Master of Science / Department of Plant Pathology / Bernd Friebe / Bikram S. Gill / Common, bread, or hexaploid wheat, Triticum aestivum L. (2n=6x=42, AABBDD), has several relatives in the Triticum/Aegilops complex of the Poaceae family in the Triticeae tribe, which are valuable sources for broadening genetic diversity and may provide genes for disease and pest resistance and general wheat improvement. Other wild relatives of wheat also may be exploited for wheat improvement, such as Haynaldia villosa (L.) Schur. (2n=2x=14, VV). It is a diploid species with resistance to powdery mildew, wheat curl mite colonization, cereal eyespot disease, rust diseases, and wheat spindle streak mosaic virus. H. villosa may harbor many other as yet unidentified traits for wheat improvement. The polyploid nature of bread wheat allows tolerance to genomic changes, because homoeologous chromosomes from other genomes compensate for missing wheat chromosomes. In this experiment, we crossed the disomic alien addition line DA4V (2n=6x=44) with a pair of H. villosa chromosomes added to the wheat chromosome complement with wheat monosomic for chromosome 4D (2n=41) to produce 4D/4V double monosomic plants. According to centric breakage-fusion mechanisms, univalents tend to break at their centromeres at meiotic metaphase I producing telocentric chromosomes with unstable or “sticky” ends that can fuse with the sticky ends of other newly formed telocentric chromosomes. This fusion results in Robertsonian whole-arm translocations that may be compensating if a short arm of one chromosome fuses with a long arm of another. Double monosomic plants were screened cytogenetically and further visualized by genomic in situ hybridization (GISH). Five transfers were identified, including T4DS.4VL and T4VS.4DL translocations, and a T4VS-W.W transfer of unknown wheat origin. These results were confirmed by GISH. The T4DS.4VL and T4VS.4DL translocations are genetically compensating and should be exploited in wheat improvement.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2018-01-24T19:09:14Z No. of bitstreams: 1 2016 - Luciane Orbem Veronezi.pdf: 3213369 bytes, checksum: 660b54c56b80e667f1e30d110c395df3 (MD5) / Made available in DSpace on 2018-01-24T19:09:14Z (GMT). No. of bitstreams: 1 2016 - Luciane Orbem Veronezi.pdf: 3213369 bytes, checksum: 660b54c56b80e667f1e30d110c395df3 (MD5) Previous issue date: 2016-07-27 / CAPES / In this study the epidemiology, clinical signs, macroscopic and microscopic lesions related to spontaneous and experimental intoxication by Vicia villosa and the evaluation of the possible toxicity of Vicia villosa in cattle were characterized. The systematic granulomatous disease (SGD associated with vetch) was verified in 7 localities distributed in Paran?, Santa Catarina and Rio Grande do Sul States, with main feed that includes oats and/or ryegrass in association and V. villosa and/or ryegrass associated with Vicia sativa, and in lower quantity. All cattle that developed the disease were cows, lactating and Dutch. The SGD associated with vetch was reproduced in six lactating Dutch cows, by supplying green V. villosa in the trough and V. villosa and ryegrass pasture. In the other experimental groups (group 3, V. villosa / V. sativa/cows, grazing; group 4, V. sativa/cows, grazing; group 5, V. villosa/V. sativa/heifers and group 6, V. villosa/heifers, grazing) do not verified clinical and pathological alterations related to the SGD associated with vetch. Six cows developed the SGD, one cow presented a leigh clinical condition (group 2/cow 6/V. villosa, pasture), four cows developed a moderated clinical condition (group 1, cows 1 and 2/V. villosa/trough and group 2, cows 4 and 5/V. villosa, grazing), and just one cow presented a severe clinical condition (group 2, cow 3/V. villosa/pasture. V. villosa was toxic to cattle and produced a chronic granulomatous disease when ingested in amounts greater than 38.2 g/kg/day for 71 days, or in direct grazing for a period of more than 28 days The main clinical signs consisted of alopecia and cutaneous crusts, pruritus, fever, conjunctivitis, diarrhea, reduction of milk production and weight loss. Macroscopically, multiple white-grayish nodules were observed, specifically in the lymph nodes, kidneys and heart, and histology revealed a granulomatous infiltrate composed of macrophages, lymphocytes, giant cells, epithelioid cells, plasma cells and eosinophils. When ingested by young cattle, the V. villosa pasture, for a period of 68 days, do not produce clinical modifications. When ingested by cows with high milk yield, the V. sativa pasture, for a period of 78 days, does not produce chronic granulomatous disease. Ingestion of V. villosa for a long time led to low production rates, such as low milk production, low reproduction rates and predisposition to infection diseases. In the bromatological analysis, the presence of phytate, in low levels, in the samples of Vicia used in experimental works, in different phases of the culture was determined. The biochemical blood and feces results showing lower amounts of serum sinc and loss of Ca and Zn feces in animals poisoned by V. villosa, that developed granulomatous disease in severe and moderate conditions, possibly indicated the presence of a metabolic disorder in the pathogen of the disease. / No presente estudo caracterizou-se a epidemiologia, os sinais cl?nicos, as les?es macrosc?picas e microsc?picas da intoxica??o espont?nea e experimental por Vicia villosa, bem como a avalia??o da poss?vel toxicidade de Vicia sativa em bovinos. A doen?a granulomatosa sist?mica (DGS ervilhaca-associada) foi verificada em 7 propriedades distribu?das nos Estados de Santa Catarina, Paran? e Rio Grande do Sul, onde a alimenta??o principal era aveia e/ou azev?m consorciados com V. villosa e/ou de azev?m consorciado com V. villosa e em menor quantidade V. sativa. Todos os bovinos que desenvolveram a doen?a eram vacas, em lacta??o e da ra?a Holand?s. A DGS ervilhaca-associada foi reproduzida em seis vacas holandesas em lacta??o, atrav?s do fornecimento de V. villosa verde no cocho e em pastagens de V. villosa e azev?m. Nos demais grupos experimentais (grupo 3, V. villosa/V. sativa/vacas, pastejo; grupo 4, V. sativa/vacas, pastejo; grupo 5, V. villosa/V. sativa/novilhas e grupo 6, V. villosa/novilhas, pastejo) n?o verificou-se altera??es cl?nicas e patol?gicas relacionadas a DGS ervilhaca-associada. Das seis vacas que desenvolveram a doen?a granulomatosa sist?mica, uma vaca apresentou quadro cl?nico leve (grupo 2/vaca 6/V.villosa, pastejo), quatro vacas desenvolveram o quadro moderado da doen?a (grupo 1, vacas 1 e 2/V. villosa/cocho e grupo 2, vacas 4 e 5/V.villosa/pastejo) e apenas em uma vaca verificou-se o quadro grave da doen?a (grupo 2, vaca 3/V.villosa/pastejo). V. villosa foi t?xica para bovinos, produzindo doen?a granulomatosa cr?nica quando ingerida em quantidades superiores a 38,2g/kg/dia, por um per?odo de 71 ou, em pastoreio direto por um per?odo superior a 28 dias. As principais manifesta??es cl?nicas consistiam em ?reas de alopecia e crostas na pele, coceira, febre, conjuntivite, diarreia, queda na produ??o de leite e perda de peso. Macroscopicamente verificaram-se m?ltiplos n?dulos branco-acinzentados principalmente em linfonodos, rins e cora??o, e na histologia estes caracterizavam um infiltrado granulomatoso composto por macr?fagos, linf?citos, c?lulas gigantes, c?lulas epitelioides, plasm?citos e eosin?filos. As pastagens de V. villosa quando ingerida por bovinos jovens, por um per?odo 68 dias n?o produziu altera??es cl?nicas. As pastagens de V. sativa quando ingerida por vacas de alta produ??o leiteira por um per?odo de 76 dias n?o produziu doen?a granulomatosa cr?nica. A ingest?o de V. villosa por per?odos prolongados conduziu a baixos ?ndices produtivos como: baixa produ??o leiteira, baixos ?ndices reprodutivos e pode predispor o surgimento de doen?as infeciosas. Na an?lise bromatol?gica determinou-se a presen?a de fitato, em pequenas quantidades, nas amostras de Vicia utilizadas na experimenta??o, em diferentes fases do seu crescimento. Os resultados das an?lises bioqu?micas de amostras de sangue e fezes evidenciaram baixas quantidades s?ricas de zinco e excessivas perdas fecais de Ca e Zn nos animais intoxicados por V. villosa que desenvolveram a doen?a granulomatosa na forma moderada e grave, indicando um poss?vel envolvimento de dist?rbios de ordem metab?lica na patogenia da doen?a.

Hairy prairie-clover (Dalea villosa Nutt. (Spreng) var. villosa), a rare plant species, grows in the Canadian Prairies. Populations of Dalea in Canada are threatened by the loss of sand dune habitat because of changes in land use and altered ecological processes such as grazing and fire. Local populations of Dalea are further threatened by one or more specific threats, including herbivory from native and domestic ungulates and invasion of habitats by exotic plants. The overall objective of this thesis was to gain more knowledge about Dalea and to determine the impact of threats and management practices to the Saskatchewan populations and their habitats. Observational studies were conducted at each of two sites in Saskatchewan supporting Dalea. First, at the Dundurn Sandhills site, structural equation modeling was used to examine landscape, ecological, and management factors associated with high rates of herbivory on Dalea and with reductions in the long-term survival and productivity of Dalea. The conditions which deer (Odocoileus hemionus and Odocoileus virginianus) or cattle (Bos taurus) were responsible for the most intense rates of herbivory to Dalea plants and patches were determined. Generally, deer appeared responsible for the most herbivory, whereas cattle grazing on Dalea increased with stocking densities. At the same time, new hypotheses about ecological processes affecting Dalea productivity in the Dundurn Sandhills were explored. In particular, it appeared that deer may be responding to cattle grazing in Dalea habitat by avoiding those areas, and that mid-season germination and recruitment of many Dalea plants may occur following precipitation events. Second, at the Mortlach site, the costs and benefits of using grazing management to control leafy spurge (Euphorbia esula L. var. esula) were assessed, especially in consideration of the potential negative effects of intense herbivory on Dalea productivity. Aspects of the grazing regime including stocking density and the livestock species influenced herbivory on Dalea and its reproductive output, but there were no apparent links between the abundance of leafy spurge abundance and the reproductive output of Dalea. The findings of these two studies are relevant for the conservation and management of Dalea in Saskatchewan.

Philosophiae Doctor - PhD / Gethyllis multifolia L. Bolus and G. villosa Thunb. are winter-growing, summerblooming,deciduous and bulbous geophytes that grow naturally in the semi-arid succulent Karoo biome of South Africa. Both species grow under full sun conditions and have four distinctive growth phases: a winter (cold and wet) growing phase, leaf senescence phase towards spring, flowering phase during the hot and dry summer months, and fruit and leaf formation phase in autumn. The medicinal uses of this genus (including G. multifolia “Kukumakranka” and G.villosa “hairy kukumakranka”) range from cures for colic, digestive disturbances,teething problems, fatigue, boils, bruises and insect bites, to being used as an aphrodisiac. Gethyllis multifolia is threatened in its natural habitat and is listed in the ‘Vulnerable’ category of the ‘Red Data List of Southern African Plants’ and the ‘IUCN-World Conservation Union List of Plants’. The literature indicate that the habitats of both species are being exposed to drier conditions and is further threatened by the encroachment of invasive indigenous plant species. It is not known to which extent these factors may pose a threat to the existence of both species. The first objective of this investigation was to determine the costs of vegetative and reproductive growth during the seasonal life cycle of the plant, using carbon(C) and nitrogen (N) as a physiological currency. The second objective was to elucidate a functional basis to explain the difference in the conservation status of both species in their natural habitat. Both species were subjected to drought and shading as environmental stresses and the plant physiological performance was investigated via photosynthetic gas exchange. The third objective of the study was to evaluate the antioxidant content (total polyphenol, flavonol/flavone and flavanone content) and antioxidant capacity [ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC) and 2,2'-azino-di-3-ethylbenzthiazoline sulphonate (ABTS) radical cation scavenging ability] of natural populations and plant samples that were exposed to photo-and-drought environmental stresses. This study was done to elucidate the antioxidant profile of plant parts of natural populations as well as providing farmers, traditional healers and pharmaceutical companies with cultivation environmental conditions to enhance the antioxidant properties of the species. This investigation also attempted to isolate and characterize, by means of thin-layer chromatography (TLC) and column chromatography (CC), natural compounds from both species to lend support to the purported antioxidant benefit of both species and to further lend support to claims made by traditional healers of the medicinal potential of the genus. This study, however, did not engage in any in vivo studies or human trials to support published literature of the medicinal benefits of the genus.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2015-12-15T13:26:48Z No. of bitstreams: 1 erickestevesdeoliveira.pdf: 1235775 bytes, checksum: d5513847048574c1ada373c0b486b859 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2015-12-15T13:42:12Z (GMT) No. of bitstreams: 1 erickestevesdeoliveira.pdf: 1235775 bytes, checksum: d5513847048574c1ada373c0b486b859 (MD5) / Made available in DSpace on 2015-12-15T13:42:12Z (GMT). No. of bitstreams: 1 erickestevesdeoliveira.pdf: 1235775 bytes, checksum: d5513847048574c1ada373c0b486b859 (MD5) Previous issue date: 2015-02-24 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / Ocimum. gratissimum L. e Mentha x villosa Huds. são duas plantas aromáticas que são amplamente utilizadas no Brasil e em outros países tanto para fins terapêuticos, como na culinária. Diversas atividades farmacológicas já foram descritas para essas duas plantas e para os componentes majoritários de seus óleos essenciais, Eugenol e Óxido de Piperitenona, respectivamente. Porém ainda há a carência de estudos sobre a atividade antiproliferativa desses óleos essenciais sobre linhagens tumorais e as implicações desses compostos sobre a produção de TGF-1, uma citocina que, em tumores já estabelecidos, apresenta efeito pró-tumoral. Esse estudo visa avaliar os efeitos dos tratamentos com os óleos essenciais de O. gratissimum e M. x villosa, sobre uma linhagem de adenocarcinoma humano de pulmão produtora de TGF-1. Métodos: A viabilidade celular foi avaliada através do ensaio de MTT em linhagens de células (A549 e J774 A.1) e em macrófagos intraperitoneais tratados com 5 a 200g/mL, por 48h. O efeito dos óleos essenciais sobre o ciclo celular foi avaliado através de marcação com iodeto de propídio e quantificação das fases de ciclo celular por citometria de fluxo. A quantificação de células em SubG1 foi utilizada como parâmetro para avaliação de apoptose, que foi confirmada através da marcação de TUNEL. A produção de TGF-1 foi avaliada por ELISA. Resultados: Os dois óleos essenciais reduziram a viabilidade da linhagem celular A549 (IC50: OG:160g/mL / MV:117g/mL), os tratamentos também induziram parada do ciclo celular em S com 24h de tratamento (controle: 12,31±0,89 / OEOG: 15,70±1,15 / OEMV: 23,35±0,75) efeito já registrado para eugenol. Também houve aumento do percentual de células em SubG1 (controle: 15,05±0,71 / OEOG: 91,94±1,71 / OEMV:24,62±1,06), indicando aumento da fragmentação de DNA, um dos sinais de apoptose, que foi confirmado pelo aumento da mediana de fluorescência na marcação de TUNEL (controle:59.1±3.4 / OEOG:68.6±3.7 / OEMV:75.3±15.7). Houve redução da produção de TGF-1 nas concentrações não letais dos óleos essenciais (10 e 50g/mL). Conclusões: Esses dados demonstram o potencial indutor de apoptose e de parada de ciclo celular, desses óleos essenciais para o tratamento de tumores, sobretudo aqueles caracterizados pela produção de TGF-1, citocina importante para a sobrevivência e proliferação de células tumorais. Contudo, essa citocina desempenha papéis importantes na homeostase do organismo, e por isso são necessários estudos que avaliem o efeito sistêmico desses óleos essenciais. / Ocimum. gratissimum L. and Mentha x villosa Huds are aromatic plants largely used, in Brazil and other countries, for therapeutic and culinary purposes. Several pharmacological properties have been described for their essential oil and their major compounds: Eugenol and piperitenone oxide respectively. However, the antiproliferative effect and the blockage of the TGF-1 production are poorly understood. This cytokine contributes for the development of late-phase tumors. This study aimed to evaluate the effects of the essential oils of Ocimum gratissimum and Mentha x villosa over a TGF-1 producer human lung adenocarcinoma cell line. Methodology: Viability was assessed through MTT assay, on cell lineages (A549 and J774 A.1) and intraperitoneal macrophages treated for 48h with essential oil concentrations ranging from 5 to 200g/mL. Cell cycle and SubG1 DNA amount was evaluated through propidium iodide staining followed by flow citometry analysis. TUNEL assay was used to quantify the DNA fragmentation. ELISA was performed to measure the TGF-1 production. Results: Both essential oils reduced A549 cell viability (IC50: EOOG:160g/mL / EOMV:117g/mL), there was also cell cycle arrest at S phase, after 24hours of treatment (control: 12,31±0,89 / EOOG: 15,70±1,15 / EOMV: 23,35±0,75). SubG1 DNA amount was also elevated after treatment (control: 15,05±0,71 / EOOG: 91,94±1,71 / EOMV:24,62±1,06), an indicative of DNA fragmentation, one of the apoptosis sign. This effect was confirmed by the elevated TUNEL labeling. The non-lethal concentrations of the essential oils (10 and 50g/mL) led to the reduction of TGF-1 production. Conclusion: These data indicates the apoptotic and cell cycle arrest effects of the essential oils, mainly over TGF-1 producing tumors. As this cytokine has a key role over the survival and proliferation of these cancer cells. The possible systemic effects of these treatments are yet to be evaluated, as the TGF-1 blockage may affect the maintenance of homeostasis.

La incorporación de leguminosas en los agroecosistemas ganaderos, presenta una serie de ventajas productivas y ambientales, respecto a los sistemas basados únicamente en gramíneas. El objetivo del trabajo fue analizar el efecto de la incorporación de leguminosas en pasturas puras de agropiro alargado (Thinopyrum ponticum) en la región semiárida. Para ello, se condujeron una serie de experimentos con el objetivo de evaluar específicamente (1) el efecto de la intersiembra con vicia (Vicia villosa) sobre la biomasa forrajera, valor nutricional de la pastura y dinámica de las fracciones orgánicas del suelo en tres ambientes del sudoeste bonaerense (Bahía Blanca, Tornquist y Cabildo), (2) el efecto de la densidad de intersiembra con vicia o trébol de olor blanco (Melilotus albus), sobre los cambios de valor nutricional en función de la composición botánica, y por último, (3) determinar el momento de corte óptimo para la confección de reservas (heno o ensilaje) y estudiar el efecto de la incorporación de grano de cereal en ensilaje de pasturas consociadas. Las mezclas forrajeras se realizaron intersembrando vicia o trébol en otoño de cada año, sobre agropiro implantado. El forraje se cortó manualmente, sobre una superficie de 0,4 m2 y la frecuencia de defoliación estuvo basada en el crecimiento de agropiro. Durante un período de dos años sobre una pastura monofítica de agropiro y una consociación con vicia (20 kg ha-1), se evaluó la biomasa forrajera y su valor nutricional (FDN, FDA, LDA, DIVMS y PB). Durante este ensayo se realizó un fraccionamiento físico del suelo, para separar las diferentes fracciones que componen la materia orgánica hasta la profundidad de 0-20 cm. En otro ensayo, se decidió estudiar el efecto de las densidades de intersiembra, se incluyeron en este caso dos especies de leguminosas (vicia o trébol). Ambas, se intersembraron separadamente a tres densidades diferentes, en función de plantas agropiro:leguminosa (80:20, 70:30 y 60:40). Por su parte, para el estudio de reservas forrajeras, en otro ensayo, se cortó una pastura pura de agropiro en cinco estados fenológicos, desde vegetativo (7/11/2012) hasta grano pastoso (14/02/2013) para la elaboración de heno y ensilaje en cada fecha. Con esta información, en un último ensayo se estudió el efecto de la inclusión de una leguminosa (trébol) y grano de avena en ensilajes de agropiro. Las mezclas de forraje y grano se realizaron en laboratorio combinando tres proporciones de trébol (T0: sin trébol, agropiro puro; T20: 20% trébol y T40: 40% trébol) y dos niveles de grano (G0: sin grano y G1: 30% de grano) en base a la MS. Cuando se intersiembra vicia o trébol sobre agropiro, se registró en todos los sitios un aumento en la producción forrajera anual que fue del 28 a 190%, salvo para la localidad de Cabildo que la producción no cambió (p=0,08). Analizando la producción de agropiro separadamente no se observa una disminución con la inclusión de vicia. Por lo tanto, se entiende que la leguminosa acompañante, explora recursos que el agropiro no hace. En cambio, se encontró que trébol ejerció una competencia con agropiro avanzada la primavera, porque a medida que aumentó la biomasa de trébol disminuyó linealmente la biomasa de agropiro. Comparando la producción forrajera de vicia y trébol, ambas especies mostraron una buena capacidad de compensación en el crecimiento, ya que no mostraron diferencias en el rendimiento a diferentes densidades. En general, la intersiembra con leguminosas aumenta el contenido de PB (entre 1,0 y 1,4% cada 10% de leguminosa en la composición), DIVMS y LDA de la pastura, mientras que disminuyó los contenidos de FDN (1,5% cada 10% de leguminosa) y FDA. Además, se identificó trasferencia de nitrógeno de vicia y trébol hacia agropiro por mayor contenido de PB del agropiro consociado, tanto en primavera como en otoño. La inclusión de vicia incrementó el contenido de Carbono orgánico particulado (COP) grueso (hasta 42%) y fino (hasta 13%) del suelo, sin modificar el contenido de carbono orgánico total. Para Cabildo y Bahía Blanca la inclusión de vicia también generó un aumento (12%) del nitrógeno de la materia orgánica particulada. Respecto a la elaboración de reservas de agropiro, se encontró que el heno mantuvo mejor el valor nutricional que el ensilaje. Aunque, la mayor fuente de variación se relacionó al estado fenológico de la pastura en el momento de corte y no al tipo de reserva elaborada. Para la elaboración de un adecuado ensilaje de agropiro, el período óptimo de corte va desde espiga embuchada a antesis, mientras que para henificación el período óptimo es mayor, desde estado vegetativo hasta antesis. La incorporación de una leguminosa como el trébol y el grano de avena al ensilaje de agropiro generó aumentos en el contenido de PB y disminuciones en FDN, FDA. La adición de grano, mejora la DIVMS en los tratamientos con menor cantidad de trébol (T0 y T20). A medida que aumentó la participación de trébol, los valores de pH se incrementaron progresivamente de 3,78 (T0) a 4 (T40). Sin embargo, la adición de grano generó una disminución de pH. El ensilaje con trébol de olor blanco hasta el 40%, no presentó un problema para la realización de un adecuado ensilaje, resultando la mejor alternativa junto con la incorporación de grano. / The inclusion of legumes in livestock agroecosystems have production and environmental advantages, compared to systems based only on grasses. The objective of this work was to analyze the effect of the inclusion of legumes in pure pastures of tall wheatgrass (Thinopyrum ponticum). A series of experiments were conducted with the objective of evaluating, (1) the effect of interseeding hairy vetch (Vicia villosa) over the forage biomass, nutritional value of pasture and dynamics of soil organic matter fractions in three representative semiarid environments of the Buenos Aires province, Argentina (Bahía Blanca, Tornquist and Cabildo), (2) the effect of the density of interseeding with hairy vetch or white sweetclover (Melilotus albus) over nutritional value depending on the botanical composition, and finally, (3) determining the optimal cutting moment for the preparation of reserves and study the effect of the incorporation of grain in silage of mixture pastures. The forage mixtures were made by interseeding hairy vetch or clover in the autumn of each year, on implanted tall wheatgrass. The forage was cut manually, on a surface of 0,4 m2 and the frequency of defoliation was based on the growth of tall wheatgrass. During a two-year period, on tall wheatgrass monoculture and mixture with hairy vetch (20 kg ha-1), biomass yield and its nutritional value (NDF, ADF, ADL, IVDMD and CP) were evaluated. During this period, soil organic matter fractions were separated by size using a wet-sieving method up to the depth of 0-20 cm. Besides, to study the effect of interseeded densities, another test was realized that included two species of legumes (hairy vetch or white sweetclover). Separately, both legumes were interseeded at three different densities based on the tall wheatgrass:legume plant relationships of 80:20, 70:30 and 60:40. Finally, for the study of reserves, a pure pasture of tall wheatgrass was cut in five phenological stages from vegetative (11/7/2012) to dough-grain (2/14/2013) for the preparation of hay and silage on each date. With this information, in another trial the effect of the inclusion of a legume (clover) and grain of oats in silages of tall wheatgrass was studied. The mixtures of forage and grain were made in the laboratory combining three proportions of clover (T0: without clover, pure tall wheatgrass, T20: 20% clover and T40: 40% clover) and two grain levels (G0: no grain and G1: 30 % grain) based on the MS. When hairy vetch or clover was interseeded over tall wheatgrass, an increase in annual yield biomass was recorded from 28 to 190%, except in Cabildo, where production did not change. Analyzing tall wheatgrass biomass separately, there is no decrease in production with hairy vetch inclusion. Therefore, the accompanying legume, seems to use the resources that tall wheatgrass leaves available. Instead, the clover competed with tall wheatgrass in late spring, thus, as the biomass of clover increased, the biomass of tall wheatgrass decreased linearly. Hairy vetch and clover plants showed growth compensation because there was no difference in forage yield at different density. In general, the hairy vetch or clover interseeding increased total CP (between 1,0 - 1,4% every 10% of legume in the composition), IVDMD and ADL, while the NDF (1,5 % every 10% of legume in the composition) and ADF decreased. In addition, the nitrogen transfer from the legume appear to be responsible for the increase in the CP content of tall wheatgrass when growing together. This was evident in both seasons spring and fall. Hairy vetch increased the content of particulate organic carbon (POC) in fraction >105μm (up to 42%) and fraction between 53 and 105 μm (up to 13%) of the soil. The total organic carbon in soil (SOC) content did not change, therefore the relationship (POC/SOC) was increased. For Cabildo and Bahía Blanca hairy vetch inclusion enhanced the N content (12%) of the particulate organic matter. Regarding the preparation of wheatgrass reserves, it was found that hay maintained its nutritional value better than silage. Although, the greatest source of variation can be attributed to the phenological stage of the pasture and to a lesser extent to the type of reserve. For the best silage of tall wheatgrass, the optimal cutting period was from booting to anthesis, whereas, for haymaking the optimal period extends from vegetative to anthesis. Inclusion of clover and grain increased silage CP content and decreased NDF and ADF. In addition, grain incorporation improved IVDMD in treatments with less clover (T0 and T20). The highest proportion of clover in the mixture reduced FDN and FDA. As clover participation was greater, the silage pH values increased progressively from 3.78 (T0) to 4 (T40). However, the addition of grain decreased pH. According to these results, a silage including white sweetclover up to 40%, was not a restriction to obtain a fairly good quality silage, being the best alternative when also combined with grain.

The Clinch River (CR) and North Fork Holston River (NFHR) contain some of the most diverse freshwater mussel assemblages in the United States; however, both rivers are experiencing declines in mussel populations. The first component of this study used histological evaluations and water quality data to determine whether mussels were negatively impacted in the CR zone of decline (ZD) and to inform future management of freshwater mussels in the river. In the 91 kilometer (km) section from Carbo, Virginia (CRKM 431) downstream to Speers Ferry, Virginia (CRKM 340), referred to as the ZD, mussel density decreased >90% from 1979 to 2014 at key sites such as Semones Island (CRKM 378.3) and Pendleton Island (CRKM 364.2). Laboratory propagated mussels were placed in cages in the river for one year from June 2012 to May 2013 at four sites within the ZD and four sites in reaches where mussel populations remain stable or are increasing, a zone of stability (ZS). The survival, growth and histological results indicated that there are continuing impacts to mussels in the ZD. Research investigating impacts to the ZD and methods to improve water quality in this zone are needed. The laboratory component of this study examined sublethal effects of potassium (K⁺), chloride (Cl⁻), and un-ionized ammonia (NH₃-N) on mussel tissues at concentrations relevant to those found in the NFHR. Historical industrial activities at Saltville, Virginia, as well as continued pollution of the NFHR from chemical waste ponds at this location, are believed to be significant contributors to mussel declines. Contaminant seepages from the waste ponds that include Cl⁻, K⁺, and NH₃-N have been shown to be toxic to adult and juvenile mussels. A three-month laboratory study was conducted to assess impacts to organ tissues (gills, digestive glands, kidneys, and gonads) of adult Villosa iris exposed to environmentally relevant concentrations of K⁻ (4 and 8 mg/L), Cl⁻ (230 and 705 mg/L), and NH₃-N (0.014 and 0.15 mg/L) using histological evaluations. No detectable differences were observed among the histological endpoints from mussels held in treatments and control (p>0.05). The study design was modified and repeated using increased concentrations of K⁺ (8, 16, and 32 mg/L) and Cl⁻ (705, 1410, and 2820 mg/L) for a two-month exposure period. Due to issues with maintaining NH3-N in mussel holding chambers, the second study did not the second study did not include NH₃-N exposures. Control mussels in both studies had a higher abundance of lipofuscin in kidneys and degraded cytoplasm in the digestive gland diverticula compared to baseline mussels, indicating that captivity influenced mussel tissues. Future studies are needed to more thoroughly address these captivity effects. Both survival and histological data in the second test showed a significant negative effect of the increased concentrations of Cl⁻ and K⁻, which were representative of those found at some sites in the NFHR downstream of Saltville, Virginia. / Master of Science

Cancer is the given name for a group of more than 100 diseases account for 13% of deaths worldwide and 15.1% in Brazil, with estimates of increased mortality. These data show the deficiency of more effective anticancer agents, leading to increased life expectancy or cure. In this context, nature is an alternative to the problem, for harboring a huge biodiversity and Brazil stands out for having the highest diversity of plant species in the world, but little explored as their biological characteristics. The genus Mentha has its very widespread species in the country, with many evidenced therapeutic activities, among them the potential anticancer action of essential oil (EO) demonstrated in two species (Mentha piperita e Mentha spicata). However, this study aimed to evaluate the antitumor activity of the essential oil of Mentha x villosa Hudson (OEMV) in vitro and in vivo, and its toxicity in vivo. For this, the EO was extracted from the leaves of Mentha x villosa, 12 of its constituents have been identified and their main constituent, the monoterpene rotundifolone was isolated. The in vitro cytotoxicity of OEMV rotundifolone and 3 were evaluated against tumor cell lines: ovarian adenocarcinoma (OVACAR-8), colon carcinoma (HCT-116) and glioblastoma (SF295) by the MTT assay. The OEMV showed cytotoxic activity in all cell lines tested, with IC50 ranging from 0.57 to 1.02 ìg/mL. However, rotundifolone did not show cytotoxicity at the tested concentrations, suggesting that the activity of the oil is not mediated by its major constituent. Considering this result, we evaluated the antitumor activity of OEMV in vivo using mice transplanted with sarcoma 180. In this study, it was showed antitumor activity in OEMV treatments performed by intraperitoneal (32.02 and 42.81% with 50 and 100 mg/kg/day, respectively) and orally (34.27 and 43.22% with 100 and 200 mg/kg/day, respectively), probably due to synergy among its constituents. No change was observed in the toxicological parameters evaluated in animals treated with OEMV: variation in body mass, organ weights (liver, spleen and kidney), blood biochemical parameters (AST, ALT, urea and creatinine) and ulcerative lesion index (this later only in the groups treated orally). Since the results demonstrated that OEMV has antitumor activity with low toxicity, it was proposed to evaluate the benefit of the association between OEMV (50 and 100 mg/kg/day) and 5-fluorouracil (5-FU, 10 mg/kg/day) reference known antineoplasic and toxicity. The association promoted tumor growth inhibition of 50.3% and 65.2% at doses of 50 and 100 mg/kg/day, respectively. The highest dose of OEMV in combination showed the same statistical difference that the level of inhibition of 5-FU at the highest dose (25 mg/kg/day). The toxicological parameters, variation of body mass and biochemical parameters (AST and ALT) showed alterations similar as the positive control. The organ weight alterations did not occur, a result that demonstrates an evident benefit of the combination compared to the group treated with 5-FU alone. The last toxicological endpoints evaluated were hematological parameters where both groups showed changes associated with differential count (lymphocytosis and neutropenia) and reduction in total leukocytes; positive factor in this result is that the reduction was not as intense as the one displayed by the control 5-FU furthermore possibly resulting in a decreased susceptibility to infection. The association promoted tumor growth inhibition 50.3% and 65.2% at doses of 50 and 100 mg/kg/day, respectively. The highest dose of OEMV in combination showed the same statistical difference that the level of inhibition of 5-FU dose (25 mg/kg/day) the toxicological parameters, variation of body mass and biochemical analysis (AST and ALT) showed alterations similar to the positive control. / O cancer e o nome dado a um conjunto de mais de 100 doencas, responsaveis por 13% das mortes no mundo e 15,1% no Brasil, com estimativas de mortalidade crescente. Esses dados refletem a carencia por agentes antineoplasicos mais efetivos, que levem ao aumento da expectativa de vida ou a cura. Nesse contexto, a natureza e uma alternativa para o problema, por abrigar uma gigantesca biodiversidade e o Brasil se destaca, por possuir a maior diversidade de especies de plantas do mundo, mas pouco exploradas quanto suas caracteristicas biologicas. O genero Mentha tem suas especies bem difundidas no pais, com diversas atividades terapeuticas comprovadas, dentre elas o potencial acao anticancer do oleo essencial (OE) comprovadas em duas especies (Mentha piperita e Mentha spicata). Portanto, o presente estudo teve como objetivo avaliar a atividade antitumoral do oleo essencial da Mentha x villosa Hudson (OEMV) in vitro e in vivo, e sua toxicidade in vivo. Para isso, o OE foi extraido a partir das folhas da Mentha x villosa, onde 12 dos seus constituintes foram identificados e isolado seu constituinte majoritario, a rotundifolona . A citotoxicidade in vitro do OEMV e da rotundifolona foi avaliada frente a 3 linhagens de celulas tumorais: adenocarcinoma ovariano (OVACAR-8), carcinoma de colon (HCT-116) e glioblastoma (SF295) atraves do ensaio do MTT. O OEMV apresentou atividade citotoxica em todas as linhagens de celulas testadas, com CI50 variando entre 0,57 e 1,02 Êg/mL. Porem, a rotundifolona nao demonstrou atividade citotoxica nas concentracoes testadas, sugerindo que a atividade do oleo nao e mediada por seu constituinte majoritario. Diante desse resultado, foi avaliada a atividade antitumoral do OEMV in vivo utilizando camundongos transplantados com sarcoma 180. Neste ensaio, o OEMV apresentou atividade antitumoral nos tratamentos realizados pela via intraperitoneal (32,02 e 42,81% com 50 e 100 mg/kg/dia, respectivamente) e pela via oral (34,27 e 43,22% com 100 e 200 mg/kg/dia, respectivamente), por provavel sinergismo entre seus constituintes. Nao foi observada nenhuma alteracao nos parametros toxicologicos avaliados em animais tratados com o OEMV: variacao de massa corporea, massa dos orgaos (figado, baco e rins), analises bioquimicas (AST, ALT, ureia e creatinina) e indice de lesao ulcerativa (este ultimo apenas nos grupos tratados por via oral). Como os resultados demonstraram que o OEMV possui atividade antitumoral, com baixa toxicidade, foi proposto avaliar o beneficio da associacao, via i.p, entre o OEMV (50 e 100 mg/kg/dia) e o 5-Fluorouracil em menor dose (5-FU, 10 mg/kg/dia), antineoplasico de referencia e conhecida toxicidade. A associacao promoveu a inibicao do crescimento tumoral de 50,3% e 65,2% nas doses de 50 e 100 mg/kg/dia, respectivamente. A maior dose do OEMV em associacao demonstrou a mesma diferenca estatistica que o indice de inibicao do 5-FU na maior dose (25 mg/kg/dia). Os parametros toxicologicos, variacao de massa corporea e analise bioquimica (AST e ALT) apresentaram alteracoes semelhantes as do controle positivo. Na massa dos orgaos nao ocorreu alteracao, resultado que demonstra um evidente beneficio da associacao quando comparada ao grupo tratado com 5-FU isolado. O ultimo parametro toxicologico avaliado foram as analises hematologicas onde ambos os grupos associados demonstraram alteracoes na contagem diferencial e reducao na contagem de leucocitos totais; o fator positivo nesse resultado e que a reducao nao foi tao intensa quanto a apresentada pelo controle positivo 5-FU, provocando possivelmente, uma menor susceptibilidade a infeccoes. Diante dos resultados, podemos afirmar que o OEMV tem atividade antitumoral in vitro e in vivo com baixa toxicidade, e que a associacao entre 5-FU e o OEMV potencializa a atividade antitumoral do antineoplasico, diminuindo alguns dos seus efeitos toxicos.

In the exploration into new therapeutic agents for human disease, medicinal herbs offer an enormous resource due to their wide range of biologically active components. However, because of these biologically active components, medicinal herbs can also have toxic side effects. The focus of this thesis is the effect of herbal therapies, both good and bad, on chronic kidney disease (CKD) and tubulointerstitial fibrosis. Tubulointerstitial fibrosis is considered one of the defining characteristics of CKD. In Chapter 1, the literature regarding the pathogenesis of tubulointerstitial fibrosis is reviewed, beginning with the mechanisms of its development, the main structural and functional features, and the molecular mediators. The structural features include activation of resident fibroblasts and transition of tubular epithelial cells into myofibroblasts, deposition of extracellular matrix proteins, increased apoptosis of normal cells of the renal nephron and development of tubular atrophy, increased renal oxidative stress, and hypoxia of renal tissues. Molecular mediators that are explored include angiotensin II, transforming growth factor-ß1 and numerous other cytokines and growth factors. Pharmacological manipulation of these features and their molecular mediators for regression of tubulointerstitial fibrosis is then discussed. Currently, the gold standard of therapy for people with CKD is blockade of the renin-angiotensin-aldosterone system with angiotensin converting enzyme inhibitors (ACEIs) and/or angiotensin receptor blockers (ARBs). Because of the complexity of the pathogenesis of renal fibrosis and the multiple mechanisms by which ACEIs and ARBs work, this portion of the thesis focuses on the qualities that additional agents should have to complement their actions. These additional agents could work by decreasing oxidative damage, by decreasing fibroblast numbers through apoptosis, through the interruption of inflammatory, fibrotic mediators, by increasing anti-inflammatory mediators or through other mechanisms. The literature review therefore continues with a discussion of the historical use of medicinal herbs in genitourinary pathologies and the known contributions that medicinal herbs can make to the treatment or development of tubulointerstitial fibrosis and CKD. From this review, a large number of herbs were identified as having traditional use in renal disorders or as being of interest, currently, to researchers of renal pathologies. However, much information is lacking regarding the mechanisms by which the hypothesised benefits occur, making it impossible to assess which herb(s) may offer valuable additive support or alternative treatments to the therapies currently given to people with CKD. Furthermore, there is a lack of information regarding toxicity of these herbs; many herbs have never been assessed in cell culture or in animal toxicity studies. It was apparent that preliminary in vitro work was necessary before in vivo pharmacological work could be undertaken. This thesis, therefore, aimed to test the following hypotheses: (1) That medicinal herbs used currently for treatment of renal dysfunction have high anti-oxidant properties that can be further enhanced by specific extraction processes; (2) That the in vitro testing of selected extracts from medicinal herbs, identified in (1), will reveal some anti-oxidant benefits or indications of toxicity that need careful analysis in animal studies; (3) That careful in vivo testing of specific toxic medicinal herbs identified in these leadup studies will define specific pathological processes that predict an outcome of CKD; and (4) That careful in vivo testing of selected medicinal herbs, used in conjunction with more conventional medicines for CKD, will show an additive benefit when used to ameliorate development of CKD induced using an established animal model. The subsequent laboratory work was designed to test the validity of these hypotheses and the results are then presented in Chapters that each comprise a publication. The aim of Chapter 2 was to present a systematic analysis of the oxidant properties of 55 medicinal herbs that have been used traditionally to treat kidney and urinary disorders or have been of recent interest to researchers of renal disorders. Since different extraction processes yield different constituents, each of the herbs was sequentially extracted with three solvents of decreasing polarity. An assay was performed on each of the fractions to determine the oxygen radical absorbance capacity. The aim of Chapter 3 was to test the benefit or otherwise of each of the three extracts of the chosen herbs using an in vitro cell study. Each extract was tested for potential toxic, apoptotic, mutagenic and antioxidant activity on normal mammalian renal tubular epithelial cells (NRK-52E). The effect of the extracts on renal fibroblasts (NRK-49F) was also analysed. Several specific hypotheses arose from the combination of the systematic analyses and the literature review regarding benefits and toxicities of a number of the extracts. The subsequent in vivo work was designed to test the validity of two of these hypotheses. The aim of Chapter 4 was to test the hypotheses developed from the results of the previous Chapter. The herb Dioscorea villosa had demonstrated extreme cytotoxicity to mammalian renal epithelial cells and had caused transdifferentiation of epithelial cells into fibroblasts. An in vivo rodent model was used to test chronic dosage with this herb and its toxicity and predisposition for induction of CKD verified. The aim of Chapter 5 was to determine whether a herbal preparation (Angelica sinensis and Astragalus membranaceus) that had some support from the literature and the results from Chapters 2 and 3, could complement the actions of ACEIs in a rodent model of renal fibrosis (unilateral ureteral obstruction). The combination of herbal medicines and the ACEI was significantly more effective than the ACEI alone in ameliorating several characteristics of CKD development. To conclude the thesis, Chapter 6 provides an overview discussion of the results and a critical analysis of the methods used. Further, Chapter 6 looks towards future experiments that are planned to further resolve issues of concern about effects on renal health from use of medicinal herbs. .

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