This work concentrated on isolation of novel hop transcription factors from bHLH, bZIP, MYB, and WRKY families involved in the regulation of lupulin flavonoid pathways, followed by their structural and functional analysis. Structural analyses included bioinformatic approaches to elucidate gene organization, domain structure of the putative protein products, and potential post-translational modifications. I performed site-directed mutagenesis to disclose the role of phosphorylation sites in HlbZIP1A stability. Further, this work determined protein-DNA interactions for obtained TFs, giving support to the binding of MYB-bHLH-WDR complexes to the promoter of chalcone synthase H1, a key enzyme of the lupulin flavonoid pathways. Employing bioinformatic approaches, quantitative RT-PCR and transient co-expression, I pointed out chalcone synthase H1 as a regulatory crossroads in the metabolic (flavonoid) responses during hop stunt viroid pathogenesis.

In the United States, hops (Humulus lupulus L.) are grown mainly in the Pacific Northwest (PNW). For this reason, most cultural information is based on the growing conditions of the PNW. Growing conditions in Virginia differ drastically and present unique disease and production challenges. Three studies were conducted with the intent of increasing hop cultivation knowledge for Virginia growers. For the first study, 13 cultivars of hops grown at the Virginia Tech hop yard were compared for growth, yield, and quality. Mean cone fresh weight per plant ranged from 12.00 g for Mt. Hood to 1002.87 g for Crystal in 2016 and from 97.98 g for Mt. Hood to 900.33 g for Cascade in 2017. In 2016, only Alpharoma, Cascade, Mt. Rainier, and Southern Cross had alpha acid levels, an indicator of cone quality, within the accepted range. In 2017, Alpharoma, Centennial, Mt. Rainier, and Nugget had alpha acid levels within the expected range. Three cultivars (Cascade, Crystal, and Ultra) were above the expected alpha acid range, which indicates more bittering potential for beer brewers. In a nutrient deficiency study, hop plants were grown in hydroponic solutions, and deficiencies were induced for nitrogen (N), phosphorus (P), and potassium (K). After visual deficiency symptoms had been induced, leaf tissue samples were taken and analyzed for nutrient content. Images were taken at each deficiency stage. For N, incipient deficiency symptoms were observed at a mean of 3.18% dry weight in leaf tissue samples. Visual symptoms included a chlorotic appearance, undersized leaves, and red petioles. Incipient symptoms for P were observed at a mean of 0.307% dry weight in leaf tissue samples. Necrotic spots, leaf cupping, and undersized leaves were apparent with this deficiency. Incipient symptoms for K were observed at a mean of 1.21% dry weight in leaf tissue samples. Symptoms included rounded leaf tips, blue veins, and marginal scorch. In the third study, a whole leaf powdery mildew (Podosphaera macularis) assay was developed and tested using five hop cultivars, Alpharoma, Cascade, Comet, Sorachi Ace, and Tahoma. Leaves were inoculated with powdery mildew (PM) using a settling tower. This method was used to rapidly assess the resistance of cultivars. Leaves were successfully inoculated and PM colonies were allowed to grow for two weeks. Images of the PM colony development on inoculated leaves were compared using ImageJ to determine percentage of coverage. Tahoma was the only cultivar found to produce a significantly different mean percent coverage (19.5%) compared with the resistant cultivars Cascade and Comet (<1%). / Master of Science in Life Sciences / Hops (Humulus lupulus) were first grown in the United States in Massachusetts in the early 1600s. Production of this crop eventually spread throughout the Northeastern US. By the mid-1800s, commercial production spread to southern states such as Virginia. Infestation of pests, e.g., hop aphid and diseases such as downy mildew in eastern states, and prohibition on the production of alcohol caused a significant shift in hop production, which favored the Pacific Northwest (PNW). As a result, in Virginia specific knowledge of hop cultivation is now lacking. Three studies were conducted to increase our knowledge of hop cultivation for the region. For the first study, 13 cultivars grown at the Virginia Tech hop yard were compared for growth, yield, and quality. The cultivars Cascade and Alpharoma had alpha acid levels equal to or higher than expected for the 2016 and 2017 seasons. The alpha acid levels are an indicator of the hop quality from the Cascade and Alpharoma cultivars. Cascade was also a top producer of hop cones with a mean fresh weight of 989.67 g and 900.33 g in 2016 and 2017, respectively. In a nutrient deficiency study, plants were grown in Hoagland hydroponic solutions, which contain all essential nutrients needed for plant growth. This was done to provide photographic documentation to assist local growers with deficiency identification. Deficiencies were induced for nitrogen (N), phosphorus (P), and potassium (K) by removing each of the specific nutrient of interest from each treatment solution. After deficiency symptoms were induced, leaf tissue samples were taken and analyzed for nutrient content. Nutrient deficiencies were documented using photographs. For N, visual symptoms included a chlorotic appearance, undersized leaves, and red petioles. Incipient symptoms for P included necrotic spots, leaf cupping, and undersized leaves. For K, leaf cupping, blue green veins, marginal scorch, and rounded leaf tips. Disease resistance of cultivars is important information for growers and can heavily influence hop yard planning. As such, in another study, an assay for powdery mildew (Podosphaera macularis) was developed that allows for rapid low cost testing of hop cultivars. The assay was tested on the following cultivars: Alpharoma, Cascade, Comet, Sorachi Ace, and Tahoma. Leaves were inoculated with powdery mildew (PM) using a settling tower. Powdery mildew colonies were allowed to grow for two weeks and were then analyzed using ImageJ software to determine percent coverage. At the end of the experiment, Tahoma had significantly greater PM coverage compared to the other cultivars, indicating that Tahoma is less resistant to the specific PM strain. / Hops (Humulus lupulus) were first grown in the United States in Massachusetts in the early 1600s. Production of this crop eventually spread throughout the Northeastern US. By the mid-1800s, commercial production spread to southern states such as Virginia. Infestation of pests, e.g., hop aphid and diseases such as downy mildew in eastern states, and prohibition on the production of alcohol caused a significant shift in hop production, which favored the Pacific Northwest (PNW). As a result, in Virginia specific knowledge of hop cultivation is now lacking. Three studies were conducted to increase our knowledge of hop cultivation for the region. For the first study, 13 cultivars grown at the Virginia Tech hop yard were compared for growth, yield, and quality. The cultivars Cascade and Alpharoma had alpha acid levels equal to or higher than expected for the 2016 and 2017 seasons. The alpha acid levels are an indicator of the hop quality from the Cascade and Alpharoma cultivars. Cascade was also a top producer of hop cones with a mean fresh weight of 989.67 g and 900.33 g in 2016 and 2017, respectively. In a nutrient deficiency study, plants were grown in Hoagland hydroponic solutions, which contain all essential nutrients needed for plant growth. This was done to provide photographic documentation to assist local growers with deficiency identification. Deficiencies were induced for nitrogen (N), phosphorus (P), and potassium (K) by removing each of the specific nutrient of interest from each treatment solution. After deficiency symptoms were induced, leaf tissue samples were taken and analyzed for nutrient content. Nutrient deficiencies were documented using photographs. For N, visual symptoms included a chlorotic appearance, undersized leaves, and red petioles. Incipient symptoms for P included necrotic spots, leaf cupping, and undersized leaves. For K, leaf cupping, blue green veins, marginal scorch, and rounded leaf tips. Disease resistance of cultivars is important information for growers and can heavily influence hop yard planning. As such, in another study, an assay for powdery mildew (Podosphaera macularis) was developed that allows for rapid low cost testing of hop cultivars. The assay was tested on the following cultivars: Alpharoma, Cascade, Comet, Sorachi Ace, and Tahoma. Leaves were inoculated with powdery mildew (PM) using a settling tower. Powdery mildew colonies were allowed to grow for two weeks and were then analyzed using ImageJ software to determine percent coverage. At the end of the experiment, Tahoma had significantly greater PM coverage compared to the other cultivars, indicating that Tahoma is less resistant to the specific PM strain.

Natural products are teeming with potential therapeutic agents. One group of compounds, polyphenols, from plants, exhibit anti-cancer, anti-inflammatory and anti-oxidant activities. Four polyphenolic compounds and their enantiomers were investigated in this thesis; the stilbene, 3-methoxypterostilbene, a structural analog of resveratrol, and the chiral prenylflavonoids from hops (Humulus lupulus L.); 8-prenylnaringenin, 6-prenylnaringenin and isoxanthohumol. A high performance liquid chromatography method for 3-methoxypterostilbene and enantiospecific liquid chromatography-mass spectrometry assays for the three prenylflavonoids were developed and validated. The methods allowed for quantification of these four polyphenols in biological samples and plant-based materials. Content analysis studies of 3-methoxypterostilbene and the three prenylflavonoids in traditional Chinese medicinal plants and hops-containing nutraceuticals were carried out, respectively. The pharmacokinetics of these four compounds were delineated through intravenous and oral administration in rats. 3-Methoxypterostilbene demonstrated greater bioavailability in rats than reported values for resveratrol. Enantiomeric differences in disposition parameters were observed for the three prenylflavonoids along with differences between compounds despite only small structural differences. The in vitro pharmacodynamics of these four compounds were elucidated including anti-oxidant, anti-inflammatory, anti-diabetic and cytochrome P450 modulation activities. All four compounds demonstrated a range of bioactivities related to chronic diseases and potential drug-botanical interactions. Further studies of polyphenols, especially clinical studies, are needed along with enantiospecific study when applicable to continue delineating the importance of bioactivity, pharmacokinetics and safety. Natural products are further developed into nutraceuticals and sold over-the-counter for both human and veterinary use but are not currently required to demonstrate efficacy prior to marketing. In the final section of this thesis, Phycox®, a multi-component veterinary nutraceutical for joint health was investigated for pharmacological activity in an in vitro model of canine osteoarthritis along with select constituents. A pilot single-dose pharmacokinetic study in dogs was also undertaken. Two liquid chromatography tandem mass spectrometry methods were developed and validated to detect constituents in serum. In vitro study results indicated that Phycox® was able to reduce inflammatory mediators similar to the NSAID, carprofen, and acute pharmacokinetic results revealed that detectable concentrations of glucosamine were evident in serum. It is suggested that further clinical studies of Phycox® are warranted to optimize its usage. / May 2016

Orientador: Rodrigo Máximo Sánchez-Román / Resumo: O cultivo de lúpulo (Humulus Lupulus L) exige um adequado fornecimento de água, principalmente na fase de desenvolvimento, o qual envolve a formação e crescimento dos ramos laterais, florescimento, formação das inflorescências e maturação dos cones. Sabendo da importância do fornecimento de água na fase de desenvolvimento, a irrigação passou a ser utilizada nas áreas de cultivo desde o século XIX. No contexto da irrigação, a qualidade da água é um fator importante e que, quando não se dá a devida atenção, pode causar efeitos deletérios no sistema de irrigação, solo e na planta. O potencial hidrogeniônico (pH) é um dos parâmetros que caracteriza a qualidade de água para irrigação podendo influenciar na microbiologia do solo e no processo de intercâmbio de cátions (solo para a solução e vice-versa). Em virtude da importância da irrigação e dos poucos estudos relacionados ao pH da água da irrigação da cultura do lúpulo, este trabalho teve como objetivo avaliar o efeito da irrigação com diferentes faixas de pH da água sob o desenvolvimento agronômico do lúpulo, variedade Cascade, cultivado em ambiente protegido. A pesquisa foi conduzida em casa de vegetação no Departamento de Bioprocessos e Biotecnologia – DBB da Faculdade de Ciências Agronômicas – FCA/UNESP, Campus de Botucatu. O delineamento estatístico utilizado foi o inteiramente casualizados (DIC) com três tratamentos e dez repetições. Os tratamentos foram constituídos por três faixas de pH da água, sendo: T1 – pH 6 a 6,5, T... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Hop cultivation (Humulus Lupulus L) requires a adequate supply of water, especially at the developmental stage, or as involved in lateral branch formation and growth, flowering, inflorescence formation and cone maturation. Knowing the importance of water supply in the development phase, irrigation has been used in cultivated areas since the 19th century. In the context of irrigation, water quality for irrigation is an important factor which, when not given due attention, can have deleterious effects on the irrigation system, soil and the plant. The hydrogen potential (pH) of water is one of the parameters that characterizes the quality of water for irrigation and can influence soil microbiology and the cation exchange process (soil for solution and vice versa). Due to the importance of irrigation and the few studies related to the water pH of the irrigation of the hop culture, this work had as objective evaluate the effect of irrigation with different water pH ranges on the agronomic development of hops, Cascade variety cultivated in protected environment. The research was conducted in a greenhouse at the Department of Bioprocesses and Biotechnology – DBB of the Faculty of Agronomic Sciences - FCA/UNESP, Botucatu Campus. The statistical design used was the completely randomized design (CRD) with three treatments and ten replications. The treatments consisted of three water pH ranges: T1 - pH 6 to 6.5, T2 - pH 6.5 to 7 and T3 - 7 to 7.5. To obtain the treatments, 0,1 mol L-1 s... (Complete abstract click electronic access below) / Mestre

Submitted by Marilene Donadel (marilene.donadel@unioeste.br) on 2018-10-31T00:41:51Z No. of bitstreams: 1 Sergio_Kupski_2015.pdf: 5793850 bytes, checksum: 1abe03c5de9e1065ce978b22b486cc50 (MD5) / Made available in DSpace on 2018-10-31T00:41:51Z (GMT). No. of bitstreams: 1 Sergio_Kupski_2015.pdf: 5793850 bytes, checksum: 1abe03c5de9e1065ce978b22b486cc50 (MD5) Previous issue date: 2015-02-26 / Supercritical fluid extraction (SFE) process of vegetable oils with CO2 is an alternative to the traditional processes, with advantages related to final product quality, extraction yield and simpler separation stages. Hops (Humulus Lupulus L.) are widely used for beer brewing and their essential oils contain substances with potential pharmaceutical use. Mathematical modeling of the mass transfer mechanisms that rule the SFE may provide information about the process parameters influence on the total yield and extraction rate. This work intends to analyze the influence of the following parameters: temperature (de 35 a 55º C), pressure (100 a 200 bar), particle size (de 3,28 X 10-4 m a 1,02 X 10-3 m), particle size distribution and raw material treatment. Extraction of hop with supercritical carbon dioxide was performed on experimental scale. The data obtained was used to adjust the extraction curves simulated with five models available at literature. Besides those, the data allowed to test a proposed model, applied for the extraction from hop bracts. Tested models adjusted the experimental data satisfactorily and the estimated parameters analysis allowed building another model. / O processo de extração supercrítica (SFE – supercritical fluid extraction) de óleos vegetais com CO2 é uma alternativa aos tradicionais, com vantagens relacionadas à qualidade do produto final, rendimento da extração e simplificação das etapas de separação. Os cones de lúpulo (Humulus lupulus L.) são amplamente utilizados na produção de cerveja e seus óleos essenciais contêm substâncias com potencial uso na indústria farmacêutica. A modelagem matemática dos mecanismos de transferência de massa que governam a SFE pode fornecer informações sobre a influência dos parâmetros de processo no rendimento total e na taxa de extração. Este trabalho propõe analisar a influência dos seguintes parâmetros: temperatura (de 35 a 55º C), pressão (100 a 200 bar), diâmetro da partícula (de 3,28 X 10-4 a 1,02 X 10-3 m), distribuição granulométrica das partículas e a forma de preparo da matéria prima. Foi realizada a extração de lúpulo com dióxido de carbono supercrítico com equipamento em escala experimental. Os dados obtidos foram utilizados para ajustar as curvas de extração simuladas utilizando cinco modelos fenomenológicos disponíveis na literatura. Além destes, os dados permitiram testar o modelo proposto, aplicado para a extração a partir das brácteas da inflorescência do lúpulo. Os modelos testados ajustaram de forma satisfatória os dados experimentais e a análise dos parâmetros estimados permitiu fundamentar um segundo modelo proposto.