Quinoa (Chenopodium quinoa Willd.) is a pseudocereal native to the Andean region of South America and a staple crop for subsistence farmers in the altiplano of Bolivia and Peru. Downy mildew is the most significant disease of quinoa caused by the pathogen Peronospora farinosa f.sp. chenopodii Byford. This disease greatly impacts quinoa crops with yield losses up to 99%. As fungicides are expensive for farmers, the development of resistant cultivars appears to be the most efficient means for controlling downy mildew. The quinoa germplasm bank contains high amounts of genetic diversity, some of which exhibit mildew resistance. Methods for evaluating mildew severity are important for finding resistant genotypes that are useful in breeding programs. The main objectives of this study were to evaluate and investigate downy mildew resistance in quinoa through several different methods. A simple inoculation method was developed for downy mildew disease assessment by placing a damp piece of cheesecloth on a leaf, pipetting a known spore solution onto the cloth, and subjecting the plants to specific humidity cycles in a growth chamber. After inoculation of five quinoa-breeding lines in a growth chamber, accession 0654 was found to be the most resistant, while genotypes NL6 and Sayana showed moderate resistance. Each of these genotypes displayed some potential for resistance breeding programs. Investigation of the growth and development of P. farinosa through resistant and susceptible quinoa genotypes revealed fewer sporangiophores, hyphal strands, and haustoria among leaf tissues of accession 0654 than in the susceptible Chucapaca cultivar. Peronospora farinosa growth was detected in leaf, petiole, and stem tissues with polymerase chain reaction (PCR) using ITSP primers designed from the internal transcribed spacer (ITS) region of the pathogen. Scanning electron microscopy (SEM) also revealed that P. farinosa penetrated stomata via appressoria, secreted extracellular matrices during sporangia germination, grew intercellularly in leaf and petiole tissues, and exited leaf tissue through stomata. Future research requiring knowledge of resistant quinoa genotypes, P. farinosa growth and development, or inoculation methods for large numbers of small quinoa plants would benefit from this report.

Molecular and morphologic studies were used to determine the evolution, classification and differentiation of Nymphaea odorata. Molecular analyses of the nuclear internal transcribed spacer (ITS) region, the chloroplast trnL-F region, and inter-simple sequence repeat (ISSR) markers determined the variation present between and within two species of Nymphaea. The ITS region resulted in a phylogeny depicting strong separation between species (N. mexicana and N. odorata) and some separation between N. odorata's subspecies. The ITS region contained polymorphisms, which upon SAHN clustering and principle coordinate (PCOA) and minimum spanning tree (MST) analyses produced groups similar to the clades in the ITS phylogeny. Sixteen accessions were chosen for trnL-F analysis, where a subspecies-specific molecular marker was found. In most accessions the marker confirmed the original subspecies classification. Molecular analyses using ISSRs characterized among population variation in N. odorata and N. mexicana using five primers. ISSR markers among populations were highly variable within a species and were used in UPGMA, PCOA and MST analysis, which resulted in separation between the subspecies. Both univariate and multivariate analyses were performed on quantitative and qualitative morphological characters. An analysis of variance resulted in six morphological characteristics that were statistically significant (P< 0.05), the majority being leaf blade characteristics. Multivariate statistics of principle component analysis and discriminate analysis resulted in groups for each subspecies, both emphasized the importance of quantitative leaf blade characteristics. Overall, both morphology and molecular characteristics supported the classification of subspecies for ssp. odorata and ssp. tuberosa, due a lack of strong segregation of characteristics. / Master of Science

The 16S, 23S, and 5S ribosomal RNA (rRNA) genes are highly conserved sequences in bacteria. For this reason, rRNA genes are often used for phylogenetic classification. On the other hand, the regions between the structural sequences, known as intergenic spacer regions (ITS), are under less evolutionary pressure to be conserved. Because they are not as highly conserved, they can be used to differentiate strains of the same bacterial specie. The purpose of this study was to evaluate the 16S-23S ITS of Flavobacterium columnare, an important pathogen of cultured fish, by comparing the 16S-23S ITS sequences from 70 isolates. We developed two PCR assays that amplify overlapping regions of one large previously identified ITS. The primers targeted the 16S sequence and isoleucine tRNA encoding sequences and the 23S sequence and alanine tRNA encoding sequences. The PCR products were cloned and sequenced. We also targeted I-CeuI restriction fragments from the ATCC type strain that were separated by pulse field gel electrophoresis and analyzed the 16S-23S ITS regions. We found that the genome of this species harbors at least 6 intergenic spacer regions that are very similar and contain the same tRNA encoding sequences. This suggests that earlier studies that used the ITS for distinguishing between strains of Flavobacterium columnare may be comparing sequences from different structural RNA operons and thus have misleading data.

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The present work aims to determine the physiological and sanitary quality of Sweet sorghum (Sorghum bicolor (L.) Moench) seeds, as well as to identify pathogens associated with seed, their transmission to seedlings and the subsequent pathogenicity of isolates obtained, In addition, molecularly identify the fungal species pathogenic to this crop. The experiments were carried out in the Teaching and Seed Research Laboratory (TSRL), of the Plant Engineering Department; In the Elocy Minussi Phytopathology Laboratory, Department of Plant Protection, and at the Biological Institute of São Paulo. Sweet sorghum seeds were used, all without chemical treatment. Sanitary quality was evaluated by sanity test, and physiological characteristics by germination and vigor tests (seedling length, dry mass, emergence, rate of emergence and accelerated aging). It was performed the test of transmission of the pathogens associated to the seeds and the subsequent pathogenicity of the obtained isolates, culminating with the molecular characterization of the identified pathogens, in which were sequenced the Internal Transcribed Spacer (ITS) genomic regions and the Elongation Factor 1 - alpha (TEF1-α) gene. The design used was the completely randomized design, with four cultivars of Sweet sorghum (BRS 506, F19, BRS 511 and BRS 509); For the evaluation of pathogenicity, the factorial scheme is represented by four cultivars and three isolates of Fusarium spp., besides the witness. The seeds of the BRS 509 cultivar were considered to have lower physiological quality than the other cultivars. The DNA sequencing allowed identifying the Fusarium thapsinum species as a pathogenic agent in the sweet sorghum crop, and proven its transmission via seeds. / O presente trabalho teve como objetivo determinar a qualidade fisiológica e sanitária de sementes de cultivares de sorgo sacarino (Sorghum bicolor (L.) Moench), bem como identificar os patógenos associados à semente, sua transmissão às plântulas e a posterior patogenicidade de isolados obtidos, além disso, identificar molecularmente as espécies fúngicas patogênicas a esta cultura. Os experimentos foram realizados no Laboratório Didático e de Pesquisas em Sementes (LDPS), do Departamento de Fitotecnia; no Laboratório de Fitopatologia Elocy Minussi, do Departamento de Defesa Fitossanitária e, no Instituto Biológico de São Paulo. Foram utilizadas sementes de sorgo sacarino, todas sem tratamento químico. A qualidade sanitária foi avaliada pelo teste de sanidade, e as características fisiológicas por meio dos testes de germinação e de vigor (comprimento de plântulas, massa seca, emergência, índice de velocidade de emergência e envelhecimento acelerado). Foi realizado o teste de transmissão dos patógenos associados à semente e a posterior patogenicidade dos isolados fúngicos obtidos, culminando com a caracterização molecular dos patógenos identificados, na qual foram sequenciadas as regiões genômicas Internal Transcribed Spacer (ITS) e o gene do fator de elongação 1-α (TEF1α). O delineamento utilizado foi o inteiramente casualizado com quatro cultivares de sorgo sacarino (BRS 506, Fepagro 19, BRS 511 e BRS 509); já para a avaliação da patogenicidade, o esquema fatorial foi representado pelas quatro cultivares e três isolados de Fusarium sp., além da testemunha. As sementes da cultivar BRS 509 foram consideradas de qualidade fisiológica inferior as demais cultivares. O sequenciamento de DNA permitiu identificar a espécie Fusarium thapsinum como agente patogênico na cultura do sorgo sacarino, sendo comprovada sua transmissão via sementes.

Innovative 3D spacer fabrics made from individual planes and connecting layers present great potential as complexly shaped textile preforms in lightweight composite applications. As one of the most flexible textile manufacturing methods, flat knitting enables the production of intricately shaped textile structures. The major advantages coupled with flat knitting techniques include the ability to produce multi-layer reinforcements, a diminishing waste, reducing production time and near-net shaping. This research includes the further development of flat knitting technology and the manufacturing processes of innovative, customized 3D spacer fabrics for high performance composite applications. Novel 3D spacer fabrics have been developed in different geometries using glass-polypropylene commingled hybrid yarns for complex shaped thermoplastic composite components. Reinforcement yarns have been integrated into spacer fabric structures with up to 4 reinforcement layers to improve mechanical performance. Furthermore, the successful addition of “sensor networks” created by integrating functional yarns into the 3D spacer fabrics could be used for structural health monitoring. Innovative integration concepts, which accommodate different positioning of the reinforcement yarns into the knit structures, can be used to adjust the mechanical properties of the finished knit composites. Moreover, the tensile properties have been accurately predicted based on the mathematical models formulated. The developed flat knitted 3D spacer fabrics are very promising for applications in lightweight composites, mechanical engineering, protective textiles, civil engineering and architectural designs. / Innovative 3D-Spacer Fabrics bestehend aus individuellen Deckflächen und Verbindungsstegen bieten ein großes Potential als komplex geformte textile Halbzeuge für Leichtbauverbundwerkstoffanwendungen. Mit Hilfe des Flachstrickens, welches einer der flexibelsten textilen Herstellungsprozesse ist, lassen sich komplex geformte textile Strukturen herstellen. Belastungsgerechte Verstärkungen, Abfallreduzierung, endkonturnahe Fertigung sind nur einige der großen Vorteile der modernen Flachstricktechnik. Die Forschungsarbeit beinhaltet die Entwicklung der Flachstricktechnologie und des Herstellungsprozesses für innovative 3D-Spacer Fabrics für Hochleistungsverbundwerkstoffe. Neuartige 3D-Spacer Fabrics wurden in unterschiedlichen Geometrien entwickelt, in dem Glas-/ Polypropylen Commingling-Hybridgarn für komplex geformte thermoplastische Verbundwerkstoffkomponenten eingesetzt wird. Verstärkungsfäden wurden für hochmechanische Belastungen in die Spacer-Fabric-Strukturen in bis zu 4 Verstärkungschichten integriert. Die erfolgreiche Umsetzung und Entwicklung von Sensornetzwerken durch die Integration von funktionalen Fäden in die 3D-Spacer Fabrics kann für die strukturelle Zustandsüberwachung genutzt werden. Die innovativen Integrationskonzepte erlauben die differenzierte Orientierung von Verstärkungsfäden in den Gestrickstrukturen, wodurch eine starke Beeinflussung der mechanischen Eigenschaften der Gestrickverbundwerkstoffe herbeigeführt wird. Darüber hinaus wurden die Zugeigenschaften basierend auf den entwickelten mathematischen Modellen vorhergesagt. Die entwickelten flachgestrickten 3D-Spacer Fabrics sind sehr vielversprechend beispielweise für die Anwendung in Leichtbauverbundwerkstoffen, im Maschinenbau, in Schutztextilien, im Bauingenieurwesen und Architekturdesign.

Heteroepitaxial SiGe(C) layers have attracted immense attention as a material for performance boost in state of the art electronic devices during recent years. Alloying silicon with germanium and carbon add exclusive opportunities for strain and bandgap engineering. This work presents details of epitaxial growth using chemical vapor deposition (CVD), material characterization and integration of SiGeC layers in MOS devices. Non-selective and selective epitaxial growth of Si1-x-yGexCy (0≤x≤0.30, 0≤y≤0.02) layers have been performed and optimized aimed for various metal oxide semiconductor field effect transistor (MOSFET) applications. A comprehensive experimental study was performed to investigate the growth of SiGeC layers. The incorporation of C into the SiGe matrix was shown to be strongly sensitive to the growth parameters. As a consequence, a much smaller epitaxial process window compared to SiGe epitaxy was obtained. Incorporation of high boron concentrations (up to 1×1021 atoms/cm3) in SiGe layers aimed for recessed and/or elevated source/drain (S/D) junctions in pMOSFETs was also studied. HCl was used as Si etchant in the CVD reactor to create the recesses which was followed (in a single run) by selective epitaxy of B-doped SiGe. The issue of pattern dependency behavior of selective epitaxial growth was studied in detail. It was shown that a complete removal of pattern dependency in selective SiGe growth using reduced pressure CVD is not likely. However, it was shown that the pattern dependency can be predicted since it is highly dependent on the local Si coverage of the substrate. The pattern dependency was most sensitive for Si coverage in the range 1-10%. In this range drastic changes in growth rate and composition was observed. The pattern dependency was explained by gas depletion inside the low velocity boundary layer. Ni silicide is commonly used to reduce access resistance in S/D and gate areas of MOSFET devices. Therefore, the effect of carbon and germanium on the formation of NiSiGe(C) was studied. An improved thermal stability of Ni silicide was obtained when C is present in the SiGe layer. Integration of SiGe(C) layers in various MOSFET devices was performed. In order to perform a relevant device research the dimensions of the investigated devices have to be in-line with the current technology nodes. A robust spacer gate technology was developed which enabled stable processing of transistors with gate lengths down to 45 nm. SiGe(C) channels in ultra thin body (UTB) silicon on insulator (SOI) MOSFETs, with excellent performance down to 100 nm gate length was demonstrated. The integration of C in the channel of a MOSFET is interesting for future generations of ultra scaled devices where issues such as short channel effects (SCE), temperature budget, dopant diffusion and mobility will be extremely critical. A clear performance enhancement was obtained for both SiGe and SiGeC channels, which point out the potential of SiGe or SiGeC materials for UTB SOI devices. Biaxially strained-Si (sSi) on SiGe virtual substrates (VS) as mobility boosters in nMOSFETs with gate length down to 80 nm was demonstrated. This concept was thoroughly investigated in terms of performance and leakage of the devices. In-situ doping of the relaxed SiGe was shown to be superior over implantation to suppress the junction leakage. A high channel doping could effectively suppress the source to drain leakage. / <p>QC 20100715</p>

CRISPR-Cas systems are adaptive immune systems in bacteria and archaea, consisting of a clustered regularly interspaced short palindromic repeats (CRISPR) array and CRISPR associated (Cas) proteins. In this work, the type I-E CRISPR-Cas system of Escherichia coli was studied. CRISPR-Cas immunity is divided into three stages. In the first stage, adaptation, Cas1 and Cas2 store memory of invaders in the CRISPR array as short intervening sequences, called spacers. During the expression stage, the array is transcribed, and subsequently processed into small CRISPR RNAs (crRNA), each consisting of one spacer and one repeat. The crRNAs are bound by the Cascade multi-protein complex. During the interference step, Cascade searches for DNA molecules complementary to the crRNA spacer. When a match is found, the target DNA is degraded by the recruited Cas3 nuclease. Host factors required for integration of new spacers into the CRISPR array were first investigated. Deleting recD, involved in DNA repair, abolished memory formation by reducing the concentration of the Cas1-Cas2 expression plasmid, leading to decreased amounts of Cas1 to levels likely insufficient for spacer integration. Deletion of RecD has an indirect effect on adaptation. To facilitate detection of adaptation, a sensitive fluorescent reporter was developed where an out-of-frame yfp reporter gene is moved into frame when a new spacer is integrated, enabling fluorescent detection of adaptation. Integration can be detected in single cells by a variety of fluorescence-based methods. A second aspect of this thesis aimed at investigating spacer elements affecting target interference. Spacers with predicted secondary structures in the crRNA impaired the ability of the CRISPR-Cas system to prevent transformation of targeted plasmids. Lastly, in absence of Cas3, Cascade was successfully used to inhibit transcription of specific genes by preventing RNA polymerase access to the promoter. The CRISPR-Cas field has seen rapid development since the first demonstration of immunity almost ten years ago. However, much research remains to fully understand these interesting adaptive immune systems and the research presented here increases our understanding of the type I-E CRISPR-Cas system.