclosed herein is a method of making a three dimensional mold comprising the steps of providing a mold substrate; exposing the substrate with an electromagnetic radiation source for a period of time sufficient to render the portion of the mold substrate susceptible to a developer to produce a modified mold substrate; and developing the modified mold with one or more developing reagents to remove the portion of the mold substrate rendered susceptible to the developer from the mold substrate, to produce the mold having a desired mold shape, wherein the electromagnetic radiation source has a fixed position, and wherein during the exposing step, the mold substrate is manipulated according to a manipulation algorithm in one or more dimensions relative to the electromagnetic radiation source; and wherein the manipulation algorithm is determined using stochastic optimization computations

The Origin, Genetic Diversity and Taxonomy of the Invasive Diatom Didymosphenia geminata (Bacilliariophyceae) in New Zealand

This thesis describes an investigation of the origin, genetic diversity and taxonomy of Didymosphenia geminata, in New Zealand. D. geminata, commonly known as Didymo or Rock Snot , is a freshwater diatom, a photosynthetic alga with a silica shell. It attaches to rocks and plants by its mucilaginous stalks, its large blooms often covering all available substrates and causing shifts in community structure. Although it was historically associated with high altitude, oligotrophic waters, it is now showing increased ecological tolerance. It has been increasing in abundance and range in Europe and North America and has been introduced into new areas including New Zealand, Iran and India. Since the first discovery of D. geminata in New Zealand in October 2004, it has spread rapidly across the South Island. I have conducted a phylogeographic study of D. geminata samples from Europe, Asia, North America and New Zealand, using D. geminata-specific primers to amplify the internal transcribed spacer (ITS) region. I have also used these primers to amplify the small nuclear ribosomal DNA (nrDNA) subunit 18S to investigate the taxonomic placement of D. geminata within the pennate diatoms. Results from this investigation indicate that D. geminata may belong to the Family Cymbellaceae. It appears that D. geminata may have been introduced though several different introduction events to North America from Europe and then to New Zealand from North America. These results can be used to inform strategies regarding the control and management of this invasive species, including lending support for continuation of the Biosecurity New Zealand program aimed at improving public, and especially freshwater users', awareness and responsibility regarding D. geminata in New Zealand. This program is especially important as dispersal appears to be human-mediated. Limiting the number and source of introductions to an area can reduce the potential for increased genetic variation and thus adaptation to new environments

Synthesizing multi-layer perceptron network with ant lion biogeography-based dragonfly algorithm evolutionary strategy invasive weed and league champion optimization hybrid algorithms in predicting heating load in residential buildings

The significance of accurate heating load (HL) approximation is the primary motivation of this research to distinguish the most efficient predictive model among several neural-metaheuristic models. The proposed models are formulated through synthesizing a multi-layer perceptron network (MLP) with ant lion optimization (ALO), biogeography-based optimization (BBO), the dragonfly algorithm (DA), evolutionary strategy (ES), invasive weed optimization (IWO), and league champion optimization (LCA) hybrid algorithms. Each ensemble is optimized in terms of the operating population. Accordingly, the ALO-MLP, BBO-MLP, DA-MLP, ES-MLP, IWO-MLP, and LCA-MLP presented their best performance for population sizes of 350, 400, 200, 500, 50, and 300, respectively. The comparison was carried out by implementing a ranking system. Based on the obtained overall scores (OSs), the BBO (OS = 36) featured as the most capable optimization technique, followed by ALO (OS = 27) and ES (OS = 20). Due to the efficient performance of these algorithms, the corresponding MLPs can be promising substitutes for traditional methods used for HL analysis

Can Very Rapid Adaptation Arise Without Ancestral Variation? Insight from the Molecular Evolution of Herbicide Resistance in Genus Amaranthus

In this dissertation I investigate the source and spread of adaptive resistance to the herbicide glyphosate by the weedy species Amaranthus palmeri. This is an ideal system for furthering scientific understanding of the dynamics of evolution because the adaptation is in response to a well understood selection pressure and is happening on an extremely short time scale. The plant genus Amaranthus contains several agriculturally important weeds, but is not closely related to any current model systems—the closest model system is Beta vulgaris(sugar beet). In the first part of this work I seek to determine the relationship between extant species of Amaranthus, particularly the relationship between weedy and non-weedy species, using Bayesian phylogenetic analysis of independent genomic loci. This phylogeny will provide context for investigating the dynamics of adaptation to glyphosate stress. The second chapter is an investigation of the sequence constraints and selection pressures acting on the gene that codes for 5-enolpyruvalshikimate-3-phosphate synthase (EPSPS) in the genus Amaranthus. The first population of A. palmeri verified as resistant to glyphosate was identified in Macon, Georgia in 2004. The mechanism of resistance was found to be proliferation in copy number of the gene encoding the enzyme target of glyphosate toxicity, EPSPS. The proliferation of genomic copies of the gene encoding a target enzyme is unique among mechanisms documented for herbicide resistance—though it has been observed as a resistance mechanism in other systems, including human cancer resistance to chemotherapy. Understanding EPSPS DNA sequence constraint will allow a better understanding of the evolutionary processes that led to a unique mechanism of herbicide resistance. In the final chapter I seek to determine if the same EPSPS copy number proliferation mechanism is responsible for glyphosate resistance in North Carolina A. palmeri and address the question of the source of the EPSPS copy number proliferation genotype. I investigate the potential of parallel evolution from ancestral variation as an explanation for observed spread of resistance by looking for genus-wide variation in EPSPS copy number and analyze population structure to determine the most probable number of adaptive events. Understanding the constraints on the EPSPS gene and protein that may have led to the observed resistance mechanism, how many times the mechanism evolved independently, and how it spread through the population(s) improves our understanding of how genomes are changed by adaptation to environmental stress. It also has the potential to provide important insights about the dynamics of herbicide resistance adaptation that can help growers make the best possible choices in weed management for protecting our food supply and our environment

An assessment of the genetic diversity and origin of the invasive weed Chromolaena Odorata (L.) King and Robinson in South Africa

Chromolaena odorata (L.) King and Robinson is an alien invasive weed to most of the Old World tropical regions of the earth, including South Africa where it is morphologically distinct from most other C. odorata plants examined from both its native and invasive range. It is thought that these morphological differences are related to difficulties encountered in successful establishment of biological control agents on the South African population of C. odorata. It has been postulated that the source population of the South African population will harbour potential biocontrol agents that will be suited to successful establishment on the South African plants. Several morphological, cytological and isozyme studies have been attempted to identify the source population of the South African population, but these have failed to identify the origin of the South African population. In this dissertation two PCR-based methods were attempted, in an investigation into whether the morphological differences and difficulties in establishment of biocontrol agents have a genetic basis. The two techniques attempted were: Inter Simple Sequence Repeat (ISSR) amplification, and DNA sequencing. Results could not be obtained using the ISSR method, and the reason for this was not discovered despite extensive trials. The internal transcribed spacer region and the external transcribed spacer region sequences were obtained from five samples, and compared. It was found that the ETS region gave more phylogenetic signal at the intraspecific level than the ITS region. However, due to difficulties in amplification of the external transcribed spacer region, work here focussed on obtaining Internal Transcribed Spacer sequences for 61 samples. Each of the samples sequenced had a unique ITS sequence, displaying a high level of intraspecific genetic diversity. The degree of this diversity is discussed with reference to the possible influences of polyploidy and concerted evolution on genetic structure. The ITS data indicated that some of the physical traits used to define ‘morphotypes’ of C. odorata were not correlated to genotype. From discussion and comparison of morphological character distributions and the ITS-based phylogeography it is suggested that the geographical origin of the South African population is Greater Antilelan, rather than from the continents of North and South America, which is where the Australasian, West African and Mauritian infestations are suggested to have originated

Artificial Neural Networks in Agriculture

Modern agriculture needs to have high production efficiency combined with a high quality of obtained products. This applies to both crop and livestock production. To meet these requirements, advanced methods of data analysis are more and more frequently used, including those derived from artificial intelligence methods. Artificial neural networks (ANNs) are one of the most popular tools of this kind. They are widely used in solving various classification and prediction tasks, for some time also in the broadly defined field of agriculture. They can form part of precision farming and decision support systems. Artificial neural networks can replace the classical methods of modelling many issues, and are one of the main alternatives to classical mathematical models. The spectrum of applications of artificial neural networks is very wide. For a long time now, researchers from all over the world have been using these tools to support agricultural production, making it more efficient and providing the highest-quality products possible

Development of transgenic black ash (Fraxinus nigra) for resistance to the emerald ash borer, and genome editing for reproductive sterility

Black ash (Fraxinus nigra Marsh.) is valued for commercial hardwood products such as cabinets, paneling, flooring, and veneer, and for food and habitat for wildlife, specifically in riparian areas. The wood is preferred by Native Americans for making splints for basketry. The emerald ash borer (EAB), an aggressive exotic wood-boring beetle from Asia, is threatening all North American ash species. Since the first EAB infestation in the United States was confirmed near Detroit, Michigan in 2002, it has spread rapidly. In order to manage the EAB and conserve Fraxinus spp., there is a need to develop ash trees with resistance to the EAB. The goal of this research was to optimize the genetic transformation and shoot regeneration system for black ash hypocotyls, develop transgenic black ash for EAB resistance and reproductive sterility, and develop an adventitious shoot regeneration protocol using black ash leaves. An in vitro system for plant regeneration from leaf explants of black ash was successfully developed. Leaf explants were transversally cut across the midrib and cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of plant growth regulators. MS medium supplemented with 22.2 μM 6-benzylaminopurine combined with 31.8 μM thidiazuron produced callus formation (100%) and adventitious shoot bud induction (28.8%). The frequency of regeneration response was significantly higher with compound leaves than single leaflets. Whole plants were acclimatized to the greenhouse. An Agrobacterium-mediated transformation system for black ash hypocotyls was optimized based upon a previously developed protocol in our lab. Sonication for 90 s followed by vacuum-infiltration for 10 min in suspension of Agrobacterium strain EHA105 (concentration at OD600 = 1.0) was found to be optimal. Silwet L-77 did not make a significant difference on transformation efficiency, but it had a negative effect at high concentration. Using this optimal transformation condition, three independent transgenic lines expressing Bacillus thuringiensis (Bt) Cry8D2 gene were obtained. The integration of the full length, intact Cry8D2 gene was confirmed by polymerase chain reaction (PCR), and its expression in mRNA and protein level was detected by qPCR and Western blot analysis, respectively. All three transgenic lines contained two copies of the transgene. An AGAMOUS (AG) homolog of black ash (FnAG) was isolated and characterized as a potential target gene for achieving transgene containment. A 729-bp coding region of FnAG was obtained by reverse transcription PCR and rapid amplification of cDNA ends. Deduced amino acid sequence showed a highly conserved MADS-domain. Phylogenetic analysis confirmed that FnAG belongs to the clade of C-lineage AG subfamily genes. Expression of FnAG transcript was detected in the reproductive tissues (female and male flowers), but rarely detected in the vegetative tissues (leaves). Transgenic Arabidopsis thaliana plants overexpressing FnAG showed ap2-like phenotypic alteration and early flowering, indicating FnAG functions in the same way as AG. To achieve transgene containment, reproductive sterility can be produced by disrupting genes involved in development of floral organs such as AG. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9 (Cas9) was used to induce mutations at three target sites within FnAG. A total of 50 transgenic lines harboring Cas9 expression cassette were obtained. Of these, only two showed one nucleotide substitution in the target site. Cas9 transgene silencing in some transgenic lines with no mutations resulted in the low mutagenesis rate

SEEING DOUBLE WITH \u3ci\u3eCANNABIS\u3c/i\u3e: HETEROPLOID POPULATIONS IN \u3ci\u3eBIPOLARIS GIGANTEA\u3c/i\u3e, CAUSAL AGENT OF BIPOLARIS LEAF SPOT

Bipolaris leaf spot (BLS) disease emerged with the reintroduction of hemp as a crop in the United States following more than 60 years of prohibition. The causal agent was identified as Bipolaris gigantea (=Drechslera gigantea), a known minor pathogen of monocots which causes devastating disease on hemp. BLS has been confirmed throughout Kentucky and reported in 15 states. Morphology and growth characteristics of isolates from eight counties across Kentucky were similar with the exception of some isolates producing protoperithecial-like structures. Phylogenetic and whole genome analysis indicated that some isolates were haploid, containing a single allele at each gene (RPB2, TEF1) and only one mating type idiomorph. Others were “heteroploid,” having two alleles at each gene, both mating type idiomorphs, and an assembled genome approximately twice the size of haploid genomes. The phylogenies suggested that most heteroploids had a genome similar to most haploids, plus a related genome that was closely related but phylogenetically distinct. Haploids and heteroploids caused indistinguishable disease symptoms on field hemp and were both equally likely to be isolated from samples. The implications of the genetic diversity of populations causing BLS are unknown, as is the influence on the implementation and development of management strategies

Molecular techniques for the identification of freshwater fish species for environmental monitoring programs

Reliable species identification methods are important for industrial environmental monitoring programs. Probe based real-time PCR (qPCR) provides an accurate, cost-effective and high-throughput method for species identification. Here we present the development and validation of species-specific primers and probe for the identification of eight freshwater fish species. The development of a fully automated species-decoder algorithm allowed for target species identification with 100% accuracy while completely removing any false-positive detection of non-target species. Furthermore, the probe-based qPCR technique utilized in this study is substantially more cost-effective and time efficient than DNA barcoding and morphological identification methods. The qPCR assays were also highly sensitive and accurately detected target species from collected environmental DNA (eDNA) samples. In summary, probe-based multiplex qPCR assays provide a rapid and accurate method for freshwater fish species identification and the methodology established in this study can be utilized for various other species identification initiatives.Master of Science (MSc) in Biolog