A vertical-style data structure and operations on data in that structure are explored and tested in the domain of sliding window average algorithms for geographical information systems (GIS) data. The approach allows working with data of arbitrary precision, which is centrally important for very large GIS data sets. The novel data structure can be constructed from existing multi-channel image data, and data in the structure can be converted back to image data. While in the new structure, operations such as addition, division, and bit-level shifting can be performed in a parallelized manner. It is shown that the computation of averages for sliding windows on this data structure can be performed faster than using traditional computation techniques, and the approach scales to larger sliding window sizes.

Upland cotton (Gossypium hirsutum L.) is the world's most prominent fiber crop. Cotton transformation is labor intensive and time consuming, taking 12 to 18 months for rooted T0 plants. One rate limiting step is the necessary production of somatic embryos. In other recalcitrant species, ectopic expression of three genes were shown to promote somatic embryogenesis: WUSCHEL (WUS), SHOOT MERISTEMLESS (STM), and BABY BOOM (BBM). WUS is responsible for maintaining stem-cell fate in shoot and floral meristems. STM is needed to establish and maintain shoot meristems. STM and WUS have similar functions but work in different pathways; overexpression of both together converts somatic cells to meristematic and embryogenic fate. BBM encodes an AP2/ERF transcription factor that is expressed during embryogenesis and ectopic expression of BBM reprograms vegetative tissues to embryonic growth. In prior studies, these genes were constitutively expressed, and cultures did not progress beyond embryogenesis because the embryogenic signal was not turned off. In our study, we set out to use these genes to increase the efficiency of cotton transformation and decrease the time it takes to regenerate a plant. A disarmed cotton leaf crumple virus (dCLCrV) vector delivers WUS, STM, or BBM into cotton tissue cultures through Agrobacterium tumefaciens infection. We propose that virus delivery of embryo-inducing genes is a better approach for transformation because A) inserts more than 800 nucleotides are unstable, and will spontaneously inactivate, B) virus DNA can migrate through plasmodesmata to cells around the infected cell, creating a gradient of embryonic potential, C) the virus DNA does not pass through the germ line and the seed will not contain virus. We propose this method of inducing embryogenesis will facilitate the stable transformation of cotton and will be beneficial to the cotton industry. Ectopic expression of AtBBM, AtSTM, and AtWUS GrWUS:meGFP from a constitutive CaMV 35S promoter produced plants with phenotypes similar to those described in previous studies overexpressing AtBBM, indicating that the AtBBM gene was functional. The cotton cotyledon infiltration of the pART27 constructs showed transformed cells in Coker 312 by GFP localization in the nucleus. Although GFP was detected, no visible embryos appeared from the cotyledon. Cotyledons infiltrated with Agrobacterium harboring overexpression vectors withered and aborted after ~2 weeks. The virus-based vector in tissue culture failed to increase transformation efficiency, resulting in no embryos. The combination of hormone concentration showed no contribution to increasing the transformation efficiency.

Conforme novas tecnologias e metodologias estão surgindo, e pesquisadores estão sedentos por ferramentas moleculares mas rápidas, mais eficientes e fáceis de usar, dominar os princípios e tecnologias do design de vetores e padronização de partes biológicas tornaram-se desafios fundamentais. Isso está abrindo espaço para o surgimento de uma disciplina inteiramente nova chamada Biologia Sintética. Esta área de estudo inovadora combina partes e módulos biológicos para criar sistemas mais confiáveis e robustos. Linhagens fúngicas são comumente alvo desses estudos, não apenas porque muitos achados fundamentais em relação à clonagem molecular surgiram das lições dadas por elas, mas também devido a um imenso e inexplorado potencial desses organismos em uma ampla gama de aplicações - desde biocombustíveis e produção de químicos finos até terapias biomédicas. Neste contexto, a presente dissertação é dividida em duas partes: a primeira diz respeito ao design e construção de uma ferramenta modular e versátil para ser aplicada em várias linhagens de fungos. Essa ferramenta é um plasmídeo binário para transformação mediada pro Agrobacterium tumefaciens, que foi construído em quatro diferentes versões contendo GFP ou mCherry como proteínas repórter e um gene sintético de resistência à higromicina como marcador de seleção. O vetor foi validado em Paracoccidioides lutzii, um patógeno oportunista humano dimórfico que é muito importante para medicina, mas ainda carecia de ferramentas genéticas eficientes. A segunda parte consiste na criação de uma biblioteca de promotores para a levedura oleaginosa Rhodosporidium toruloides, um promissor hospedeiro para a produção de bioprodutos a partir de biomassa, uma vez que pode eficientemente consumir açúcares C5 e C6 e aromáticos derivados da lignina. Vinte e nove promotores foram testados em um cassete de duplo-repórter - compreendendo ambas as proteínas fluorescentes GFP e mRuby - utilizando citometria de fluxo para análise de células únicas. A coleção de promotores apresentados neste trabalho é a maior disponível para R. toruloides até o momento e foi um avanço indispensável para superar a10 escassez de ferramentas para este organismo. Notavelmente, também apresentamos os primeiros promotores bidirecionais descritos para essa levedura e otimizamos o protocolo de transformação. Portanto, a Biologia Sintética foi eficientemente aplicada para expandir a coleção de partes biológicas padronizadas e otimizar vetores para transformação e manipulação genética de fungos. Estas ferramentas são de valor imediato e são aplicáveis a desafios muito distintos, mas igualmente importantes: a busca de novas soluções para a saúde humana e para uma economia bio-sustentável. / As new technologies and methodologies are surfacing, and researchers are now eager for fast, enhanced and easy-to-use molecular tools, mastering the principles and technologies of vector design and standardization of biological parts have become fundamental challenges. This is making room for the rise of an entirely novel discipline called Synthetic Biology. This innovative field of study combines biological parts and modules to create more reliable and robust systems. Fungal strains are commonly the target of these studies, not only because several fundamental findings regarding molecular cloning arose from lessons given by them, but also due to an immense and much unexplored potential of those organisms in a wide range of applications - ranging from biofuels and fine chemicals production to biomedical therapies. In this context, the present dissertation is divided in two parts: the first one concerns the design and construction of a modular and versatile tool to be applied in several fungal strains. This tool is a plasmid binary vector for Agrobacterium tumefaciens-mediated transformation, which was built in four different versions containing either GFP or mCherry as reporter proteins and a synthetic hygromycin resistance gene as selection marker. The vector was validated in Paracoccidioides lutzii, a dimorphic human opportunist pathogen that is very important for health care but was still lacking efficient genetic tools. The second part consists in the creation of a promoter library for the oleaginous yeast Rhodosporidium toruloides, a promising host for the production of bioproducts from biomass since it can efficiently consume C5 and C6 sugars and lignin-derived aromatics. Twenty-nine promoters were tested with a dual-reporter cassette - comprising both GFP and mRuby fluorescent proteins - using flow cytometer for single-cell analysis. The assortment of promoters presented in this work is the largest set available for R. toruloides until now and was an imperative advancement to overcome the scarcity of tools for this organism. Remarkably, we also presented the first bidirectional promoters described for this yeast and optimized the transformation protocol. Thus, we efficiently applied Synthetic Biology to expand the collection of standard biological parts and to12 optimize vectors for fungal transformation and genetic manipulation. These tools are of immediate value and are applicable for very distinct but equally important challenges: the pursuit of new solutions for human health and for a sustainable biobased economy