Philosophiae Doctor - PhD / Electroanalytical and optical properties of nanoscale materials are very important for biosensing applications as well as for understanding the unique one-dimensional carrier transport mechanism. One-dimensional semiconductor nanomaterials such as semiconductor quantum dots are extremely attractive for designing high-density protein arrays. Because of their high surfaceto-volume ratio, electro-catalytic activity as well as good biocompatibility and novel electron transport properties make them highly attractive materials for ultra-sensitive detection of biological macromolecules via bio-electronic or bio-optic devices. A genosensor or gene based biosensor is an analytical device that employs immobilized deoxyribonucleic acid (DNA) probes as the recognition element and measures specific binding processes such as the formation of deoxyribonucleic acid-deoxyribonucleic acid (DNA-DNA), deoxyribonucleic acid- ribonucleic acid (DNA-RNA) hybrids, or the interactions between proteins or ligand molecules with DNA at the sensor surface.In this thesis, I present four binary and two ternary-electrochemically and optically modulated selenide and telluride quantum dots, all synthesised at room temperature in aqueous media. Cationic gallium (Ga3+) synthesized in form of hydrated gallium perchlorate salt[Ga(ClO4)3.6H2O] from the reaction of hot perchloric acid and gallium metal was used to tailor the optical and electrochemical properties of the selenide and telluride quantum dots. The synthesized cationic gallium also allowed successful synthesis of novel water soluble and biocompatible capped gallium selenide nanocrystals and gallium telluride quantum dots. Cyclic voltammetric studies inferred that presence of gallium in a ZnSe-3MPA quantum dot lattice improved its conductivity and significantly increased the electron transfer rate in ZnTe-3MPA.Utraviolet-visible (UV-vis) studies showed that incorporation of gallium into a ZnSe-3MPA lattice resulted in a blue shift in the absorption edge of ZnSe-3MPA from 350 nm to 325 nm accompanied by decrease in particle size. An amphiphilic bifunctional molecule, 3-Mercaptopropionic acid (3-MPA) was used as a capping agent for all quantum dots. It was found that 3-MPA fully solubilised the quantum dots, made them stable, biocompatible, non agglomerated and improved their electron transfer kinetics when immobilized on gold electrodes.Retention of the capping agent on the quantum dot surface was confirmed by Fourier transform infrared spectroscopy (FTIR) which gave scissor type bending vibrations of C-H groups in the region 1365 cm-1 to 1475 cm-1, stretching vibrations of C=O at 1640 cm-1, symmetric and asymmetric vibrations of the C-H in the region 2850 cm-1 to 3000 cm-1 as well as stretching vibrations of –O-H group at 3435 cm-1. The particle size and level of non-agglomeration of the quantum dots was studied by high resolution transmission electron microscopy (HRTEM). The optical properties of the quantum dots were studied using UV-vis and fluorescence spectroscopic techniques.Quantum dot/nanocrystal modified gold electrodes were prepared by immersing thoroughly cleaned electrodes in the quantum dot/nanocrystal solution, in dark conditions for specific periods of time. The electrochemical properties of the modified electrodes were characterized by cyclic voltammetry (CV), square wave voltammetry (SWV), electrochemical impedance and spectroscopy (EIS). Six sensing platforms were then prepared using quantum dot/nanocrystal, one of which was used for detection of dopamine while the rest were used for detection of a DNA sequence related to 5-enolpyruvylshikimate-3-phosphate synthase, a common vector gene in glyphosate resistant transgenic plants.The first sensing platform, consisting of ZnSe-3MPA modified gold electrode (Au|ZnSe-3MPA) gave rise to a novel method of detecting dopamine in presence of excess uric acid and ascorbic acid. Using a potential window of 0 to 400 mV, the ZnSe-3MPA masked the potential for oxidation of uric and ascorbic acids, allowing detection of dopamine with a detection limit of 2.43 x 10-10 M (for SWV) and 5.65 x 10-10 M (for steady state amperometry), all in presence of excess uric acid (>6500 higher) and ascorbic acid (>16,000 times higher). The detection limit obtained in this sensor was much lower than the concentration of dopamine in human blood(1.31 x 10-9 M), a property that makes this sensor a potential device for detection of levels of dopamine in human blood.The other sensing platforms were prepared by bioconjugation of amine-terminated 20 base oligonucleotide probe DNA (NH2-5′-CCC ACC GGT CCT TCA TGT TC-3′) onto quantum dot modified electrodes with the aid of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The prepared DNA electrodes were electrostatically hybridized with different sequences which included 5′-GAA CAT GAA GGA CCG GTG GG-3′ (complementary target), 5′-CATAGTTGCAGCTGCCACTG-3′ (non complementary target) and 5′-GATCATGAAGCACCGGAGGG-3′ (3-base mismatched target).The hybridization events were monitored using differential pulse voltammetry (DPV) and SWV by monitoring the guanine oxidation signal or using EIS by monitoring changes in the charge transfer resistance. The quantum dot genosensors were characterized by low detection limits (in the nanomolar range), long linear range (40 - 150 nM) and were able to discriminate among complementary, non-complementary and 3-base mismatched target sequences.

Esta dissertação é resultado da pesquisa etnográfica desenvolvida junto a os atores envolvidos na gestão ambiental da Unidade de Conservação da Natureza (UC) chamada Floresta Nacional de Passo Fundo (Mato Castelhano/RS). Este universo compõe-se de cientistas, servidores públicos, agricultores e índios kaingang, em diálogo sobre o uso e a preservação da floresta. Nos encontros do Conselho Gestor da UC são expressas visões de mundo que se refletem em formas diferenciadas de apropriação dos elementos do ambiente próprias de cada grupo. Em um contexto de introdução de lavouras transgênicas nas propriedades próximas à área protegida, o estudo visa a apreender pontos de vista sobre a "natureza" e a "sociedade" e sobre o ambiente de Mato Castelhano entre os participantes do Conselho Gestor da UC, pesquisadores, especialistas da área ambiental e agricultores, abordando ainda a perspectiva de índios kaingang acampados na BR-285. Foram realizadas entrevistas semi-estruturadas com índios e conselheiros e observação participante de suas atividades e reuniões, além da análise de documentos. Evidencia-se que o diálogo em relação aos elementos objetos de manejo e conservação é dificultado pela desconsideração da complexidade da maneira de conceber o ambiente entre os diferentes atores locais. / This dissertation results from an ethnographic field work among actors who are involved in a process of public management of an Environmental Protected Area, which is called National Forest of Passo Fundo (Mato Castelhano/RS). This universe is composed of scientists, public servants, farmers and a kaingang indigenous group, dialoging about the use of "nature" and its conservancy. In the gatherings of the Management Council of the National Forest are expressed different perspectives in what concerns the appropriation of the area and its elements, which are related to each one's ways to engage themselves in that environment. In a context of transgenic agriculture in the farms around the protected area, the study focus the apprehension of points of view related to "nature" and "society", as well as the environment of Mato Castelhano, among members of the Council, professional researchers, experts on environment issues, farmers, and includes the perspective of the kaingang indians. In a qualitative approach, were made analysis of documents, semi-directive interviews and participant observation during ordinary meetings and tasks of the councilors, and within the indigenous encampment on the highway BR-285. The research evidences that the dialog about use and conservancy of "natural" elements do not reach to consider the different ways of conceive environment among the local actors.

Esta dissertação é resultado da pesquisa etnográfica desenvolvida junto a os atores envolvidos na gestão ambiental da Unidade de Conservação da Natureza (UC) chamada Floresta Nacional de Passo Fundo (Mato Castelhano/RS). Este universo compõe-se de cientistas, servidores públicos, agricultores e índios kaingang, em diálogo sobre o uso e a preservação da floresta. Nos encontros do Conselho Gestor da UC são expressas visões de mundo que se refletem em formas diferenciadas de apropriação dos elementos do ambiente próprias de cada grupo. Em um contexto de introdução de lavouras transgênicas nas propriedades próximas à área protegida, o estudo visa a apreender pontos de vista sobre a "natureza" e a "sociedade" e sobre o ambiente de Mato Castelhano entre os participantes do Conselho Gestor da UC, pesquisadores, especialistas da área ambiental e agricultores, abordando ainda a perspectiva de índios kaingang acampados na BR-285. Foram realizadas entrevistas semi-estruturadas com índios e conselheiros e observação participante de suas atividades e reuniões, além da análise de documentos. Evidencia-se que o diálogo em relação aos elementos objetos de manejo e conservação é dificultado pela desconsideração da complexidade da maneira de conceber o ambiente entre os diferentes atores locais. / This dissertation results from an ethnographic field work among actors who are involved in a process of public management of an Environmental Protected Area, which is called National Forest of Passo Fundo (Mato Castelhano/RS). This universe is composed of scientists, public servants, farmers and a kaingang indigenous group, dialoging about the use of "nature" and its conservancy. In the gatherings of the Management Council of the National Forest are expressed different perspectives in what concerns the appropriation of the area and its elements, which are related to each one's ways to engage themselves in that environment. In a context of transgenic agriculture in the farms around the protected area, the study focus the apprehension of points of view related to "nature" and "society", as well as the environment of Mato Castelhano, among members of the Council, professional researchers, experts on environment issues, farmers, and includes the perspective of the kaingang indians. In a qualitative approach, were made analysis of documents, semi-directive interviews and participant observation during ordinary meetings and tasks of the councilors, and within the indigenous encampment on the highway BR-285. The research evidences that the dialog about use and conservancy of "natural" elements do not reach to consider the different ways of conceive environment among the local actors.

Esta dissertação é resultado da pesquisa etnográfica desenvolvida junto a os atores envolvidos na gestão ambiental da Unidade de Conservação da Natureza (UC) chamada Floresta Nacional de Passo Fundo (Mato Castelhano/RS). Este universo compõe-se de cientistas, servidores públicos, agricultores e índios kaingang, em diálogo sobre o uso e a preservação da floresta. Nos encontros do Conselho Gestor da UC são expressas visões de mundo que se refletem em formas diferenciadas de apropriação dos elementos do ambiente próprias de cada grupo. Em um contexto de introdução de lavouras transgênicas nas propriedades próximas à área protegida, o estudo visa a apreender pontos de vista sobre a "natureza" e a "sociedade" e sobre o ambiente de Mato Castelhano entre os participantes do Conselho Gestor da UC, pesquisadores, especialistas da área ambiental e agricultores, abordando ainda a perspectiva de índios kaingang acampados na BR-285. Foram realizadas entrevistas semi-estruturadas com índios e conselheiros e observação participante de suas atividades e reuniões, além da análise de documentos. Evidencia-se que o diálogo em relação aos elementos objetos de manejo e conservação é dificultado pela desconsideração da complexidade da maneira de conceber o ambiente entre os diferentes atores locais. / This dissertation results from an ethnographic field work among actors who are involved in a process of public management of an Environmental Protected Area, which is called National Forest of Passo Fundo (Mato Castelhano/RS). This universe is composed of scientists, public servants, farmers and a kaingang indigenous group, dialoging about the use of "nature" and its conservancy. In the gatherings of the Management Council of the National Forest are expressed different perspectives in what concerns the appropriation of the area and its elements, which are related to each one's ways to engage themselves in that environment. In a context of transgenic agriculture in the farms around the protected area, the study focus the apprehension of points of view related to "nature" and "society", as well as the environment of Mato Castelhano, among members of the Council, professional researchers, experts on environment issues, farmers, and includes the perspective of the kaingang indians. In a qualitative approach, were made analysis of documents, semi-directive interviews and participant observation during ordinary meetings and tasks of the councilors, and within the indigenous encampment on the highway BR-285. The research evidences that the dialog about use and conservancy of "natural" elements do not reach to consider the different ways of conceive environment among the local actors.

Genetically modified (GM) crops are commercially cultivated worldwide but there are concerns on their possible negative impacts on soil biodiversity. A glasshouse study was conducted to determine effects of Bt maize residues on soil microbial diversity. Residues of Bt maize (PAN 6Q-308B) and non-Bt maize (PAN 6Q-121) were incorporated into the soil and corresponding maize seeds planted. The treatments were replicated three times. Fertilizer and water application were similar for both treatments. Rhizosphere and bulk soil was destructively sampled from each treatment and analyzed for microbial community level physiological profiles using Biolog plates with 31 different carbon substrates. Absorbance in the Biolog plates was recorded after 72 h of incubation at 20oC. Arbuscular mycorrhizal fungi spore counts were also determined. Field studies were conducted at the University of Free State and University of Fort Hare Research Farms to determine the effects of growing Bt maize on soil microbial diversity. One Bt maize cultivar (PAN6Q-308B) and non-Bt maize (PAN6Q-121) were grown in a paired experiment at University of Free State farm, while two Bt maize (DKC61-25B and PAN6Q-321B) and their near-isogenic non-Bt maize lines (DKC61-24 and PAN6777) were grown in a randomized complete block design with three replicates. Fertilization, weed control and water application, were similar for both Bt maize cultivars and their non-Bt maize counterparts. Rhizosphere soil samples were collected by uprooting whole plants and collecting the soil attached to the roots. The samples were analysed for microbial diversity and for arbuscular mycorrhizae fungal spore counts. Principal component analysis showed that soil microbial diversity was affected more by sampling time whereas genetic modification had minimal effects. Presence of residues also increased the diversity of microorganisms. Mycorrhizal fungal spores were not affected by the presence of Bt maize residues. Growing Bt maize had no effect on the soil microbial diversity in the rhizosphere.

The theoretical part of the diploma thesis summarizes basic facts related to the global climate change and provides up-to-date knowledge on the issue of genetically modified plants more resistant to stress factors of environment, including specific examples of strategies regarding preparation of genetically modified plants resistant particularly to abiotic stress. Although genetically modified plants and global climate changes belong to very important and current issue which public and foremost young generation should be sufficiently aware of, contemporary biology books and high school framework educational program pay to the issue very little attention. Therefore, the goal of this thesis is not only to summarize the basic facts and current knowledge regarding this issue but mainly to handle the issue in didactic level in the form of activating learning tasks. There were 28 learning tasks created in the thesis that gradually in unconventional way familiarize students with the issue of genetically modified plants and with the impacts of climate changes. Students must actively acquire information from accompanying materials to address the tasks properly, think critically over them, utilizing acquired knowledge and experience from the past to some extent. Fourteen tasks were chosen from the file created which...

RNA interference (RNAi) is a conserved mechanism in a wide range of eukaryotes. Introduction of synthetic dsRNA could specifically target suppression of a gene or could result in off-target silencing of another gene due to sequence similarity. To verify if the observed phenotype in an RNAi transgenic line is due to silencing of a specific gene or if it is due to another nontarget gene, a synthetic gene complementation approach could be used. Synthetic gene complementation described in this method uses the technology of synthesizing a variant of a native gene (used in RNAi silencing) to maximize the difference in DNA sequences while coding for the exact same amino acids as the original native gene. This is achieved through the use of alternate codons. The new variant gene is expressed in the original RNAi transgenic lines and analyzed for complementation of the RNAi phenotype. Complementation of the RNAi-induced phenotype will indicate gene-specific silencing and not off-target silencing.

<p dir="ltr">Bacterial Wilt, caused by <i>Ralstonia solanacearum</i>, is among the most devastating plant diseases in the world. This pathogen causes significant loss in crops such as tobacco, potato, and tomato. <i>R. solanacearum</i> root infection and xylem colonization determine disease outcome. To date, little is known about the defense mechanism mediated by roots to prevent <i>R. solanacearum</i> vascular colonization during the initial infection stages. Plant early responses are important since they may impact disease outcomes<i>.</i><i> </i>Here, we report the formation of root hairs and primary root growth inhibition in tomato seedlings as <i>Ralstonia</i>-induced phenotypes that depend on tomato genotype and <i>Ralstonia</i> species. The <i>Ralstonia</i>-induced root phenotypes are independent of a functional type III secretion system and exopolysaccharide production (EPS). We also found that <i>R. solanacearum</i><i> </i>K60 infection increased auxin levels throughout the root meristem in wilt-susceptible tomato roots. Our data suggest proper auxin signaling and transport are important for susceptibility to <i>R. solanacearum</i> K60. Blocking auxin transport pharmacologically or genetically led to fewer wilting symptoms, suggesting that auxin is important during early infection stages and disease outcomes in tomato. We previously found that a tomato mutant defective in auxin transport and signaling, known as <i>diageotropica</i> (<i>dgt</i>), has enhanced resistance to <i>R. solanacearum</i> K60. We characterized the resistant response in the <i>dgt</i> mutant, and we found that the resistant response in the <i>dgt</i> mutant may be due to increased lignin content preventing pathogen vasculature colonization. <i>DGT</i> encodes a cyclophilin protein that regulates auxin transport and signaling. Mutations in the cyclophilin DGT promote resistance to <i>R. solanacearum</i> K60. DGT has been reported to regulate auxin transport and signaling. However, the molecular mechanism regarding how DGT mediates these processes is still unknown. We used Yeast Two-Hybrid to identify candidate protein interactors, and we found that SlbZIP1/SlbZIP29, Sl14-3-3, and SlMYB110 may interact with DGT to regulate both development and defense responses. Understanding the role of DGT, auxin, and lignin in defense responses to <i>R. solanacearum</i> K60 in tomato is necessary for Solanaceae crop improvement.</p>

<div>Anthropogenically driven global change is disrupting ecosystems and habitats of many plant species, straining the ability of native species to survive and reproduce. The overarching goal of this research was to holistically work towards restoration of a threatened tree species by connecting research from different disciplines. In order to do so, the threatened butternut tree (<i>Juglans cinerea</i>) and its hybrids were used as a case study. Hybridization can incorporate stress tolerance in plants and could be a potential restoration tool. Evidence in some wild butternut populations indicates that naturalized hybrids of butternut with Japanese walnut (<i>Juglans ailantifolia</i>) may be more tolerant to butternut canker disease (BCD) than butternut, but this has not been formally tested. Thus, chapter 2 examined potential BCD tolerance within and between unadmixed and hybrid butternut inoculated with two BCD fungal isolates. Differences in canker growth were observed by fungal isolate, which could help to explain some differences in BCD severity found among butternut populations. Smaller and fewer cankers and greater genetic gains were detected in hybrid families, demonstrating that hybrids warrant further evaluation as a possible breeding tool for developing BCD-resistant butternut trees.</div><div>However, even with increased disease tolerance, hybrids must possess similar ecophysiological tolerances to their native progenitor to be an effective replacement. Butternut is extremely cold hardy, but Japanese walnuts are native to a warmer ecosystem, indicating potential disparities in extreme temperature tolerances between the two species and their hybrids. Thus, samples from mature trees were subjected to cold and heat treatments to compare relative extreme temperature tolerances within butternut and between butternut, Japanese walnut, and their hybrids. Within butternut, trees from colder areas exhibited less cold damage than those from warmer areas. Differences in heat damage among provenances occurred but did not follow a clear trend. Butternut exhibited greatest cold tolerance, Japanese walnut exhibited greatest heat tolerance, and hybrids were intermediate. Thus, the utility of hybrids for restoration could be limited at the extremes of the species’ distributions.</div><div>A second, but different type of freeze test was conducted for chapter 4 using seedlings to gain a more nuanced understanding of cold tolerance within butternut and between butternut and its hybrids. No survival or damage differences were detected in butternut provenances, although seedlings from the coldest provenances experienced more delayed budbreak at the two warmest treatments than those from warmer provenances. Interspecific differences were not observed in dieback but were detected in survival and budbreak. The hybrids had greater survival than butternut from warmer provenances at the lowest temperature treatment (-38 °C), but given that temperatures that low are extremely unlikely to occur in those provenances, it is not anticipated to give the hybrids an advantage if planted in those areas. However, the hybrids’ earlier budbreak could limit the success of restoration with these hybrids in the coldest extents of butternut’s range. </div><div>If hybrids, as well as genetically modified (GM) trees, are successfully developed for effective disease tolerance and to serve as an ecologically suitable replacement, success of restoration using hybrids will ultimately depend on those directly responsible for replanting efforts. A survey was administered to land managers in 46 organizations in Indiana to gauge perceptions of hybrid and GM trees, as well as current use of hybrid trees. Land managers had stronger concern for ecological, rather than economic, issues. Agreement was highest for using hybrid and GM trees for “conservation and restoration of at-risk species”, “timber production”, and “non-timber products (fruit, syrup, etc.)”. However, perceptions varied by characteristics, such as concern type, age, and the type of land they managed. Ecological concern and the type of land being managed most strongly predicted current hybrid use. Overall, results indicate the majority of land managers in Indiana would likely be agreeable to recommendations towards using hybrids. However, most nonetheless had strong ecological concerns about their suitability as a native replacement. It is important to note, though, that consistent with the results of previous studies, great variation was seen within the performance and characteristics of the butternut hybrids in chapters 2-4. Thus, it may be possible with careful selection and breeding to harness this variation to develop disease tolerant and ecologically similar hybrids acceptable to land managers.</div>

Text in English / South Africa is one of the biggest producers of genetically modified crops in the world. However, recent studies in South Africa show a low public willingness to consume genetically modified crops and accept modern agricultural biotechnology. The study analysed public perception towards consuming genetically modified crops and the acceptance of modern agricultural biotechnology in South Africa. 220 participants (N = 220) were sampled from the city of Kempton Park and the Chi-square formula was used to determine how well the sample represented the population under study. Data was collected using a 7-point Likert scale questionnaire designed following the guidelines for developing a theory of planned behaviour questionnaire in Ajzen (1991, 2001). Data analyses were carried out using the Statistical Package for Social Sciences (SPSS). The Cronbach’s alpha and Exploratory Factor Analysis were both used to determine the internal consistency and validity of the questionnaire. Correlations, independent sample t-tests, ANOVA, linear regression, and path analysis were also conducted. Findings of the study confirmed that there is low public willingness to consume genetically modified crops and to accept modern agricultural biotechnology in South Africa. / Development Studies / M.A. (Development Studies)