The dwarf wedgemussel (Alasmidonta heterodon) is a federally endangered freshwater mussel that once ranged from New Brunswick to North Carolina, but now only exists in isolated populations throughout its diminished range. Laboratory propagation in conjunction with augmentation or reintroduction is considered a critical component of dwarf wedgemussel restoration. My thesis aimed to I) develop in-vitro propagation techniques including methods to minimize microbial contamination for two Alasmidonta species and II) compile and assess critical fish assemblage information at existing dwarf wedgemussel presence and absence locations to inform future restoration activities. For the first objective (Chapter 2) I assessed three methods of contamination mitigation (media change frequency, concentration of the antifungal Amphotericin B, and method of antifungal replenishment). Across all experiments, higher levels of contamination severity had a negative impact on transformation success. In my experiments, dishes that had media changes every other day (vs. those changed daily and every 3 days) had the highest contamination and the lowest glochidia transformation success. Treatments with the lowest (0 µg/mL) concentration of the antifungal, Amphotericin B, and got a low-dose replenishment of Amphotericin B (vs frequent media changes) had the highest transformation success. The fungus was identified as Candida parapsilosis, a common fungus found in aquatic and human environments; future propagation efforts should use mitigation methods that are specific to the fungal contaminant. In the assessment of host fish near dwarf wedgemussel locations (Chapter 3), two fish repeatedly emerged as indicators of dwarf wedgemussel occurrence and abundance: the tessellated darter (Etheostoma olmstedi) and brown trout (Salmo trutta). Tessellated darter was positively related to dwarf wedgemussel which supports the importance of tessellated darter as a host fish in the wild. The negative relationship between dwarf wedgemussel and brown trout may indicate a difference in habitat between the species or brown trout’s predation upon wild hosts. Results from this project will be used in conjunction with habitat and genetic information to inform future restoration plans in the northeast and add to the growing body of literature on in-vitro propagation of freshwater mussels.

In the summers of 1989 and 1990, researchers in Va. and Md. began to observe lateseason reductions in com vigor in areas treated with nicosulfuron or primisulfuron for postemergence johnsongrass control. Symptoms observed included chlorosis, reddening of the leaves and shortening of the internodes. The nature and time of symptom expression were consistent with those caused by maize chlorotic dwarfvirus (MCDV) and maize dwarf mosaic virus (MDMV) infection of com. It was hypothesized that postemergence johnsongrass control increased the incidence and severity of MCDV and MDMV in virus-susceptible corn hybrids due to increased feeding by vectors of these viruses on treated corn. Field experiments were conducted in 1991 and 1992 to evaluate the effect of postemergence johnsongrass control with broad casted nicosulfuron, postemergence directed imazethapyr, mechanical control and no control on virus disease incidence and severity in a virus-susceptible ('Southern States 565') and a virus-tolerant ('Southern States 844) corn hybrid. Visual injury evaluations taken 10 weeks after treatment showed that the virus-susceptible com hybrid sustained significantly more injury, averaged across johnsongrass control methods, than did the virus-tolerant corn hybrid. Within the virus-susceptible com hybrid, where johnsongrass was controlled, regardless of method, significantly more injury was observed relative to the nontreated check. Further, averaged across johnsongrass control treatments, the virus-tolerant corn hybrid yielded significantly higher compared to the virus-susceptible com hybrid. Experiments conducted in 1993 and 1994 utilized cages as a means of preventing insect movement from the infected johnsongrass to the crop. Blackfaced leafhopper evaluations suggested that the cages significantly reduced leafhopper movement from the infected johnsongrass to the corn, however complete exclusion was not achieved. Results of corn tissue assays showed that MCDV and MDMV were being transmitted, however no treatment differences were detected. Two experiments were conducted in 1994 to analytically test the hypothesis and to determine the time course of MCDV and MDMV double infection of corn tissue. Johnsongrass control treatments evaluated included broadcast nicosulfuron and no treatment. Postemergence johnsongrass control increased MCDV and MDMV incidence 9 to 21 days after treatment. Further, significantly more double infections of MCDV and MDMV were observed 14 to 21 days after treatment in experimental units receiving the nicosulfuron application. / Ph. D.

Gray leaf spot, caused by Cercospora zeae-maydis, can be a yield-limiting factor in maize where continuous minimum tillage practices are followed. Commercial corn hybrids were evaluated for response to gray leaf spot for seven years at two Virginia locations (Shenandoah and Wythe Counties) and one year at a third location in Virginia (Montgomery County). Yield losses, when comparing resistant to susceptible classes, were approximately 2,000 kg ha⁻¹ at Wythe County in 1982, 750 kg ha⁻¹ at Shenandoah County in 1984, and 2,150 kg ha⁻¹ at Montgomery County in 1988. The inheritance of reaction to gray leaf spot was studied using a 14 inbred diallel in Montgomery and Wythe Counties, Virginia in 1987 and 1988 planted in randomized complete block designs. Resistance was found to be highly heritable and controlled by additive gene action. Inbreds producing high yielding, resistant, and agronomically superior hybrids were identified (B68, NC250, Pa875, Va14, Va17, and Va85); and several hybrids between these lines had high levels of resistance, high yield, and good general agronomic characters (B68 x KB1250, KB1250 x Pa875, and NC250 x Pa875). Currently available inbreds could be used to produce hybrids with higher levels of resistance than hybrids currently available to growers, and these could serve as a basis for gray leaf spot breeding programs. Lesion size measurements were not correlated with disease scores. Late-season photosynthesis rates were associated positively with resistance. The hybrids of some inbreds were found to produce high levels of pigment (believed to be anthocyanins) around the gray leaf spot lesions. These did not limit the size of the individual lesion later in the season. Some pigment(s)-producing genotypes were found to be resistant when the pigment character was expressed. This type of resistance must prevent or inhibit infection of the leaf but not later colonization, once established. Maize dwarf mosaic virus (MDMV) also limits maize production in some areas where johnsongrass (Sorghum halepense L.) is a problem. Resistance to MDMV was found to be mainly additive and highly heritable. However, a strong specific combining ability component was found, indicating that the background of the material receiving resistance genes may have a strong effect on the expression of resistance. Inbreds capable of producing high-yielding, resistant, and agronomically acceptable hybrids are available (B68, NC250, A632, Pa875, Va17, and Va85); and several hybrids between these lines have high levels of resistance, high yield, and good general agronomic characters (B68 x KB1250, KB1250 x Pa875, and NC250 x Pa875). / Ph. D.

Barley yellow dwarf (BYD), caused by Barley yellow dwarf virus and Cereal yellow dwarf virus, and is a yield limiting disease of small grains. A research study was initiated in 2015 to identify the implications of BYD on small grain crops of North Dakota. A survey of 187 small grain fields was conducted in 2015 and 2016 to assess cereal aphid diversity; cereal aphids identified included, Rhopalosiphum padi, Schizaphis graminum, and Sitobion avenae. A second survey observed and documented field absence or occurrence of cereal aphids and their incidence. Results indicated prevalence and incidence differed among respective growth stages and a higher presence of cereal aphids throughout the Northwest part of North Dakota than previously thought. Field and greenhouse screenings were conducted to identify hard red spring wheat and durum responses to BYD. Infested treatments in the greenhouse had significantly lower number of spikes, dry shoot mass and yield.

Três experimentos foram desenvolvidos em duas etapas, utilizando-se as dependências e instalações da Embrapa - Agroindústria Tropical, em Fortaleza, CE, com os seguintes objetivos: avaliar quanto aos aspectos físicos, físico-químicos, químicos e bioquímicos de pedúnculos de cajueiro anão precoce CCP-76 durante o desenvolvimento e maturação; caracterizar quantitativa e qualitativamente as alterações nos constituintes da parede celular do pedúnculo associadas aos processos de desenvolvimento e maturação, e verificar o efeito da aplicação pós-colheita de cálcio, em diferentes concentrações, sobre as características físicas, físico-químicas, químicas e bioquímicas dos pedúnculos submetidos a armazenamento refrigerado sob atmosfera modificada. Os cajus utilizados neste trabalho, foram obtidos na área comercial de clones de cajueiro anão precoce instalada na Empresa Mossoró Agro-industrial S.A. O clone selecionado para este estudo foi o CCP-76 por ser o mais cultivado para produção de pedúnculos para comercialização in natura. As colheitas foram feitas em junho e agosto de 1997 para os experimentos 1 e 2 respectivamente, e em novembro de 1998 para o experimento 3. Os cajus, depois de colhidos, foram imediatamente transportados para o laboratório, onde se deu início aos estudos conforme o experimento em questão. Para os experimentos 1 e 2, os cajus foram selecionados em sete estádios de desenvolvimento e maturação de acordo com a coloração externa do pedúnculo e da castanha: 1. verde com castanha verde; 2. verde com castanha madura e seca; 3. verde claro; 4. Início de coloração amarela; 5. Amarelo com início de cor laranja; 6. laranja claro e 7. laranja escuro. No primeiro experimento, foram realizadas análises físicas, físico-químicas, químicas e bioquímicas dos pedúnculos: peso total (castanha + pedúnculo) peso do pedúnculo e o da castanha, diâmetros e comprimento, firmeza, clorofilas, carotenóides, antocianinas, pH, sólidos solúveis, acidez, vitamina C, cálcio, amido, açúcares, compostos fenólicos, pectinas, atividade das enzimas poligalacturonase, pectinametilesterase, polifenoloxidase e peroxidase. No segundo experimento, a partir de material de parede celular extraído, foram realizadas as seguintes análises: celulose, hemicelulose, uronídeos totais, grau de esterificação de pectinas e cálcio ligado. No terceiro experimento, os cajus foram colhidos no estádio de maturação comercial (estádio 7) e submetidos a seleção, préresfriamento, desinfecção em água clorada, secagem ao ar, nova seleção e tratamento com CaCI2 a 0,5%, 1% e 2% com espalhante adesivo, sendo que a testemunha foi apenas em água contendo o espalhante. Após secagem ao ar, os cajus foram acondicionados em bandejas, as quais foram envolvidas em PVC armazenadas em câmara com temperatura de 5°C ± 1°C e umidade relativa de 88% ± 3%. A cada 5 dias, por um período de 25 dias, foram tomadas amostras para execução das seguintes análises: perda de peso, firmeza, senescência, antocianinas, pectinas, compostos fenólicos, vitamina C, pH, sólidos solúveis, acidez, açúcares solúveis, cálcio, atividades de pectinamestilesterase e poligalacturonase. A maior relação sólidos solúveis/acidez foi atingida no estádio 7, indicando que este é o ideal para a colheita de pedúnculos ocasião em que alcança sua máxima qualidade para o consumo. Os pedúnculos de caju apresentaram amaciamento acentuado durante o desenvolvimento e maturação, observando-se paralelamente redução no teor de pectina total e aumento na percentagem de solubilização. A firmeza dos pedúnculos apresentou correlação significativa e inversa com a solubilidade das pectinas e com a atividade das enzimas PME e PG. A solubilidade da pectina, por sua vez, mostrou correlação significativa e positiva com a atividade de ambas as enzimas, que se correlacionaram entre si positivamente. O ambiente refrigerado aliado a atmosfera modificada, pode ser usado com sucesso no retardamento do processo de senescência do pedúnculo de caju, uma vez que aumenta substancialmente sua vida pós-colheita sem causar injúrias fisiológicas por até 20 dias. Pedúnculos tratados com cálcio foram mais firmes do que os não tratados, sendo que a partir de 0,5% não se observou um aumento do efeito. Doses crescentes de cálcio proporcionaram teores médios de cálcio total, solúvel e insolúvel superiores aos da tetemunha. O tratamento com cálcio não inibiu a atividade da pectinametilesterase. Porém foi decisivo na atividade de poligalacturonase, uma vez que os pedúnculos não tratados obtiveram valores médios de atividade dessa enzima superiores aos tratados. / Three experiments were carried out in two stages, using the EMBRAPA (Tropical and Agricultural Industry) - plants and annexes in Fortaleza, CE aiming the following: to evaluate the physícal, physicochemical, chemical and biochemical characteristícs of cashew apples of the clone CCP-76 during maturation and developing stages; to characterize qualitative and quantitative changes in the cell wall contents of the cashew apples due to developing and maturation processes; and to verify the post harvest application effect of calcium under different concentrations on cashew apples. The physical, physicochemical, chemical and biochemical characteristics of cashew apples stored under refrigeration and modified atmosphere were studied. The cashew apples used for this work were obtained from the clone experimental areas of early dwarf cashew trees of Mossoró Agricultural Company S.A. The selected clone for this study was the CPP-76 due to be recommended for commercial planting in function of its production aspects and cashew apple quality for consumption in natura. The harvests were done in June and August 1997 for the 1st and 2nd experiments, respectively and in November 1998 for the 3rd experíment. The cashew apples, after being harvested were transported immediately to the laboratories, for the starting of the due experiment. For the experiments 1 and 2, the cashew apples were selected in seven developing and maturation stages according to the cashew apple size and skin externai color as the following: 1- green cashew apple and nut; 2- green cashew apple with ripen dry nut; 3- light green cashew apple, 4- yellowish, 5- onset of the orange color, 6- light orange color, 7- dark orange color. For the first experiment the following physical, physicochemical, chemical and biochemical analyses were realized: total weight (cashew apple + nut), nut weights, cashew apple weights, diameters and lengths, firmness, chlorophylls, carotenoids, anthocyanins, pH, total soluble solids, titratable acidity, vitamin C, calcium, starch, sugars, phenolic compounds, pectins, polygalacturonase, pectinmethylesterase, polyphenoloxidade e peroxidase activities. In the second experiment, the following analyses on the cell wall material extracted, were realized: cellulose, hemicellulose, total uronides, pectin esterification degree and bound calcium. For the third experiment, the cashew apples were harvested on the commercial maturation stage and submitted to a selection, pre-cooling, chlorinated water washing, air drying, new selection and treatment with CaCI2 0.5%, 1% and 2% with thickener being the testimony, water and thickener only. After air drying, the cashew apples were placed on trays, involved with PVC film and stored at 5°C ± 1°C and relative humidity 88% ± 3%. Samples were taken for analyses at each 5 days during a 25 day period and the following analyses carried out: weight loss, firmness, senescence, anthocyanins, pectins, tannins, vitamin C, pH, total soluble solids, titratable acidity, calcium, soluble sugars, polygalacturonase, pectinmethylesterase activities. The major ratio soluble solids/acidity was reached in the stage 7, indicating that stage as the ideal for cashew apple harvesting, occasion that the edible quality peak is reached. The cashew apples showed a sharp softness during the development and maturation in parallel to a decrease in total pectin levei and increase in soluble pectin/total pectin ratio. There was an inverse and significative correlation between cashew apples firmness and pectin solubility, PME and PG activities. On the other hand, pectin solubility showed a significative and positive correlation with the activity of both enzymes, which correlated, positively to each other. The refrigeration added to the modified atmosphere may be used with success to delay cashew apple senescence process once they increase substantially its post harvest shelf life without causing physiological damage up to 20 days of storage. The cashew apples treated with calcium showed better firmness than those without treatment did, although, under calcium concentration up to 0,5% that effect was not observed anymore. Increased calcium dosages gave mean levei of total, soluble and insoluble calcium higher than the testimony. The calcium treatment did not inhibit the pectinmethylesterase activity. However, calcium was decisive for polygalacturonase activity, once the cashew apples that did not receive calcium treatment, obtained mean values for that enzyme activity higher than those treated.

Queratinócitos são células bastante atrativas para a transferência gênica ex vivo e liberação sistêmica, uma vez que as proteínas secretadas por estas células podem atingir a circulação via um mecanismo similar ao processo natural. No presente trabalho, queratinócitos transduzidos mediante um vetor retroviral com o gene do hormônio de crescimento de camundongo (mGH) foram submetidos a um tratamento de aderência ao colágeno e à análise clonal, com o intuito de enriquecer esta população de queratinócitos em células-tronco. O principal resultado foi um aumento da viabilidade celular in vitro dos queratinócitos tratados, que poderá se refletir num aumento da durabilidade da secreção do hormônio in vivo, quando realizado o implante de culturas organotípicas em camundongos anões imunodeficientes (lit/scid). Foi também utilizado um modelo de terapia gênica in vivo, baseado na eletrotransferência de DNA plasmidial (naked DNA), contendo o gene do hormônio de crescimento humano (hGH), no músculo quadríceps de camundongos anões (lit/lit) e anões imunodeficientes (lit/scid). Foram padronizadas as condições de eletroporação em 8 pulsos de 50 V e 20 ms com 0,5 s de intervalo, utilizando um plasmídeo com o promotor da ubiquitina C e a sequência genômica do hGH. A administração de uma dose única de 50 g do plasmídeo pUC-UBI-hGH em camundongos lit/scid, seguida de eletroporação, propiciou pela primeira vez na literatura obtenção de níveis sustentáveis na circulação de 1,5-3,0 ng hGH/ml, durante 60 dias. Esses animais tratados com DNA apresentaram um aumento de peso altamente significativo (P<0,001) de 33,1%, comparado a uma perda de peso, não significativa, no grupo controle (camundongos injetados com salina + eletroporação). Os músculos quadríceps dos animais tratados apresentaram um aumento de 48%, quando comparados aos dos animais do grupo controle (P<0,001). Outro estudo de eletrotransferência foi realizado comparando a utilização da sequência genômica do hGH (gDNA) com a complementar (cDNA), em plasmídeos sob o controle do promotor de citomegalovírus (CMV). Foi observado que o cDNA do hGH foi expresso de maneira mais eficiente na circulação de camundongos lit/scid, por um período de 21 dias. Foram também construídos vetores lentivirais com os genes do hGH (cDNA e gDNA) e do mGH (cDNA), que serão utilizados num próximo estudo, tanto para a transdução de queratinócitos, como para a eletrotransferência in vivo. Vetores lentivirais serão de fato necessários para futuros estudos devido a sua reduzida toxicidade em comparação com os retrovirais. Os resultados deste trabalho, assim como as perspectivas de estudo abertas, têm como meta estabelecer condições para que a terapia gênica seja num futuro próximo uma alternativa viável e segura para o tratamento da deficiência de GH e de outras doenças sistêmicas. / Keratinocytes are very attractive cells for ex vivo gene transfer and systemic delivery, since proteins secreted by these cells may reach the circulation via a mechanism which mimics the natural process. In this study, retrovirally transduced keratinocytes with the mouse growth hormone (mGH) gene underwent a treatment for adhesion to collagen and clonal analysis, in order to enrich in stem cells the keratinocyte population. The main result was an in vitro increase of cell viability for treated keratinocytes, which should result in an increase of the in vivo sustainability of hormone secretion, when implanting organotypic cultures onto immunodeficient dwarf (lit/scid) mice. A model for in vivo gene therapy based on the electrotransfer of human growth hormone (hGH)-coding naked DNA in the exposed quadriceps muscle of dwarf (lit/lit) and immunodeficient dwarf (lit/scid) mice was also utilized. Electroporation conditions were optimized, setting up eight 50-V pulses of 20 ms at a 0.5 s interval, using a plasmid containing the ubiquitin C promoter and the genomic hGH sequence. Administration of a single dose of 50 g of this plasmid led, for the first time in the literature, to sustained levels of circulating hGH of the order of 1.5-3 ng/ml, up to 60 days. The DNA-treated animals presented a highly significant weight gain (P<0.001) of 33.1%, compared to a non-significant weight loss of 4.2% for the control group (saline injected mice + electroporation). The injected quadriceps muscles of the DNA-treated mice showed a 48% weight increase, when compared to the control group (P<0.001). Another electrotransfer study was carried out comparing the use of the genomic hGH sequence (gDNA) with the complementary sequence (cDNA), cloned under the control of the cytomegalovirus (CMV) promoter. It was observed that hGH was more efficiently expressed in the circulation of lit/scid mice, for 21 days, when injecting cDNA. Lentiviral vectors containing the hGH (cDNA and gDNA) and the mGH (cDNA) genes were also constructed and will be used in a next study, both for the transduction of keratinocytes or for in vivo electrotransfer. Lentiviral vectors will in fact be necessary for future studies, considering their reduced toxicity when compared to retroviral vectors. The results of this work, as well as opening perspectives of new studies, will establish in the near future conditions for gene therapy as a feasible and safe option for the treatment of GH deficiency and other systemic diseases.

Le monde grec antique nous a transmis l'image d'une civilisation imprégnée de beauté à travers ses œuvres artistiques, cette image étant renforcée par la richesse et la qualité de ses productions littéraires. La quête de la beauté suprême atteint son apogée durant la période classique, et dans ce contexte où tout semble tendre vers cet idéal, la laideur d'apparence est très peu évocatrice; les quelques personnages grecs laids ou difformes auxquels nous pouvons penser, Thersite, Socrate ou Héphaïstos, semblent constituer la minorité d'exceptions qui confirme la règle tellement ils sont présentés comme des cas à part. Cette image que nous avons des Grecs est évidemment trompeuse: les maladies, les difformités et les différentes formes de laideur devaient naturellement faire partie de leur vie quotidienne. Travailler sur ce sujet encore peu exploré nous a paru fort intéressant; pour tenter de saisir ce que les Grecs eux-mêmes ont peu exprimé, nous avons couvert le plus d'aspects possibles en ayant recours à l'ensemble des textes de la période classique sans oublier l'iconographie, indispensable pour une étude sur l'esthétique. / The ancient Greek world passed on to us the image of a civilization filled with beauty through its artistic works, this image being strengthened by the richness and quality of its literary productions. The quest for supreme beauty reached its peak during the classical period, and in this context where everything seems to tend towards this ideal, physical ugliness is not something we generally equate with Greek thought; a few ugly or deformed Greek characters of whom we can think, Thersite, Socrates or Hephæstus, are so isolated that they seem to be the exception rather than the rule. Thus, this image is clearly incorrect since sickness, deformity, and other kinds of ugliness were natural parts of their lives. This little investigated subject is full of interest to us. In our efforts to seize what the Greeks themselves failed to express, we covered every relevent aspect possible by using all the texts of the classical period, not leaving the iconography behind, which is indispensable for a study on aesthetics.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-13T14:20:21Z No. of bitstreams: 1 Iwanne Lima Coelho.pdf: 1395783 bytes, checksum: bfb69bfcc0a83cb3f0e7ff59aade864a (MD5) / Made available in DSpace on 2017-03-13T14:20:21Z (GMT). No. of bitstreams: 1 Iwanne Lima Coelho.pdf: 1395783 bytes, checksum: bfb69bfcc0a83cb3f0e7ff59aade864a (MD5) Previous issue date: 2013-07-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The occurrence of stem bleeding, caused by Ceratocystis paradoxa (Anamorphic: Thielaviopsis paradoxa), compromises the productivity of green dwarf coconut and poses a threat to the entire production chain of green coconut. There are no fungicides registered in the Ministério da Agricultura Pecuária e Abastecimento (MAPA) for the management of this disease, the indiscriminate use of the fungicide thiophanate methyl may result in the emergence of resistant pathogenic strains, environmental contamination and to human health. Biological control using yeast, promising biocontrol agents, may represent an efficient and applicable solution when aggregated to crop management. Work on yeasts not recovered in control of the causal agent of this disease, as well as studies related to its field control. The study aimed to select yeasts from fruits of the green dwarf coconut, with potential for biocontrol of stem bleeding coconut on the seedlings of green dwarf coconuts. Thirty-seven yeasts, isolated from immature coconuts, were evaluated in vivo for their potential to reduce the symptoms of stem bleeding in seedlings of green dwarf coconuts, packed in a greenhouse under 40 ± 2 ° C and 60 % RH, for two methods of application: spray (10 mL de 1x107 cells mL-1) and injection (1 mL de 1x107 cells mL-1); and in vitro (28 ± 2 °C, 12 h photoperiod), as for inhibition of mycelial growth, volatile metabolic action, parasitism, antibiosis and "killer" activity. Fifteen yeast strains in vivo reduced the percentage of the area damaged by C. paradoxa isolated Moju - PA. In vitro, the isolates identified as Pichia guilliermondii (LEV 25) and Candida dosseyi (LEV47), stood out as potentially promising antagonists, because of the diameter of the inhibited mycelial growth , competing for space and nutrients, and reduced production of conidia by the action of volatile metabolites on pathogenic isolates of Moju - PA, Neópolis- SE and Sousa –PB. This is the first report of reduction of the symptoms of stem bleeding in seedlings of green dwarf coconuts by the antagonistic action of yeast on C. paradoxa. The isolates of P. guilliermondii and C. dosseyi have potential as a component of integrated management of coconut stem bleeding / A ocorrência da resinose, causada por Ceratocystis paradoxa (Anamorfo: Thielaviopsis paradoxa), compromete a produtividade do coqueiro-anão-verde e representa ameaça para toda a cadeia produtiva do coco-verde. Não há registros no Ministério da Agricultura Pecuária e Abastecimento (MAPA) de fungicidas para o manejo dessa doença, o uso indiscriminado do fungicida tiofanato metílico pode resultar no surgimento de linhagens patogênicas resistentes, contaminação ambiental e à saúde humana. O controle biológico utilizando-se leveduras, promissores agentes de biocontrole, pode representar uma solução eficiente e aplicável quando agregada ao manejo da cultura. Não foram recuperados trabalhos sobre leveduras no controle do agente causal dessa enfermidade, assim como estudos referentes ao seu controle em campo. O trabalho teve por objetivo selecionar leveduras, obtidas de frutos do coqueiro-anão-verde, com potencial para o biocontrole da resinose do coqueiro, em mudas de coqueiro-anão-verde. Trinta e sete leveduras, isoladas de cocos imaturos, foram avaliadas in vivo quanto ao potencial de redução dos sintomas de resinose em mudas de coqueiro-anão-verde, acondicionadas em telado sob 40±2 °C e 60% UR, por dois métodos de aplicação: aspersão (10 mL de 1x107 células mL-1) e injeção (1 mL de 1x107 células mL-1); e in vitro (28±2 °C, 12 h fotoperíodo), quanto inibição de crescimento micelial, ação de metabólitos voláteis, parasitismo, antibiose e atividade “killer”. Quinze cepas de leveduras, reduziram in vivo o percentual de área lesionada por C. paradoxa isolado de Moju-PA. In vitro, os isolados identificados como Pichia guilliermondii (LEV 25) e Candida dosseyi (LEV 47), destacaram-se como os antagonistas potencialmente promissores, pois inibiram o diâmetro de crescimento micelial, competindo por espaço e nutrientes, e reduziram a produção de conídios, pela ação de metabólitos voláteis sobre os isolados patogênicos de Moju-PA, Neópolis-SE e da Sousa-PB. Este é o primeiro relato da redução dos sintomas de resinose em mudas de coqueiro-anão-verde pela ação antagônica de leveduras sobre C. paradoxa. Os isolados de P. guilliermondii e C. dosseyi apresentam potencialidade como componente do manejo integrado da resinose do coqueiro.

Queratinócitos são células bastante atrativas para a transferência gênica ex vivo e liberação sistêmica, uma vez que as proteínas secretadas por estas células podem atingir a circulação via um mecanismo similar ao processo natural. No presente trabalho, queratinócitos transduzidos mediante um vetor retroviral com o gene do hormônio de crescimento de camundongo (mGH) foram submetidos a um tratamento de aderência ao colágeno e à análise clonal, com o intuito de enriquecer esta população de queratinócitos em células-tronco. O principal resultado foi um aumento da viabilidade celular in vitro dos queratinócitos tratados, que poderá se refletir num aumento da durabilidade da secreção do hormônio in vivo, quando realizado o implante de culturas organotípicas em camundongos anões imunodeficientes (lit/scid). Foi também utilizado um modelo de terapia gênica in vivo, baseado na eletrotransferência de DNA plasmidial (naked DNA), contendo o gene do hormônio de crescimento humano (hGH), no músculo quadríceps de camundongos anões (lit/lit) e anões imunodeficientes (lit/scid). Foram padronizadas as condições de eletroporação em 8 pulsos de 50 V e 20 ms com 0,5 s de intervalo, utilizando um plasmídeo com o promotor da ubiquitina C e a sequência genômica do hGH. A administração de uma dose única de 50 g do plasmídeo pUC-UBI-hGH em camundongos lit/scid, seguida de eletroporação, propiciou pela primeira vez na literatura obtenção de níveis sustentáveis na circulação de 1,5-3,0 ng hGH/ml, durante 60 dias. Esses animais tratados com DNA apresentaram um aumento de peso altamente significativo (P<0,001) de 33,1%, comparado a uma perda de peso, não significativa, no grupo controle (camundongos injetados com salina + eletroporação). Os músculos quadríceps dos animais tratados apresentaram um aumento de 48%, quando comparados aos dos animais do grupo controle (P<0,001). Outro estudo de eletrotransferência foi realizado comparando a utilização da sequência genômica do hGH (gDNA) com a complementar (cDNA), em plasmídeos sob o controle do promotor de citomegalovírus (CMV). Foi observado que o cDNA do hGH foi expresso de maneira mais eficiente na circulação de camundongos lit/scid, por um período de 21 dias. Foram também construídos vetores lentivirais com os genes do hGH (cDNA e gDNA) e do mGH (cDNA), que serão utilizados num próximo estudo, tanto para a transdução de queratinócitos, como para a eletrotransferência in vivo. Vetores lentivirais serão de fato necessários para futuros estudos devido a sua reduzida toxicidade em comparação com os retrovirais. Os resultados deste trabalho, assim como as perspectivas de estudo abertas, têm como meta estabelecer condições para que a terapia gênica seja num futuro próximo uma alternativa viável e segura para o tratamento da deficiência de GH e de outras doenças sistêmicas. / Keratinocytes are very attractive cells for ex vivo gene transfer and systemic delivery, since proteins secreted by these cells may reach the circulation via a mechanism which mimics the natural process. In this study, retrovirally transduced keratinocytes with the mouse growth hormone (mGH) gene underwent a treatment for adhesion to collagen and clonal analysis, in order to enrich in stem cells the keratinocyte population. The main result was an in vitro increase of cell viability for treated keratinocytes, which should result in an increase of the in vivo sustainability of hormone secretion, when implanting organotypic cultures onto immunodeficient dwarf (lit/scid) mice. A model for in vivo gene therapy based on the electrotransfer of human growth hormone (hGH)-coding naked DNA in the exposed quadriceps muscle of dwarf (lit/lit) and immunodeficient dwarf (lit/scid) mice was also utilized. Electroporation conditions were optimized, setting up eight 50-V pulses of 20 ms at a 0.5 s interval, using a plasmid containing the ubiquitin C promoter and the genomic hGH sequence. Administration of a single dose of 50 g of this plasmid led, for the first time in the literature, to sustained levels of circulating hGH of the order of 1.5-3 ng/ml, up to 60 days. The DNA-treated animals presented a highly significant weight gain (P<0.001) of 33.1%, compared to a non-significant weight loss of 4.2% for the control group (saline injected mice + electroporation). The injected quadriceps muscles of the DNA-treated mice showed a 48% weight increase, when compared to the control group (P<0.001). Another electrotransfer study was carried out comparing the use of the genomic hGH sequence (gDNA) with the complementary sequence (cDNA), cloned under the control of the cytomegalovirus (CMV) promoter. It was observed that hGH was more efficiently expressed in the circulation of lit/scid mice, for 21 days, when injecting cDNA. Lentiviral vectors containing the hGH (cDNA and gDNA) and the mGH (cDNA) genes were also constructed and will be used in a next study, both for the transduction of keratinocytes or for in vivo electrotransfer. Lentiviral vectors will in fact be necessary for future studies, considering their reduced toxicity when compared to retroviral vectors. The results of this work, as well as opening perspectives of new studies, will establish in the near future conditions for gene therapy as a feasible and safe option for the treatment of GH deficiency and other systemic diseases.

Spatially-resolved studies of nearby star-forming galaxies are essential to understand various physical and chemical phenomena at play in the interstellar medium in the galaxies, and consequently to obtain a comprehensive picture of galaxy formation and evolution. In this thesis, I perform spatially-resolved analyses of chemical abundances and star-formation in nearby star-forming galaxies - blue compact dwarf galaxies (BCDs) and spiral galaxies. I map various properties of H II regions and the surrounding gas within three BCDs, using integral field spectroscopic (IFS) data from the Gemini Multi-Object Spectrograph-North. While answering questions related to chemical homogeneity, ionisation mechanisms and stellar populations within BCDs, I address more profound issues, which go beyond the characterisation of studied BCDs and aim to explain global phenomena with broader implications. The BCD NGC 4449 hosts a metal-poor central star-forming region, which I explain by various scenarios related to the interplay between star-formation, metal-distribution and gas dynamics within galaxies. The BCD NGC 4670 shows an unusual negative relationship between the nitrogen-to-oxygen ratio and oxygen abundance at spatially-resolved scales. I explore this relation with chemical evolution models and by comparison to other star-forming galaxies and suggest that nitrogen enrichment, variations in star-formation efficiency or hydrodynamical effects may be responsible for the observed relation. For another BCD, SBS 1415+437, the spatially-resolved abundances on average agree with the integrated abundance, implying that low-redshift spatially-resolved results may be directly compared with unresolved high-redshift results. I study spiral galaxies to address long-standing issues related to the reliability of metallicity calibrators and the Schmidt Law of star-formation. Using IFS data of twenty-four spiral galaxies taken with the Multi-Unit Spectroscopic Explorer, I find that the current strong-line metallicity calibrators for H II regions are unsuitable for regions dominated by diffuse ionised gas (DIG). I devise new recipes for estimating the metal-content of the DIG. For another set of nine spiral galaxies, I use multi-wavelength data to show that the spatially-resolved Schmidt relation is very sensitive to the consideration of diffuse background, which is a component unrelated to the current star-formation. Removal of this component from the SFR tracers and the atomic gas results in similar local and global Schmidt relation. To conclude, the spatially-resolved analyses presented in this thesis have led to discoveries and further questions, which I will address in my ongoing and future works.